EA045393B1 - EFFECTIVE PRODUCTION OF Pichia YEAST AND ITS USE TO IMPROVE PLANT AND ANIMALS HEALTH - Google Patents

Description

Cross-reference to related patent applications

The application for this invention claims the priority of provisional patent applications

U.S. No. 62/537670, filed July 27, 2017, and No. 62/563,992, filed September 27, 2017, each of which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

In the agriculture, horticulture, livestock and aquaculture industries, several common problems hinder farmers' ability to increase yield while keeping costs low. These include infections and infestations caused by bacteria, fungi and other pests and pathogens; high costs of feed and chemical fertilizers, including their impact on the environment and health; and the difficulty of dissolving essential nutrients, particularly phosphate, into usable forms, but not limited to.

In agriculture and horticulture, farmers relied on the use of synthetic chemicals and chemical fertilizers to increase yields and protect crops from pathogens, pests, and diseases; however, when used excessively or incorrectly, these substances can leach into surface waters, flow into groundwater, and evaporate into the air. Responsible management of these substances as sources of air and water pollution is a critical environmental and commercial concern. Even when used correctly, over-dependence and long-term use of some chemical fertilizers and pesticides cause detrimental changes to soil ecosystems, reduce stress tolerance, increase pest resistance, and impede the growth and viability of plants and animals.

For livestock and aquaculture farming, the cost of feeding can be significant. For example, feeding fish populations, especially in the case of large-scale farming, accounts for the bulk of the ongoing costs of aquaculture production. Fish food must contain highly digestible sources of protein along with a full complement of other components necessary for proper nutrition, including amino acids, carbohydrates, vitamins, minerals, cholesterol and essential fatty acids. Traditionally, feed sources have included fishmeal derived from, for example, wild or farmed fish, such as fishmeal, shrimp mixture and squid meal; However, due to the increasing costs in farming and fish production for the preparation of traditional feeds derived from fish and industrial fish oil, many aquaculture farms have begun to use grain-based feeds for feeding fish. Ingredients currently used to produce fish feed include corn, soybeans, whole wheat and sorghum. The health effects of grain-based diets on the fish themselves are not fully known, but are likely to cause adverse effects in the long term.

Large-scale livestock operations typically rely on feed grains such as corn and soybeans to provide the necessary carbohydrates, fiber and protein for optimal animal growth. These foods are not optimal for animals whose stomachs are best suited to digest grass and other high cellulose plants, and this can lead to a number of problems. For example, livestock farms relying on grain-based feeds often have to rely on antibiotics to prevent the spread of disease, as an inappropriate diet coupled with the cramped living conditions of highly concentrated feed farms results in animals with weakened immune systems.

In addition to the cost of feeding and protecting both crops and farmed animals, nutrient deficiencies, particularly phosphorus deficiency, are often an ongoing problem in these industries. Phosphorus in the form of phosphates is essential for the biosynthesis of genetic material and ATP, as well as many other functions for the growth and viability of all organisms. To prevent or treat phosphorus deficiency in plants, growers often apply phosphorus-containing fertilizers, usually to the soil. Application of these compounds to the soil, however, does not guarantee that the plant will be able to absorb and use phosphorus. Some forms of phosphorus, such as phytates, are insoluble or otherwise not absorbable by plant roots. Phytate serves as a storehouse for phosphorus, energy, cations, and myoinositol (a cell wall precursor), which remains in a form that cannot be used by plants and nonruminant animals.

For farmed animals, such as those grazing in areas where soils have low levels of plant bioavailable phosphorus, phosphorus deficiency can lead to problems including infertility, decreased milk production (eg, in cattle), and inadequate bone mineralization (eg, in fish, mammals and birds). Dietary supplementation (eg with inorganic feed phosphates) is the most common way to combat these problems, but can be expensive when used on a large scale.

To meet global needs for sustainable food and consumer products, microorganisms such as bacteria, yeast and fungi, and their

- 1 045393 waste products are becoming increasingly useful in many situations, including agriculture and horticulture, husbandry, forestry and the remediation of soils, waters and other natural resources. For example, farmers are learning to use biological agents, such as living microorganisms, biological products derived from them, and combinations thereof, as biopesticides and biofertilizers. These biological agents have important advantages over other traditional pesticides and fertilizers, including: 1) they are less harmful than conventional chemicals; 2) they are more effective and specific; 3) they often biodegrade quickly, resulting in less environmental pollution.

The economic costs of producing food on a large scale, as well as the adverse health and environmental impacts of current production methods, continue to negatively impact the economic feasibility and efforts to feed a growing world population.

Thus, there is a constant need for improved, non-toxic and environmentally friendly ways to grow crops, livestock and fish at low cost. In particular, there is a need for products to treat and prevent the spread of diseases and infestations in agriculture, livestock and aquaculture; supplementing soils to enhance crop growth and reduce the cost of feeding livestock and farmed fish, all without compromising the health and vitality of crops, animals and human consumers.

Summary of the invention

The present invention proposes microorganisms, as well as their metabolic products, such as bio-surfactants, metabolites and/or enzymes. The present invention also provides methods for using these microorganisms and their metabolic products, as well as methods and systems for their production. Advantageously, the microorganism-based products and methods of the present invention are environmentally friendly, safe for household use, and cost-effective.

In preferred embodiments, the present invention provides microorganism-based compositions containing cultured microorganisms and/or their metabolic products, as well as methods for preparing such compositions and methods for their use in agriculture, horticulture, animal husbandry and aquaculture. In general, microorganism-based compositions can be used to prevent and/or treat infection or infestation in plants and animals; improving soil and increasing the growth of crops and/or increasing the growth and health of livestock and fish.

More specifically, microorganism-based compositions can be used to protect plants, humans or animals by controlling infections, infestations and/or diseases caused by pathogenic uni- or multicellular organisms, including, but not limited to, fungi, bacteria, parasites and nematodes them; increasing productivity and product quality; soil enrichment; as an additive to livestock feed and/or as an additive to fish and aquatic animal feed. Methods for such use are also provided in this document.

In specific embodiments of the invention, the microorganism of the claimed microbial composition is a biologically pure yeast selected from the Pichia clade, such as, for example, P. anomala (Wickerhamomyces anomalus), P. kudriavzevii (Wickerhamomyces kudriavzevii), and/or P. guilliermondii ( Meyerozyma guilliermondii), and/or combinations thereof. Inclusion of other yeasts of the Pichia clade or yeasts closely related to it (eg, belonging to the same genus or family) and/or their products is also intended.

Compositions based on microorganisms of the claimed invention can be obtained through cultivation processes ranging from small-scale to large-scale. These cultivation processes include, but are not limited to, submerged culture, solid state fermentation (SSF), and combinations thereof.

The yeast in the composition may be in active or inactive form. In addition, the composition may also contain a fermentation broth obtained by culturing yeast, which may include, in particular, cellular components and microbial waste products, such as bio-surfactants, metabolites and/or enzymes. Predominantly direct use of composition, i.e. without further stabilization, preservation or storage, maintains high microbial viability, reduces the possibility of contamination by foreign agents and unwanted microorganisms, and maintains the activity of microbial waste products.

In some embodiments, the microorganism-based composition further comprises a carrier. The carrier may be any suitable carrier known in the art that enables delivery of yeast or yeast products to target plants, soil, animals, fish, etc. in such a way that the product remains viable, or, in the case of inactive yeast, retains the components necessary to ensure the effectiveness

- 2 045393 activity.

In some embodiments, the composition further comprises an adjuvant capable of increasing the effectiveness of the composition. Thus, for example, fulvic acid, humic acid or humate can act as auxiliary substances that are particularly useful in embodiments describing compositions for soil improvement.

The microbial composition may be formulated as, for example, a liquid suspension, emulsion, lyophilized powder or spray-dried solid, granules or gel.

In some embodiments, the compositions of the claimed invention have advantages over, for example, only purified metabolites of microorganisms, for example, due to the use of a complete culture of microorganisms. These benefits may include one or more of the following: high concentrations of mannoprotein as part of the outer surface of the yeast cell wall; the presence of beta-glucan in yeast cell walls; the presence of bio-surfactants in the culture and the presence of solvents (e.g. lactic acid, ethyl acetate, etc.) and other metabolites (e.g. vitamins, minerals, carbohydrates and protein sources) in the culture. These benefits are present whether using active or inactive yeast.

In some embodiments, the microorganisms, as well as metabolites and other waste products of the microorganisms, act synergistically with each other.

In one embodiment, the claimed invention relates to methods for producing a waste product of Pichia yeast by cultivating a strain of microorganism according to the claimed invention under conditions suitable for growth and production of waste products; and, optionally, purification of waste products. In some embodiments, the waste product is not purified, but is instead used in its original form, for example, including the fermentation broth in which it was obtained. Examples of waste products in accordance with the claimed invention include enzymes, acids, solvents, ethanol, proteins, amino acids, bio-surfactants and other substances. In specific embodiments, methods for producing a phytase enzyme are provided.

The present invention further relates to methods for increasing productivity in an industry selected from agriculture, horticulture, animal husbandry and aquaculture, wherein an effective amount of a microorganism-based composition of the claimed invention is applied to a specific or targeted area associated with the selected industry. The site of application will depend on the industry and may include, for example, a plant and/or its environment or an animal and/or its environment.

Application of the claimed methods may involve applying the microorganism-based composition directly to the plant, animal and/or their environment. Microorganisms may be alive (or viable) or inactive at the time of use.

In the case of live microorganisms, the microorganisms can grow in situ at the site of application and produce any active compounds or waste products in situ. Consequently, high concentrations of microorganisms and beneficial waste products can be achieved easily and continuously at the processing site (eg, CAFO or fish farm).

To this end, methods may include adding materials to enhance microbial growth during use (eg, adding nutrients to promote microbial growth). In one embodiment, nutrient sources may include, for example, nitrogen, nitrate, phosphorus, magnesium and/or carbon.

In one embodiment, the methods further include the step of culturing the microorganism-based composition prior to use. Preferably, the microbial composition is cultured at or near the site of use, for example, less than 100 miles from the site.

In some embodiments, methods are provided for the treatment and/or prevention of infection, infestation and/or disease in agriculture, horticulture, animal husbandry and/or aquaculture, in which the microorganism-based composition of the claimed invention is applied to a plant, animal and/or their environment. In some embodiments, the composition is applied directly to an organism or pest that is causing or has caused an infection, infestation and/or disease.

In some embodiments, methods for enriching soil are provided in which a microorganism-based composition of the claimed invention is applied to soil. Preferably, the methods can increase the yield and improve the quality of crops and products grown in the soil. In addition, the methods can help in phosphate conservation by recovering unusable phosphate present in the soil and converting it into a form that can be easily absorbed by plants.

In some embodiments, the claimed invention provides low-cost materials and methods for feeding livestock and farmed fish using the claimed composition based on

- 3 045393 microorganisms. The composition can be used as a highly nutritious source of supplemental nutrition for livestock, for example, by introducing the composition into feeders along with traditional livestock feed and the livestock consuming the composition. The microorganism-based composition can be used as a highly nutritious supplemental food source for fish, for example, by introducing the composition into a fish pond or aquarium and the fish consuming the composition. Advantageously, the present invention can be used to reduce the cost of feeding livestock and farmed fish while increasing the growth and health of the animals, for example by supporting their growth and their immune system.

In some embodiments, the claimed invention provides materials and methods for treating and/or preventing nutritional deficiencies in plants and/or animals. In the event of a plant being nutrient deficient, the methods may include applying the inventive microorganism-based compositions to the soil around the plant. In case of nutritional deficiency in an animal, the methods may include applying the claimed microorganism-based compositions to the animal's feed and/or drinking water. In one embodiment, the nutrient deficiency is a phosphorus deficiency.

Advantageously, the present invention can be used without releasing large amounts of inorganic compounds into the environment. In addition, the compositions and methods use components that are biodegradable and toxicologically safe. Thus, the present invention can be used to improve productivity in agriculture, horticulture, animal husbandry and/or fish farming as a green treatment.

Brief description of graphic materials

The figure shows a graph illustrating the phosphate concentrations used to determine the phytase production activity of Pichia anomala.

Detailed Description of the Invention

The present invention provides microorganism-based compositions containing cultivated microorganisms and/or their metabolic products, as well as methods for the preparation and use of such compositions. Microorganism-based compositions can be used in agriculture, horticulture, animal husbandry and aquaculture, for example, for the prevention and/or treatment of infection or infestation in plants and animals; improving soil and increasing the growth of crops and/or increasing the growth and health of livestock and fish.

In specific embodiments of the invention, the microorganism of the claimed microbial composition is a biologically pure yeast selected from the Pichia clade, such as, for example, Pichia anomala (Wickerhamomyces anomalus), Pichia kudriavzevii (Wickerhamomyces kudriavzevii), and/or Pichia guilliermondii (Meyerozyma guilliermondii) , and/or combinations thereof. Inclusion of other yeasts of the Pichia clade or yeasts closely related to it (for example, P. occidentalis) and/or their products is also provided.

The claimed invention also relates to methods for increasing productivity in industries selected from agriculture, horticulture, animal husbandry and aquaculture, wherein the method includes applying a microorganism-based composition according to the claimed invention to a specific area associated with the industry. For example, the application site may be a plant, an animal, and/or the environment surrounding the plant or animal.

Selected definitions.

As used herein, the term microorganism-based composition means a composition that contains components that are derived from the activity of microorganisms or other cell cultures. Thus, a microorganism-based composition may contain microorganisms themselves and/or waste products of microorganisms. Microorganisms can be in a vegetative state, in the form of spores, in the form of mycelium, in any other form of propagules of microorganisms or mixtures thereof. Microorganisms may be in the form of plankton, or in the form of biofilm, or a mixture of both. The waste products may be, for example, metabolites (eg, bio-surfactants), cell membrane components, expressed proteins and/or other cellular components. Microorganisms can be whole or lysed. Cells may be completely absent or present, for example, at a concentration of 1x10, 1x105, 1x10, ^1x107 , 1x108, ^1x109 , ^1x1010 or 1x1011 or more cells or propagules per ml of composition.

As used herein, the term propagule means any part of a microorganism from which a new and/or mature organism can develop, including, but not limited to, cells, conidia, cysts, spores (e.g., reproductive spores, endospores, and exospores), mycelium, buds, and seeds. them.

The claimed invention further provides products based on microorganisms, which are products that must be put into practice to achieve the desired result. The microorganism-based product may simply be a microorganism-based composition collected through the process of culturing the microorganisms. Alternatively,

- 4 045393 microorganism-based product may contain additional ingredients that have been added. These additional ingredients may include, for example, stabilizers, buffers, suitable vehicles (for example, water or salt solutions), added nutrients to support the continued activity of microorganisms, non-nutrient growth enhancers, and/or agents that facilitate the tracking of microorganisms and /or compositions in the environment in which they are used. The microorganism-based product may also contain mixtures of microorganism-based compositions. The microorganism-based product may also contain one or more components of the microorganism-based composition that have been processed in some manner, such as, but not limited to, filtration, centrifugation, lysing, drying, purification, and the like.

As used herein, the term collected refers to the removal of part or all of the microorganism-based composition from the growth container.

As used herein, the term isolated or purified molecule or compound is substantially free of other compounds, such as cellular material, with which it is naturally associated. A purified or isolated polynucleotide (ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)) does not contain genes or sequences that flank it in its natural state. A purified or isolated polypeptide does not contain the amino acids or sequences that flank it in its natural state.

As used herein, organic culture means a culture isolated from materials with which it is associated in nature. In a preferred embodiment, the culture is isolated from all other living cells. In other preferred embodiments, the biologically pure culture has advantageous characteristics over a culture of the same microorganism that exists in nature. Advantageous characteristics may be, for example, increased production of one or more desired waste products.

In certain embodiments, the purified compounds comprise at least 60% by weight (dry weight) of the compound of interest. Preferably, the drug represents at least 75% by weight, more preferably at least 90% by weight, and most preferably at least 99% by weight of the compound of interest. For example, a purified compound is a compound that represents at least 90, 91, 92, 93, 94, 95, 98, 99, or 100% by weight of the desired compound. Purity is measured by any suitable standard method, such as column chromatography, thin layer chromatography or high performance liquid chromatography (HPLC).

The term metabolite refers to any substance produced during metabolism (i.e., a waste product) or a substance necessary to participate in a particular metabolic process. A metabolite may be an organic compound that is a parent (eg, glucose), an intermediate (eg, acetyl-CoA), or an end product (eg, n-butanol) of metabolism. Examples of metabolites include, but are not limited to, biopolymers, enzymes, acids, solvents, alcohols, proteins, vitamins, minerals, trace elements, amino acids and bio-surfactants.

As used herein, the term modulate is interchangeable with the term alter (eg, increase or decrease). Such changes are detected by standard methods known in the art, such as those described herein.

The ranges presented in this document are considered to be representative of all values within the stated range. For example, the range from 1 to 20 means any value, combination of values, or subrange from the group including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, as well as all intermediate decimal values between the above integers, such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8 and 1.9. With respect to subbands, covered subbands are specifically provided that are within any endpoint of the range. For example, a covered subrange of the exemplary range of 1 to 50 may comprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and from 50 to 10 in the other direction.

As used herein, the term reduce refers to a negative change of at least 1, 5, 10, 25, 50, 75 or 100%.

As used herein, the term reference refers to a standard or reference value.

As used herein, the term surfactant refers to a surfactant that reduces the surface tension (or interfacial tension) between two liquids or between a liquid and a solid. Surfactants act as detergents, wetting agents, emulsifiers, foaming agents and dispersants. The term bio-surfactant is a surfactant produced by a living organism.

As used in this document, the term agriculture means the cultivation and propagation of

- 5 045393 production of plants and/or fungi for food, fiber, biofuels, medicines, cosmetics, food additives, decorative purposes and other uses. According to the claimed invention, agriculture may also include horticulture, landscaping, horticulture, plant conservation, fruit growing and tree growing. In addition, agriculture as defined herein further includes soil science (e.g., pedology and edaphology), as well as agronomy or soil care, monitoring and management, and crop production.

As used herein, the term livestock refers to any domesticated animal raised under agricultural or industrial conditions to produce agricultural commodities such as food, fiber, and labor. The term animal husbandry includes the breeding, rearing, feeding, housing, maintenance and/or slaughter of these animals. Livestock may be raised in natural settings, such as an open field, farm, or animal feed facility. Types of animals covered by the term livestock may include, but are not limited to, alpacas, cattle, dairy cattle, bison, swine, sheep, goats, horses, mules, donkeys, camels, chickens, turkeys, ducks, geese, guinea fowl and squabs.

As used herein, an animal feed facility or AFO refers to a land or base (not including a fish farming base) where animals have been, are, or will be kept, or kept and fed or maintained for a total of 45 days or more in any 12 -month period and where crops, vegetation, forages or harvest residues are not maintained in a normal growing season in any part of the base. AFOs primarily use artificial structures and equipment (for feeding, temperature control, manure storage and management, etc.) at planting and processing sites.

The term CAFO or concentrated AFO is an AFO that concentrates large numbers of animals in relatively small and confined spaces that meet certain size thresholds as defined by the Environmental Protection Agency.

As used in this document, the terms aquaculture, aquaculture, aquatic farming, aquaculture or fish farming mean the breeding, rearing and harvesting of aquatic animals in aquaculture. Aquaculture can be intensive (relying on technology to raise fish in artificial pens at high densities) or extensive (performed in the ocean or in natural and artificial lakes, bays, river fjords or other bodies of water). Aquaculture farming involves the production of seafood from fish and shellfish hatcheries that are grown to market size in pens, ponds, tanks, aquariums, cages or canals. In addition, aquaculture farming includes marine farming, which entails the cultivation of marine organisms in open seawater or closed seawater areas. In addition, aquaculture involves restocking or augmentation, in which fish and shellfish from a hatchery are released into the wild in an attempt to restore populations in wild or coastal habitats. Moreover, aquaculture includes raising ornamental fish for sale to aquariums, as well as keeping ornamental fish housed in aquariums. Species that may be cultured include freshwater or saltwater fish and shellfish, and may also include ornamental fish, edible fish, sport fish, live bait, crustaceans, shellfish, algae, sea vegetables, or caviar.

As used herein, the term aquaculture is any aquatic environment where aquaculture occurs or may occur. Fish farms as defined herein may include all types of aquatic environments or areas of aquatic environments, whether artificial or natural, including ponds, irrigation canals, rivers, lakes, oceans, fjords, tanks, aquariums, cages or canals.

As used herein, the term pest is any non-human organism that is destructive, disease-causing, and/or harmful to humans or human needs (eg, agriculture, horticulture, livestock management, aquaculture). Pests can cause infections, infestations and/or disease. Pests can be single- or multicellular organisms, including, but not limited to, viruses, fungi, bacteria, parasites and/or nematodes.

As used herein, the term treatment refers to eradicating, reducing, ameliorating, reversing or preventing the extent, manifestation or symptom of a condition or disorder to any extent and includes, but does not require, complete cure of the condition or disorder. Treatment may be a cure, amelioration, or partial relief of the disorder. Treatment may also include prevention of a condition or disorder, which as used herein means delaying the onset or progression of a particular manifestation or symptom of the condition or disorder.

- 6 045393

Compositions based on microorganisms.

The claimed invention proposes compositions based on microorganisms containing beneficial microorganisms, as well as their metabolic products, such as bio-surfactants, metabolites, acids, solvents and/or enzymes. The claimed invention also provides methods for using these compositions to improve productivity in agriculture, horticulture, livestock and aquaculture. In addition, the claimed invention proposes materials and methods for producing compositions based on microorganisms.

It is preferable that the microorganism-based compositions prepared in accordance with the claimed invention are non-toxic (for example, toxicity if ingested is greater than 5 g/kg body weight) and can be applied in high concentrations without causing irritation, for example, to the skin or gastrointestinal tract of humans or animals. Thus, the claimed invention is particularly suitable when the use of microorganism-based compositions occurs in the presence of living organisms, such as livestock and fish, produced by humans and for human consumption.

In some embodiments, the microorganisms of the claimed invention are biologically pure killer yeasts. In particular, killer yeast belonging to the genus Pichia is used in the claimed invention. Even more specifically, in one embodiment, the microorganisms include Pichia anomala (Wickerhamomyces anomalus), Pichia guilliermondii (Meyerozyma guilliermondii), Pichia kudriavzevii (Wickerhamomyces kudrizvzevii), and/or combinations thereof.

The microorganisms and microorganism-based compositions of the claimed invention have a number of beneficial properties that are suitable for increasing productivity in agriculture, horticulture, livestock farming and aquaculture. For example, in one embodiment, the composition contains a bio-surfactant. Bio-surfactants may be present in the composition as a result of the growth of microorganisms present in the composition, or bio-surfactants may be produced separately from other microorganisms and added to the composition in original and/or purified form. As an example, the waste products of microorganisms in their original form may contain the fermentation broth in which they were obtained.

Bio-surfactants inhibit microbial adhesion to various surfaces, prevent the formation of biofilms, and can have powerful emulsifying and demulsifying properties. In addition, bio-surfactants can reduce the surface and interfacial tension of water, for example, in fish farms and aquariums.

In some embodiments, the bio-surfactants of the claimed composition are glycolipid bio-surfactants, such as mannosilerythritol lipids, sophorolipids, rhamnolipids, or trehalose lipids. Bio-surfactants can also be lipopeptides such as surfactin, iturin, fengycin and lichenisin.

In a specific embodiment, the composition contains one or more glycolipids, such as mannosilerythritol lipids (MEL), which are produced in large quantities, for example, by Pseudozyma yeast, and sophorolipids (SLP), which are produced, for example, by Starmerella yeast and Pichia yeast.

MEL and SLP exhibit excellent surface and interfacial tension reducing properties, highly effective emulsifying and demulsifying properties, as well as versatile biochemical and physiological effects through, for example, their antifungal, antibacterial, antiparasitic and/or antiviral properties.

In some embodiments, the concentration of one or more bio-surfactants in the composition is from 0.001 to 90% by weight (wt%), preferably from 0.01 to 50 wt%, and more preferably from 0.1 to 20 wt%. Bio-surfactants may additionally be present in an amount of about 0.01 to about 500 g/L, about 0.5 to about 50.0 g/L, about 1.0 to about 10.0 g/L, or about 2 .0 to about 5.0 g/l.

In some embodiments, the composition further contains enzymes formed by microorganisms. These enzymes may be present in the composition as a result of the growth of microorganisms therein, or may be obtained separately and added in their original form and/or purified form to the composition. By way of example, enzymes in their original form may contain the fermentation broth in which they were produced.

In a specific embodiment, the composition contains the enzyme phytase. Preferably, the cultivation methods described herein, in addition to the particular combination of yeast species described, work synergistically to produce phytase in unexpectedly high concentrations and with unexpected efficiency.

In some embodiments, the concentration of phytase (or other enzyme) in the composition is from 0.001 to 90% by weight (wt%), preferably from 0.01 to 50 wt%, and more preferably from 0.1 to 20 wt%. Phytase (or other enzyme) may additionally be present in an amount of from about 0.01 to about 500 g/L, from about 0.5 to about 50.0 g/L, from about 1.0 to about 10.0 g/L or from about 2.0 to about 5.0 g/L.

In some embodiments, the composition further contains additional microbial products in their original or purified form, such as enzymes, biosurfactants, solvents, acids, proteins, minerals and/or vitamins.

The metabolites in their original form may be in the form of, for example, a liquid mixture containing a precipitate of the metabolite in a fermentation broth obtained by culturing the microorganism. This solution in its original form may contain from about 25 to about 75%, from about 30 to about 70%, from about 35 to about 65%, from about 40 to about 60%, from about 45 to about 55%, or about 50% pure metabolite.

In some embodiments, the microbial composition of the claimed invention may comprise a fermentation broth containing a live and/or inactive culture and/or microbial metabolites produced by the microorganism and/or any residual nutrients. The fermentation product can be used directly without extraction or purification. If desired, extraction and purification can be easily achieved using standard methods or extraction and/or purification techniques described in the literature.

Advantageously, in accordance with the claimed invention, the microorganism-based composition may contain a broth in which the microorganisms have been grown. The composition may be, for example, at least 1, 5, 10, 25, 50, 75 or 100 wt.% broth. The amount of biomass in the composition may be, for example, from 0 to 100% by weight, including all percentages in between.

The biomass content of the fermentation broth can be, for example, from 5 to 180 g/L or more. In one embodiment, the solids content of the broth is between 10 and 150 g/L.

Fermentation by microorganisms.

The claimed invention uses methods for cultivating microorganisms and obtaining metabolites of microorganisms and/or other waste products of microorganisms. More specifically, the claimed invention provides materials and methods for the production of biomass (eg, viable cellular material), extracellular metabolites (eg, small molecules and isolated proteins), residual nutrients and/or intracellular components (eg, enzymes and other proteins).

The present invention further utilizes cultivation processes that are suitable for culturing microorganisms and producing microbial metabolites at any desired scale, from small scale (eg, laboratory setup) to large scale (eg, industrial application). These cultivation processes include, but are not limited to, submerged culture/fermentation, solid state fermentation (SSF), and combinations thereof.

As used herein, the term culture refers to growing cells under controlled conditions. Cultivation can be aerobic or anaerobic.

The microorganism cultivation vessel used in accordance with the claimed invention may be any fermenter or culture reactor for industrial use. In one embodiment, the container may have functional controls/sensors or may be associated with functional controls/sensors to measure important factors in the cultivation process such as pH, oxygen, pressure, temperature, agitator power, humidity, viscosity and/or or microbial density and/or metabolite concentration.

The container may also be capable of monitoring the growth of microorganisms within the container (eg, measuring cell numbers and growth phases). Alternatively, a 24-hour sample may be taken from the container and counted by methods known in the art, such as dilution culture.

In preferred embodiments, a microbial growth apparatus comprising a plurality of microbial growth vessels produces high-density new microorganisms and/or microbial products of interest at a desired scale. The plant for growing microorganisms may be located at or near the site of use. The plant produces compositions based on microorganisms at high densities during batch, semi-continuous or continuous cultivation.

Distributed microbial cultivation facilities may be located in the location where the microbial product will be used (eg, in a field or fish farm). For example, the microbial growth facility may be located less than 300, 250, 200, 150, 100, 75, 50, 25, 15, 10, 5, 3, or 1 mile from the site of use, or may be located directly at the site of use.

In some embodiments, production may or may not be achieved using local and/or distributed fermentation methods, meaning that conventional methods may also be used in accordance with the claimed invention. Predominantly local and/or distributed microbial cultivation facilities provide a solution to the current problem of relying on large industrial producers whose quality

- 8,045,393 products suffer from processing delays, supply chain bottlenecks, improper storage and other unforeseen circumstances that prevent timely delivery and use of the useful product.

Plants for growing microorganisms produce new compositions based on microorganisms containing the microorganisms themselves, metabolites of the microorganisms and/or other components of the broth in which the microorganisms are grown. If desired, the compositions may have a high density of vegetative cells, inactive cells, propagules, or a mixture of vegetative cells, inactive cells and/or propagules.

Advantageously, the compositions can be tailored for use in a specified location. In one embodiment, the microbial growth facility is located at or near the site where the microbial products will be used. Plants for growing microorganisms can be operated off-grid using, for example, solar, wind and/or hydroelectric energy.

Microbial growth facilities provide manufacturing versatility through the ability to tailor microbial products to improve interaction with destination geographies. For example, the systems according to the claimed invention are capable of using the capabilities of natural microorganisms and their metabolic products. Local microorganisms can be identified based on, for example, salt tolerance, ability to grow at high temperatures and/or ability to produce certain metabolites.

Because the microorganism-based product is formed at or near the point of application, without the need for stabilization, preservation, long-term storage and extensive transportation processes typical of conventional production, a much higher density of live (or inactive) microorganisms can be obtained, requiring much less volume microorganism-based product for use at the point of application or provides a much higher density of application of microorganisms where needed. This reduces the possibility of contamination by foreign agents and unwanted microorganisms, maintains the activity of microbial waste products, and also allows for the efficient use of a reduced bioreactor (e.g., smaller fermentation tank and smaller volume of feed material, nutrients, pH adjusting agents and defoamers, etc. ) without the need to stabilize cells. Locally produced, high-density and stable cultures of microorganisms are more effective in the field than those that have undergone vegetative cell stabilization or have been in the supply chain for some time.

Local production of a microorganism-based product also facilitates the inclusion of fermentation broth in the product. The broth may contain agents formed during fermentation that are particularly suitable for topical use. This further facilitates the portability of the product.

Reducing transportation times allows new batches of microorganisms and/or their metabolites to be produced and delivered at a specific time and in volume, as required by local demand. Local production and delivery within, for example, 24 hours of fermentation provides pure compositions with high cell density and significantly reduces shipping costs. With the prospect of rapid advances in the development of more efficient and potent microorganisms for inoculation, consumers will greatly benefit from this ability to rapidly deliver microorganism-based products.

In one embodiment, the culture method, performed using conventional methods or using local or distributed systems, uses a culture medium containing molasses, urea and peptone.

In one embodiment, the molasses concentration is from 2 to 6%, preferably 4%. In one embodiment, the urea concentration is from 0.01 to 1.0%, preferably 0.2%. In one embodiment, the peptone concentration is from 1.0 to 5%, preferably 2.5%.

In one embodiment, the method includes supplementing the cultivation with a nitrogen source. The nitrogen source may be, for example, potassium nitrate, ammonium nitrate, ammonium sulfate, ammonium phosphate, ammonia, urea and/or ammonium chloride. These nitrogen sources may be used independently or in combination of two or more.

The cultivation method can provide oxygenation to the growing culture. In one embodiment, slow air movement is used to remove oxygen-poor air and introduce oxygen-rich air. The oxygenated air may be ambient air, replenished daily through mechanisms including turbine agitators to mechanically mix the liquid and air distributors to introduce gas bubbles into the liquid to dissolve oxygen in the liquid.

The method may further include supplementing the cultivation with a carbon source. The carbon source is usually a carbohydrate such as glucose, sucrose, lactose, fructose, trehalose,

- 9 045393 mannose, mannitol and/or maltose; organic acids such as acetic acid, fumaric acid, citric acid, propionic acid, malic acid, malonic acid and/or pyruvic acid; alcohols such as ethanol, isopropyl, propanol, butanol, pentanol, hexanol, isobutanol and/or glycerin; fats and oils such as soybean oil, rice bran oil, olive oil, corn oil, sesame oil and/or flaxseed oil. These carbon sources may be used independently or in combination of two or more.

In one embodiment, growth factors and micronutrients for microorganisms are included in the culture medium. This is especially preferable when cultivating microorganisms that are not able to produce all the vitamins they need. Inorganic nutrients, including trace elements such as iron, zinc, copper, manganese, molybdenum and/or cobalt, can also be included in the culture medium. In addition, sources of vitamins, essential amino acids and micronutrients may be included, for example, in the form of flour or meal, such as corn meal, or in the form of extracts, such as yeast extract, potato extract, beef extract, soybean extract, extract banana peels and the like or in peeled forms. Amino acids, such as those required for protein biosynthesis such as L-alanine, may also be included.

In one embodiment, inorganic salts may also be included. Suitable inorganic salts may be potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, magnesium chloride, ferrous sulfate, ferric chloride, manganese sulfate, manganese chloride, zinc sulfate, lead chloride, copper sulfate, calcium chloride, calcium carbonate and/or carbonate sodium Said inorganic salts may be used independently or in combination of two or more.

In some embodiments, the culture method may further include adding additional acids and/or antimicrobials to the liquid culture medium before and/or during the culture process. Antimicrobial agents or antibiotics may be used to protect the crop from contamination. For example, Streptomyces erythromycin, hops or hop acid and/or small amounts, for example 50-100 ppm, of sophorolipids or other bio-surfactants as antibacterial agents can be added to the growth medium. In addition, when gas is generated during culture, defoamers may be added to prevent the formation and/or accumulation of foam.

The pH value of the mixture must match the microorganism of interest. Buffers and pH adjusters such as carbonates and phosphates can be used to stabilize the pH within a preferred range. pH control can also be used to prevent crop contamination. For example, cultivation may be initiated at a low pH value that is suitable for yeast growth (e.g., 3.0-3.5), and then, after yeast accumulation, increased (e.g., to 4.5-5.0) and stabilized over a period of time remaining fermentation period. When metal ions are present in high concentrations, the use of a chelating agent in the liquid culture medium may be required.

Microorganisms can be grown in planktonic form or as a biofilm. In the case of biofilm, the container may contain a substrate on which microorganisms in the biofilm state can be grown. The system may also have, for example, the ability to apply stimuli (such as mechanical stimulation) that stimulate and/or improve biofilm growth characteristics.

In one embodiment, the method of cultivating microorganisms is carried out at a temperature of from about 5 to about 100°C, preferably from 15 to 60°C, more preferably from 25 to 50°C. In a further embodiment, cultivation may be carried out continuously at a constant temperature. In another embodiment, the cultivation may be subject to temperature changes.

In one embodiment, the equipment used in the culture method and process is sterile. The culture equipment, such as the reactor/vessel, may be separate from, but connected to, the sterilization device, such as an autoclave. Culture equipment may also have a sterilization unit that sterilizes in situ before inoculation begins. The air can be sterilized by methods known in the art. For example, ambient air may pass through at least one filter before entering the container. In other embodiments, the culture medium may be pasteurized or optionally not heated at all, and low water activity and low pH may be used to control bacterial growth.

In other embodiments, the culture system may be self-sterilizing, i.e. the cultured organism is capable of preventing contamination by other organisms through the production of antimicrobial waste products or metabolites.

In one embodiment, surfactants, enzymes, metabolites and/or other proteins are obtained by cultivating a strain of microorganism according to the claimed invention under conditions suitable for growth and their production; and, optionally, purifying a surfactant, enzyme, metabolite, or other protein. Any

- 10 045393 from useful waste products, which are known to be obtained using Pichia yeast, can be obtained and purified in accordance with the claimed invention. These include bio-surfactants, enzymes such as exo-β-1,3-glucanase, chitinases and phytase, solvents, proteins, lipids, carbon sources, and minerals and vitamins such as vitamins B1, B2, B3 (PP) , B5, B7 (H), B6 and E, but are not limited to them.

The microbial waste product produced by the microorganisms of interest may be retained in the microorganisms or secreted into the growth medium. In another embodiment, a method for obtaining a microbial waste product may further include the steps of concentrating and purifying the microbial waste product of interest. In an additional embodiment, the growth medium may contain compounds that stabilize the activity of the microbial waste product.

The biomass content of the fermentation broth can be, for example, from 5 to 180 g/L or more. In one embodiment, the solids content of the broth is between 10 and 150 g/L.

When it is time to harvest the microorganism-based product from the growth container or containers, the microorganisms and/or the broth resulting from the growth of the microorganisms can be removed from the growth container and transferred, for example, through a pipeline for immediate use. These microorganisms may be in active or inactive form or may contain a combination of active and inactive microorganisms.

The composition (microorganisms, broth or microorganisms and broth) may also be placed in containers of appropriate size, taking into account, for example, the intended use, the intended method of use, the size of the fermentation vessel and any method of transport from the microorganism growth facility to the point of use. Thus, the containers in which the microorganism-based composition is placed may have a volume, for example, from 1 to 1000 gallons or more. In certain embodiments, the containers have a volume of 2, 5, 25 gallons, or larger.

Other components may be added when the harvested product is placed into containers and/or piped (or otherwise transported for use). Additives may include, for example, buffers, carriers, other microorganism-based compositions produced in the same or a different facility, viscosity modifiers, preservatives, microbial growth nutrients, tracking agents, pesticides, and other ingredients specific to the intended use.

In one embodiment, the entire microorganism culture composition is removed upon completion of the culture (eg, after achieving the desired cell density or density of the specified metabolite in the broth). In this batch culture mode, a completely new batch is started to be grown after the first batch is received.

In another embodiment, only a portion of the fermentation product is removed at a time. In this embodiment, the biomass with viable cells remains in the vessel as an inoculum for a new culture batch. The composition that is removed may be a cell-free broth or may contain cells. Thus, a semi-continuous system is created.

Preparation of products based on microorganisms.

One of the microbial products of the claimed invention is simply a fermentation broth containing the microorganism and/or microbial metabolites produced by the microorganism and/or any residual nutrients. The fermentation product can be used directly without extraction or purification. If desired, extraction and purification can be easily achieved using standard methods or extraction and/or purification techniques described in the literature.

Microorganisms in a microorganism-based product may be in active or inactive form. Microorganism-based products may contain combinations of active and inactive microorganisms.

Products based on microorganisms can be used without further stabilization, preservation and storage. Advantageously, direct use of these microorganism-based products maintains high microbial viability, reduces the possibility of contamination by foreign agents and unwanted microorganisms, and maintains the activity of microbial products.

The microorganisms and/or the broth resulting from the growth of the microorganisms can be removed from the growth vessel and transferred, for example, through a pipeline for immediate use. In other embodiments, as described above, the composition (microorganisms, broth, or microorganisms and broth) can be placed in appropriately sized containers.

After collecting the microorganism-based composition from the growth container, additional

- 11 045393 components can be added when the collected product is placed in containers and/or piped (or otherwise transported for use). Additives may be, for example, buffering agents, carriers, adjuvants, other microorganism-based compositions produced in the same or a different plant, viscosity modifiers, preservatives, microbial growth nutrients, tracking agents, biocides, other microorganisms, non-biological surfactants , emulsifiers, lubricants, buffering agents, solubility regulators, pH adjusters, stabilizers, ultraviolet stable agents and other ingredients specific to the intended use.

In one embodiment, the composition may further contain buffering agents, including organic acids and amino acids or salts thereof, to stabilize the pH around a preferred value. The pH value of the microorganism-based composition should correspond to the microorganism of interest.

Suitable buffers include citrate, gluconate, tartrate, malate, acetate, lactate, oxalate, aspartate, malonate, glucoheptonate, pyruvate, galactarate, glucarate, tartronate, glutamate, glycine, lysine, glutamine, methionine, cysteine, arginine, and mixtures thereof, but not limited to them. Phosphoric and phosphorous acids or their salts can also be used. Synthetic buffers are suitable, but it is preferable to use natural buffers such as organic acids and amino acids or their salts.

In a further embodiment, pH adjusters include potassium hydroxide, ammonium hydroxide, potassium carbonate or bicarbonate, hydrochloric acid, nitric acid, sulfuric acid, or mixtures thereof.

In one embodiment, additional components may be included in the microorganism-based composition, such as an aqueous salt preparation such as sodium bicarbonate or carbonate, sodium sulfate, sodium phosphate or sodium biphosphate.

Advantageously, the microorganism-based product may contain a broth in which the microorganisms have been grown. The product may be, for example, at least 1, 5, 10, 25, 50, 75 or 100% by weight broth. The amount of biomass in the product may be, for example, from 0 to 100% by weight, including all percentages in between.

In some embodiments, the microorganism-based composition of the claimed invention further comprises a carrier. The carrier may be any suitable carrier known in the art that enables delivery of yeast or yeast products to target plants, soil, animals, fish, etc. in such a way that the product remains viable, or, in the case of inactive yeast, retains the components necessary for effectiveness.

In some embodiments of the invention, particularly in the context of agriculture, the microorganism-based composition may further contain an adjuvant to enhance the effectiveness of the composition. In one embodiment, the adjuvant is selected from one or more of fulvic acid, humic acid or humate.

If desired, the composition may be stored until use. The storage time is preferably short. Thus, the storage time may be less than 60, 45, 30, 20, 15, 10, 7, 5, 3, 2, 1 day or 12 hours. In a preferred embodiment, if live cells are present in the product, the product is stored at a cool temperature , such as, for example, less than 20, 15, 10 or 5°C. On the other hand, the bio-PVA composition can usually be stored at ambient temperature.

In some embodiments, the compositions of the claimed invention have advantages over, for example, only purified microbial metabolites, due to, for example, the use of a complete culture of microorganisms. These benefits may include one or more of the following: high concentrations of mannoprotein as part of the outer surface of the yeast cell wall; the presence of beta-glucan in yeast cell walls; the presence of bio-surfactants in the culture; and the presence of solvents and other metabolites (eg, lactic acid, ethanol, ethyl acetate, etc.) in the culture. These benefits are present whether using active or inactive yeast.

The microbial composition may be formulated as a microbial product in the form of, for example, a liquid suspension, emulsion, lyophilized powder or spray-dried granule, solid or gel.

Other formulations which are indicated as being suitable for a particular application.

In one embodiment, particularly for use in livestock applications, the microbial product may be formulated as a feed pellet containing uniform concentrations of the desired microorganism or microbial waste product, such as purified phytase, in the pellet. Methods known in the art for producing feed briquettes can be used to produce them, including pressure grinding. Preferably, the briquette process is cold granulation or a process that does not use heat or steam.

- 12 045393

In one embodiment, particularly for use in aquatic applications, strains of microorganisms are cultured to obtain an inactive microorganism-based composition. The composition is prepared by culturing the desired microorganism, destroying the microorganism using a microfluidizer (or any other method known in the art so as not to cause protein denaturation), pasteurized and added to the food product in concentrated form. In one embodiment, inactivation occurs by pasteurizing temperature (65 to 70°C for a period of time sufficient to inactivate 100% of the yeast cells) and increasing the pH to about 10.0. This causes partial hydrolysis of the cells and allows the release of some nutritional components. The composition is then neutralized to a pH of about 7.07.5 and the various hydrolysis components are mixed. The resulting microorganism-based product can then be used, for example, in fish feed and for water treatment in fish farming.

The microbial products of the present invention can be used in a variety of unique conditions due to, for example, the ability to effectively deliver 1) fresh fermentation broth with active metabolites; 2) a mixture of cells and/or propagules and fermentation broth; 3) a composition with a high cell density, including vegetative cells and/or propagules; 4) microorganism-based products that do not require time to obtain them, and 5) microorganism-based products to remote locations.

The microorganism-based products of the present invention are particularly advantageous over traditional products in which the cells have been separated from the metabolites and nutrients present in the fermentation culture medium.

Strains of microorganisms grown in accordance with the claimed invention.

Microorganisms grown in accordance with the systems and methods of the present invention may be, for example, bacteria, yeast and/or fungi. These microorganisms may be natural or genetically modified microorganisms. For example, microorganisms can be transformed with specific genes to exhibit specific characteristics. Microorganisms can also be mutants of the desired strain. As used herein, the term mutant means a strain, genetic variant, or subtype of a reference microorganism, wherein the mutant has one or more genetic variations (e.g., a point mutation, a missense mutation, a nonsense mutation, a deletion, a duplication, a frameshift mutation, or a repeat expansion) compared with a reference microorganism. Procedures for generating mutants are well known in the field of microbiology. For example, UV mutagenesis and nitrosoguanidine are widely used for this purpose.

In some preferred embodiments, the microorganism is any yeast known as killer yeast. As used herein, the term yeast killer means a strain of yeast characterized by the secretion of toxic proteins or glycoproteins to which the strain itself is immune. Exotoxins released by killer yeast can kill other strains of yeast, fungi, or bacteria. For example, microorganisms that can be controlled by killer yeasts include Fusarium and other filamentous fungi. Such yeasts may include, but are not limited to, Wickerhamomyces, Pichia, Hansenula, Saccharomyces, Hanseniaspora, Ustilago Debaryomyces, Candida, Cryptococcus, Kluyveromyces, Torulopsis, Williopsis, Zygosaccharomyces and others.

In a specific embodiment, the claimed invention uses killer yeast belonging to the genus Pichia. Even more specifically, microorganisms of the claimed invention include Pichia anomala (Wickerhamomyces anomalus), Pichia guilliermondii (Meyerozyma guilliermondii), Pichia kudriavzevii (Wickerhamomyces kudriavzevii) and/or combinations thereof. These three Pichia microorganisms and their metabolites and other waste products (sometimes referred to herein as 3P or Triple-P) work synergistically with each other to produce desired results when used to improve plant and animal health as described herein.

This yeast has a number of useful characteristics useful for the present invention, including its ability to produce useful metabolites. For example, Pichia anomala are characterized by exo-β-1,3-glucanase activity, which makes them capable of killing or inhibiting the growth of a wide range of pathogenic fungi. In addition, when cultivated for 5-7 days, Pichia anomala produces bio-surfactants that are capable of reducing surface/interfacial tension of water and also exhibit antimicrobial and antifungal properties.

Pichia kudriavzevii produce metabolites with antibacterial activity against several animal and human pathogens, such as Escherichia coli, Enterococcus faecalis, Klebsiella sp., Staphylococcus aureus, Pseudomonas aeruginosa and Pseudomonas alcaligenes. Moreover, the toxins produced by P. kudriavzevii are capable of combating many other gram-positive and gram-negative bacteria that can cause bacterial diseases in plants.

Pichia guilliermondii strains produce hydrolytic enzymes, including β-1,3-glucanases and chitinases. These enzymes are known to have nematicidal (e.g.

- 13 045393

Meloidogyne incognita) and antifungal (eg against B. cinereal) properties.

In addition to various waste products, these yeasts are capable of producing phytase, a phosphatase enzyme that catalyzes the hydrolysis of phytic acid (phytate or myoinositol hexakisphosphate). Phytic acid is an indigestible organic form of phosphorus that, when hydrolyzed, releases a usable form of inorganic phosphorus.

Additionally, Pichia yeast produces a range of proteins (constituting up to 50% of dry cell biomass), lipids and carbon sources, as well as a full spectrum of minerals and vitamins (B1; B2; B3 (PP); B5; B7 (H); B6; E ).

Other strains of microorganisms may also be used in accordance with the claimed invention, including, for example, other strains of microorganisms capable of accumulating significant quantities of, for example, enzymes, acids, proteins, bio-surfactants, minerals and vitamins that are useful for increasing productivity in agriculture, horticulture, livestock and aquaculture.

Ways to improve productivity in agriculture, horticulture, livestock and aquaculture.

In some embodiments, the claimed invention provides environmentally friendly, cost-effective materials and methods for increasing productivity in agriculture, horticulture, livestock and/or aquaculture. More specifically, the claimed invention provides methods for improving the health and quality of cultivated plants, fungi and/or animals using the microorganism-based products of the claimed invention. In one embodiment, methods are provided for feeding livestock and/or farmed fish.

Specifically, the claimed invention relates to methods for increasing productivity in an industry selected from agriculture, horticulture, animal husbandry and aquaculture, wherein an effective amount of the microorganism-based composition of the claimed invention is applied to a specific or targeted area associated with the selected industry. The site of application will depend on the industry and may include, for example, a plant and/or its environment or an animal and/or its environment.

As used herein, the terms applying a composition or product to a target or site or treating a target or site refer to bringing a composition or product into contact with a target or site such that the composition or product can affect that target or site. Exposure may be due, for example, to the growth of microorganisms and/or the action of a metabolite, enzyme, biosurfactant or other waste product. Application or treatment may include applying the microorganism-based composition directly to the plant, fungi, animal and/or their environment.

Use may further include contacting the composition or product directly with a plant, a part of a plant, and/or the plant's environment (eg, soil) or directly with an animal, a body part of an animal, and/or an animal's environment (eg, an animal's food source). The claimed microbial product may, for example, be sprayed as a liquid or dry powder, dust, granules, microgranules, solid, wettable powder, bulk powder, emulsions, microcapsules, oils, gels, pastes or aerosols.

In one embodiment, the claimed invention provides for the use of microorganism-based products in the field under agricultural and horticultural conditions. The microorganism-based products of the claimed invention can be applied, for example, through an irrigation system, as a spray, as a seed treatment, to soil surfaces, to crops, to plant or fungal surfaces, and/or pest surfaces. Mechanical applications are also facilitated by conventional equipment, while robotic applications are facilitated by airborne or ground-based drones. Any part of the plant may be selected for treatment according to the claimed invention, including, but not limited to, fruits, flowers, bulbs, leaves, grains, roots, seeds and/or stems.

In one embodiment, the claimed invention provides for the use of microorganism-based products in the field under livestock conditions. Microorganism-based products may be administered directly to animals, for example, as an oral, intravenous, subcutaneous or topical drug or supplement, or added to animal feed and/or water. Microorganism-based products can also be applied to the animals' habitat, such as as a spray or coating, to the ground or surfaces of pens, or directly to pest surfaces.

In one embodiment, the claimed invention provides field application of microorganism-based products in livestock production. Microorganism-based products can be applied to the fish farm, for example, by pumping, spraying, pouring or injecting the compositions into the water of the fish farm, or, for example, by coating or spreading the composition onto various surfaces, walls or pens of the fish farm. In addition, the compositions can be administered to animals as such, for example, as a food source.

Microorganisms may be alive (or viable) or inactive at the time of use. In the case of live microorganisms, the microorganisms can grow in situ at the site of application and produce any active compounds or waste products in situ. Consequently, high concentrations of microorganisms and beneficial waste products can be achieved easily and continuously at the processing site (eg, CAFO or fish farm).

To this end, methods may include adding additional materials to enhance microbial growth during use (eg, adding nutrients to enhance microbial growth). In one embodiment, nutrient sources may include, for example, nitrogen, nitrate, phosphorus, magnesium and/or carbon.

In one embodiment, the methods further include culturing the microorganism-based composition prior to use. Preferably, the microbial composition is cultured at or near the site of use, for example, less than 100 miles from the site.

In some embodiments, methods are provided for preventing and/or treating infection or infestation caused by pathogenic unicellular or multicellular organisms; improving soil and increasing the growth of crops and/or increasing the growth and health of livestock and fish.

In one embodiment, a method is provided for treating and/or preventing an infection, infestation, disease and/or the spread thereof. In specific embodiments, pathogenic infections, infestations, and/or diseases that are caused by pathogenic unicellular or multicellular organisms, including, but not limited to, fungi, bacteria, parasites, and/or nematodes, that are pathogenic to plants, fungi, and/or agricultural crops; livestock and other terrestrial animals and/or fish and other aquatic organisms.

In one embodiment, the method includes applying a microbial composition containing Pichia yeast and/or its metabolic products to the site, wherein the composition kills and/or competitively inhibits the growth of pathogenic fungi; kills and/or competitively inhibits the growth of pathogenic gram-positive and gram-negative bacteria; kills disease-causing helminth parasites and/or kills disease-causing nematodes. Combinations of these yeasts, each with unique killing abilities, create an effective synergistic agent for biological control, such as in the organic agriculture, organic livestock and organic aquaculture industries.

Preferably, the methods enhance the immune system and/or protect plants, livestock and farmed fish from pathogens without the use of harsh chemicals or antibiotics.

In a specific embodiment, the method is used to improve agricultural productivity by preventing and/or treating infections, infestations and/or diseases of crops, plants and/or fungi in agricultural settings.

Examples of bacterial infections affecting plants against which the claimed invention is effective include Pseudomonas syringae pathovars; Ralstonia solanacearum; Agrobacterium tumefaciens; Xanthomonas oryzae pv. oryzae; Xanthomonas campestris pathovars., Xanthomonas axonopodis pathovars; Erwinia amylovora; Xylella fastidiosa; Dickeya (dadantii and solani); Pectobacterium carotovorum (and Pectobacterium atrosepticum); Clavibacter michiganensis (michiganensis and sepedonicus), Pseudomonas savastanoi and Candidatus Liberibacter asiaticus, but not limited to.

Examples of viral infections affecting plants against which the claimed invention is effective include Carlavirus, Abutilon, Hordeivirus, Potyvirus, Mastrevirus, Badnavirus, Reoviridae Fijivirus, Oryzavirus, Phytoreovirus, Mycoreovirus, Rymovirus, Tritimovirus, Ipomovirus, Bymovirus, Cucumovirus, Luteovirus, Begomovirus, Rhabdoviridae , Tospovirus, Comovirus, Sobemovirus, Nepovirus, Tobravirus, Benyvirus, Furovirus, Pecluvirus and Pomovirus, but are not limited to.

Nematodes can also be treated using the claimed invention. Examples of nematodes are the cyst-forming nematodes of the genera Heterodera (eg, H. glycines, H. avenae and H. shachtii) and Globodera (eg, G. rostochiens and G. pallida); bristle root nematodes of the genus Trichodorus; stem nematodes of the genus Ditylenchus; potato nematode Heterodera rostochiensis; root nematodes of the genus Meloidogyne (for example, M. javanica, M. hapla, M. arenaria and M. incognita); root-attacking nematodes of the genus Pratylenchus (for example, P. goodeyi, P. penetrans, P. bractrvurus, P. zeae, P. coffeae, P. bractrvurus and P. thornei); citrus nematodes of the genus Tylenchulus and stinging nematodes of the genus Belonalaimus.

Other plant or crop diseases against which the claimed methods and compositions are effective include pests and/or pathogens causing stunting, plant cancer, rot, wilt, rust, anthracnose, bacterial blight, clubroot, corn smut, galls, rhizoctinia , downy and true powdery mildew, scab, leaf spot, fungal mold, mosaic virus, blistering and leaf curl.

In another embodiment, the method is used to improve productivity in livestock production by preventing and/or treating infections, infestations and/or diseases of livestock and other

- 15 045393 terrestrial animals.

Examples of diseases against which the claimed methods and compositions are effective include candidiasis, trichophytosis, complex bovine respiratory infection, ascochytasis (clostridial), bovine respiratory syncytial virus, bovine viral diarrhea, bacteriosis caused by Haemophilus somnus, infectious bovine rhinotracheitis, disease caused by Pasteurella Haemolytica and Pasteurella Multocida, rabies, fowl pox, influenza, Newcastle disease, contagious catarrh of the upper respiratory tract, avian cholera, colibacillosis, bacteriosis caused by Bordetella bronchiseptica, aspergillosis, mycoplasmosis, eastern equine encephalitis, botulism , hemorrhagic enteritis, bacteriosis due to Salmonella, ulcerative or necrotic enteritis, pullorous disease, avian typhus, disease caused by Coccidia, worms (eg roundworm, whipworm, kidneyworm), acne, bacteriosis due to Staphylococcus, bacteriosis due to Streptococcus, parvovirus, leptospirosis and others, but not limited to them.

In yet another embodiment, the method is used to improve productivity in aquaculture by preventing and/or treating infections, infestations and/or diseases of fish and other aquatic and/or marine organisms in the fish farm. It is preferable that the yeast microorganism-based composition according to this embodiment is applied to the fish farm in an inactive state.

Examples of pathogenic microorganisms against which the claimed methods and compositions are effective include viruses (e.g., Aquabirnavirus, Betanodavirus, Orthomyxovirus, Alphavirus, rhabdoviruses, and Ranavirus), bacteria (e.g., Pseudomonas fluorescens, Aeromonas, Edwardsiella, Flavobacterium, Francisella, Photobacterium, Piscirickettsia, Pseudomonas, Tenacibaculum, Vibrio, Yersinia, Lactococcus, Renibacterium and Streptococcus), fungi (e.g. Saprolegnia, Aspergillus, Penicillium, Exophiala, Ichthyophonus, Branchiomyces, Dermocystidium, Prototheca, Oscillatoria, Phoma herbarum and Paecilomyces), water mold (e.g. Saprolegnia sp .) and parasites (e.g., but not limited to, nematodes, tapeworms, roundworms, leeches, aphids, multicellular parasites such as copepods, unicellular parasites such as Ichthyophthirius multifilii, and helminths such as Eustrongylides).

Viral diseases of aquatic organisms that can be alleviated or prevented using the claimed invention include infectious pancreatic necrosis, nervous system necrosis virus, salmon anemia virus, pancreatic diseases, infectious necrosis of salmonid hematopoietic tissue, and hemorrhagic septicemia virus, and epizootic hematopoietic necrosis of salmonids. , but are not limited to them.

Bacterial diseases of aquatic organisms that can be mitigated or prevented using the claimed invention include fin rot, waterfish, septicemia caused by motile aeromonas, furunculosis, bacteriosis caused by Chryseobacterium, catfish intestinal septicemia, disease caused by Edwardsiella or causing suppuration, columnar disease fish, false columnar disease of fish, Flavobacteriia disease, or rainbow trout fry disease, bacterial gill disease, Francisella disease, winter canker disease, Pasteurella disease, Piscirickettsia disease or rickettsial septicemia, Pseudomonas septicemia, or carp rubella, disease caused by Tenacibaculum maritimum, disease caused by Vibrio, disease caused by Yersinia or intestinal disease red mouth of salmonids, disease caused by Lactococcus garvieae, disease caused by Nocardia, bacterial kidney disease, disease caused by Staphylococcus, disease caused by Streptococcus, and hemorrhagic septicemia, but not limited to them. Other diseases or infestations of aquatic organisms that may be mitigated or prevented using the claimed invention include gill rot, diseases caused by Ichthyophonus, Saprolegniaceae, velvet disease, head erosion and writhing.

In additional embodiments of the claimed invention, methods are proposed for increasing productivity in agriculture by enriching the soil, and compositions based on microorganisms according to the claimed invention are applied to the soil. By applying a composition containing one or more cultured Pichia yeasts to the soil and, optionally, one or more adjuvants, the claimed methods can increase the yield and improve the quality of produce and plant products grown due to, for example, the presence of beneficial microbial metabolites such as phytase , amino acids, proteins, vitamins and microelements. In a specific embodiment, the adjuvants are fulvic acid, humic acid or humate.

Preferably, the presence of phytase allows the treatment and/or prevention of nutrient deficiencies in plants. In particular, the present invention can be used for the treatment and/or prevention of phosphorus deficiency in plants when applied to the soil around the plants.

In a further embodiment, the present invention can be used in phosphate storage. When phosphates are present in the soil in insoluble forms, they remain there

- 16 045393 unsuitable for use by the root system of plants. By adding Triple-P product to soil, phosphates can be converted into more soluble forms that can be extracted by plants.

The claimed invention additionally provides low-cost materials and methods for feeding livestock using the claimed composition based on microorganisms. In one embodiment, the microorganism-based composition is formulated with or applied to the animal's food and/or the animal's drinking water as a dietary supplement.

The microorganism-based composition can be used as a highly nutritious source of supplemental nutrition for livestock, for example, by introducing the composition into feeders along with traditional livestock feed and the livestock consuming the composition. In one embodiment, the composition can be mixed with feed components and formulated into uniform, homogenized briquettes. As a dietary supplement, microorganism-based products can provide phytase and additional sources of amino acids, proteins, vitamins and micronutrients, among other benefits.

In some embodiments, the microbial composition may further be used to treat and/or prevent nutritional deficiencies in livestock production. In particular, the claimed invention can be used as a food additive for the treatment and/or prevention of phosphorus deficiency in livestock by supplementing their feed source with phytase.

Advantageously, the claimed invention can be used to reduce the cost of feeding livestock while increasing the growth and health of livestock, for example, by supporting their growth and their immune system and providing additional sources of phosphorus, amino acids, proteins, vitamins and other micronutrients.

In other embodiments, the claimed invention provides low-cost materials and methods for feeding fish using the claimed microbial-based composition. The microorganism-based composition can be used as a highly nutritious food additive for fish, for example, by introducing the composition into the fish's environment and ingesting the composition and/or feed by the fish. The composition can also be applied to or combined with standard fish food and fed to farmed fish. Preferably, the claimed invention can be used to reduce the cost of feeding farmed fish.

In some embodiments, the claimed invention relates to methods for increasing aquaculture productivity by increasing nutrient uptake by farmed fish. In particular, when absorbed by fish, the bio-surfactants that are produced by the microorganisms of the claimed invention help improve the overall health and quality of farmed fish by increasing the absorption of nutrients, including fat-soluble vitamins, within the digestive tract of the fish.

The composition can be applied to the environment surrounding the fish, such as water in a fish farm, in the form of, for example, a liquid solution or in the form of a dry powder, meal, flake or solid. Preferably, fish that ingest the claimed compositions experience increased weight gain compared to fish fed conventional fishmeal, with much of the weight gain resulting from an increased percentage of body protein accompanied by a lower percentage of body fat.

In another embodiment, the claimed invention provides methods for supplementing fish feeding, as well as methods for reducing the cost of feeding fish. The microorganism-based composition of the claimed invention can be applied to the fish environment as a sole source of cellular protein containing inactive yeast cells that provide a variety of proteins, carbohydrates, lipids, fatty acids, amino acids, vitamins and minerals in concentrated quantities. In some embodiments, the microbial composition may be used to treat and/or prevent nutritional deficiencies in fish. In particular, the claimed invention can be used as a food additive for the treatment and/or prevention of phosphorus deficiency in farmed fish, supplementing their food source with phytase.

Due to the rising costs of traditional fish and industrial fish oil feeds, many aquaculture farms have begun to use grain-based fish feeds. Ingredients currently used to produce fish feed include corn, soybeans and sorghum. The health effects of grain-based diets on the fish themselves are not fully known, but are likely to cause adverse effects in the long term.

In addition, the success of, for example, the seafood industry is based in part on the attractive health benefits of human consumption of seafood, such as high levels of omega-3 fatty acids. As farmed fish transition from an animal diet or omnivory to a strict plant-based diet, the nutrient content of the final fish product may be altered, for example, the fatty acid content of the fish product may be reduced. The impact of such a change on the seafood industry, as well as human nutrition, could be widespread.

Thus, acting as an additive to existing fish feeds, the claimed invention

- 17 045393 can be used to reduce the rising costs of using traditional fish-based fish feeds. The claimed invention has the potential to reduce the cost of fish feeding while providing a return to traditional fish-based fish feeds, all without compromising the health and quality of farmed fish.

Examples

A deeper understanding of the present invention and its many advantages can be obtained from the following examples given by way of illustration. The following examples illustrate some methods, applications, embodiments and variations of the present invention. They should not be construed as limiting the invention. Numerous changes and modifications may be made to the invention.

Example 1: Cultivation of Pichia Triple-P Yeast Product.

A fermentation system and method is proposed for use in accordance with the claimed invention to reduce the cost of cultivating Pichia yeast, as well as to create an inexpensive and effective production of this yeast on an industrial scale.

Triple-P is produced by yeast culture using unconventional methods and under aerobic conditions that are effective in promoting growth and metabolite production. Preferably, each of the Pichia strains is cultivated separately, after which the resulting cultures are mixed using a mechanical stirrer to form the Triple-P product.

Preferably, the systems and methods of the claimed invention are inexpensive to produce at any size and can produce virtually unlimited quantities of yeast-based products. Additionally, these systems may be operated by, for example, a farmer or producer who has little or no previous experience with fermentation systems.

Fermentation is carried out in a scale-up reactor as a batch process, preferably without stirring. A portable reactor with distribution capability is used, containing a vertical parallelepiped tank. The system is equipped with a mixing system consisting of two pipe systems, each equipped with a pumping mechanism to pump the crop from the bottom of the tank and introduce it into the upper parts of the tanks. An air compressor is used to add filtered air to the system to aerate the moving crop.

The addition of filtered air to the tanks was controlled using a sparging mechanism, which is carried out through a bubbler. Filtered air for bubbling is generated by a large volume water pumping system. In total, there are two 72-inch bubblers per tank.

The reactor has a working volume of 550-1,000 L for growing yeast to produce cells and metabolites; however, the size, material (eg metal) and configuration of reactors may vary (depending on requirements).

Culture production costs are reduced because the reactors are not sterilized using traditional methods. Instead, to produce Pichia, killer yeasts are capable of self-sterilization, i.e. their waste products fight against contamination of the crop by any possible contaminating microorganisms. In addition, a method for sanitizing empty vessels can be used, which involves treating the internal surfaces with 1% hydrogen peroxide and rinsing with a disinfectant solution and hot water under high pressure.

Inoculation of the reactor can be done with 20-30 L of an actively growing culture to ensure the presence of desired toxins and/or other waste products in the inoculum and prevent the proliferation of possible contaminants, or with a concentrated Pichia supernatant. In addition, inoculation can begin at a pH level of 2.5-3.5 and increase to 4.5-5.0 after 12 hours during the start of the fermentation cycle.

Fermentation temperature and pH are not critical, but in general the temperature should be in the range of about 25 to less than 37°C, preferably from about 25 to 30°C. The pH level should be in the range of about 3.0 to about 5.0, preferably from about 3.5 to about 4.5. Stabilizing the pH during fermentation is not necessary, but it is recommended to increase the pH to 3.5-4.0 if a decrease below 3.0 occurs.

If necessary, adjusting or maintaining the pH during the fermentation process can be accomplished using manual or automatic methods conventional in the art, for example, using automatic pH controllers for base addition. Preferred bases used to adjust pH include, but are not limited to, NaOH and KOH.

To obtain Pichia by fermentation, either a complex medium such as NYDB (nutrient broth, 10 g, yeast extract, 8 g, dextrose 20 g per 1 liter of water), PDB (potato dextrose broth), ME (malt extract), or a medium with a known chemical composition with the addition of a various carbon source, such as glucose, sucrose, sorbitol, molasses, etc.

However, if necessary, the cost of the medium can be further reduced by including only the three main components as outlined below.

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Component Concentration (g/l) Weight (g/l) Yeast extract 5 5,000 Glucose thirty 30,000 Urea 1 1000

Typical culture times range from 48 to 72 hours, and CFU concentrations range from 0.6 to 1.0 billion cells/ml.

The finished crop is an end product for many applications. However, if necessary, yeast cells can be collected by centrifugation, filtration or sedimentation. The resulting yeast paste (wet biomass) can be preserved by adding salt, glycerol, lactose, trehalose, sucrose, amino acid to increase shelf life.

Most commercial yeast products are produced by high-speed centrifugation to separate the yeast cells from a liquid medium. The collected yeast cells can be dried by freeze drying, fluid bed drying or spray drying. Dried products will be rehydrated in water in preparation for use. These stages can damage cell walls and cause cell death.

If yeast cells are to be stored for a long time before use, they should be placed in appropriate containers and stored at 4°C. With this method, the yeast cells settle to the bottom of the containers after two days. The supernatant medium can be removed by draining, leaving a small amount of supernatant to coat the yeast cells. Under these conditions and after six months of storage, the viability of Pichia cells will not decrease sharply.

Example 2 Calculation of phytase production using Pichia anomala.

Pichia anomala was grown in a fermentation reactor at pH adjusted to 5.0 for 72 h in the following growth medium: molasses (4%), urea (0.2%), peptone (2.5%).

Phytic acid was obtained from sesame seeds by breaking/crushing the seeds. The seed parts (5 g) were then added to 10 ml of water and mixed. The medium and cells were tested for the presence of phytase. 0.2 M glycine buffer was used to adjust the pH to 4 and the pH of phytic acid was also adjusted to 4.

After 3 days, phytase activity was measured by preparing 6 different standards:

0) 550 µl water,

1) 10 µl 50 mM potassium phosphate and 540 µl water,

2) 20 μl of 50 mM potassium phosphate and 530 μl of water,

3) 30 μl of 50 mM potassium phosphate and 520 μl of water,

4) 40 µl 50 mM potassium phosphate and 510 µl water,

5) 50 µl of 50 mM potassium phosphate and 500 µl of water.

phytic acid controls were prepared using 500 μL phytic acid, 25 μL 0.2 M glycine buffer, and 25 μL water.

phytase control samples were prepared using 25 μL phytase, 25 μL 0.2 M glycine buffer, and 500 μL water.

phytase samples were prepared using 500 μL phytic acid, 25 μL 0.2 M glycine buffer, and 25 μL phytase.

A 4 ml solution of acetone - ammonium molybdate - H ₂ SO ₄ (50 ml - 25 ml - 25 ml) was added to each of the 6 standards.

Each of the samples and controls was transferred to a water bath for 30 min at 37°C. After 30 minutes, 4 ml of acetone-ammonium molybdate-H ₂ SO ₄ solution was quickly added to each of the samples and controls to precipitate phosphate. They were then centrifuged and 300 μl of the supernatant was dissolved in 600 μl of water.

Next, 500 μl of all samples, controls and standards were loaded into well plates of the Varioscan device and absorbance was measured at 400 nm.

The values of the standards were measured 3 times and their average values were taken. The value of standard 0 was subtracted from the value of each of standards 1-5 to arrive at a number that correlated with the absorbance of the specific phosphate concentration. The average optical density for each standard is given in Table. 1. The average optical density of samples and controls is given in table. 2.

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Claims (7)

Table 1 Average optical density (OD) and levels (mM) of phosphate _____________measured for standards 0-5________________________ Standard Optical Density (OD) Phosphate (mM) 0 0 0 1 0.0456 0.9091 2 0.1426 1.8182 3 0.1696 2.7273 4 0.2566 3.6364 5 0.2726 4.5455 table 2 Average optical density (OD) of samples and controls. Sample/Control Optical Density (OD) Phytic acid control 0.26 Phytase control sample 0.047 Phytase sample 0.319 To determine the millimoles of phosphate released, the average absorbance of the phytase controls was added to the average absorbance of the phytic acid controls and subtracted from the average absorbance of the phytase sample. The resulting number was 0.012 OD. Based on the above figure, the value of released phosphate was 0.198 mM. units/ml enzyme activity (mM phosphate) x (dilution factor) (time) x (amount of enzyme added) Finally, according to the above equation (1), the phytase concentration in the reactor was calculated as 792 U/mL. Example 3: Application of Triple-P to Tomato Plants. Tomato plants were treated with Triple-P. An increase in the height of tomato plants by 20-25% was observed. In addition, both the number and size of fruits produced increased by 50%. CLAIM 1. A method for increasing productivity in agriculture and horticulture, characterized in that the method includes applying a composition containing the yeasts Pichia anomala, Pichia kudriavzevii and Pichia guilliermondii and a fermentation broth in which each of the yeasts was cultivated to the soil in which the plants are grown, while the method increases productivity due to one or more of the following consequences of its application: increasing yield, reducing nutrient deficiencies in the plant, inhibiting the growth of phytopathogenic fungi, inhibiting the growth of phytopathogenic bacteria and control of nematodes. 2. The method according to claim 1, characterized in that the nutrient deficiency is a phosphorus deficiency. 3. The method according to claim 1, characterized in that the composition additionally contains at least one adjuvant selected from fulvic acid, humic acid or humate. 4. The method according to claim 1, characterized in that the composition additionally contains nutrients for the growth of microorganisms. 5. The method according to claim 1, characterized in that the composition additionally contains yeast waste products selected from the group consisting of enzymes, acids, solvents, alcohols, proteins, vitamins, minerals, trace elements, amino acids and bio-surfactants. 6. The method according to claim 1, characterized in that the composition additionally contains the enzyme phytase. 7. The method according to claim 1, characterized in that the composition additionally contains a carrier. -