WO2017087234A1 - Méthodes et compositions destinées au traitement et à la prévention de la formation de daggins - Google Patents

Méthodes et compositions destinées au traitement et à la prévention de la formation de daggins Download PDF

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WO2017087234A1
WO2017087234A1 PCT/US2016/061143 US2016061143W WO2017087234A1 WO 2017087234 A1 WO2017087234 A1 WO 2017087234A1 US 2016061143 W US2016061143 W US 2016061143W WO 2017087234 A1 WO2017087234 A1 WO 2017087234A1
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composition
animal
microorganisms
pen
coating
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PCT/US2016/061143
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English (en)
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Michael Keoni MANION
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Empire Technology Development Llc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K1/00—Housing animals; Equipment therefor
    • A01K1/015—Floor coverings, e.g. bedding-down sheets ; Stable floors
    • A01K1/0152—Litter
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/02—Algae
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/04—Preserving or maintaining viable microorganisms
    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
    • D06M16/00—Biochemical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. enzymatic
    • D06M16/003—Biochemical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. enzymatic with enzymes or microorganisms
    • 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
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Dags comprise a mixture of excrement, soil, and chaff. Livestock affected by heavy dag burdens can incur cost penalties at the abattoir and increase the risk of bacterial contamination of meat resulting from knives becoming contaminated with fecal organisms during slaughtering. Where the breech region of an animal is not appropriately maintained it is susceptible to soiling by fecal matter and urine, which create insect attractant sites where insects may lay eggs leading to flystrike. Dags are difficult to prevent, costly to remove, produce discomfort in the animal during removal, and are associated with an increased incidence of flystrike.
  • compositions and methods for the treatment and prevention of dag formation and related conditions are provided herein.
  • compositions comprising
  • microencapsulated enzyme-producing microorganisms are provided, as are methods of making and using the same.
  • the disclosure provides a composition comprising a dormant enzyme- producing microorganism.
  • the composition includes a coating encapsulating the microorganism.
  • there is a coating configured to release the microorganism upon hydration.
  • the microorganism is activated by hydration to produce one or more enzymes.
  • the enzyme-producing microorganism is a prokaryotic organism. In some embodiments, the enzyme-producing microorganism is a eukaryotic organism. In some embodiments, the enzyme-producing microorganisms are a combination of prokaryotic and eukaryotic organisms.
  • the prokaryotic organism is one or more selected from the group consisting of Bacillis subtilis, non-pathogenic Escherichia coli, actinomycetes, and Archaea.
  • the eukaryotic organism is one or more selected from the group consisting of fungi, algae, and protozoa.
  • the one or more enzymes is selected from the group consisting of hemicellulases, cellulases, amylases, glucanases, papain, pectinases, xylanases, proteases (e.g., keratinases, trypsin), nucleases, lipases, laccases (e.g., ligninases), peroxidases (e.g., lignin peroxidases), hydrolases, oxidoreductases, and lyases.
  • the coating encapsulating the microorganism comprises one or more materials that degrade upon hydration.
  • the material that degrades upon hydration is one or more materials selected from the group consisting of soil, biomass, manure, alginate, ethyl cellulose, polyvinyl alcohol (PVA), chitosan, and gelatin.
  • the coating further comprises a biodegradable binder, including one or more selected from the group consisting of polylactic acid (PLA), polyvinyl alcohol (PVA), starch, natural resins, lignin, and other bioadhesives such as proteins (e.g., casein).
  • a biodegradable binder including one or more selected from the group consisting of polylactic acid (PLA), polyvinyl alcohol (PVA), starch, natural resins, lignin, and other bioadhesives such as proteins (e.g., casein).
  • the thickness of the coating material ranges from about 10 nm to about 1 mm. In some embodiments, the thickness of the coating material ranges from about 10 nm to about 100 nm. In some embodiments, the thickness of the coating material ranges from about 100 nm to 200 nm. In some embodiments, the thickness of the coating material ranges from about 200 nm to about 300 nm. In some embodiments, the thickness of the coating material ranges from about 300 nm to about 400 nm. In some embodiments, the thickness of the coating material ranges from about 400 nm to about 500 nm. In some embodiments, the thickness of the coating material ranges from about 500 nm to about 600 nm.
  • the thickness of the coating material ranges from about 600 nm to about 700 nm. In some embodiments, the thickness of the coating material ranges from about 700 nm to about 800 nm. In some embodiments, the thickness of the coating material ranges from about 800 nm to about 900 nm. In some embodiments, the thickness of the coating material ranges from about 900 nm to about 1 mm.
  • the coating material is crosslinked with one or more crosslinking agents selected from the group consisting of aldehydes (e.g., formaldehyde), alcohols (e.g., ethanol, methanol), and oxidizing agents (e.g., potassium permanganate).
  • aldehydes e.g., formaldehyde
  • alcohols e.g., ethanol, methanol
  • oxidizing agents e.g., potassium permanganate
  • the microorganism is present in a dehydrated state.
  • the composition is in the form of microparticles.
  • each microparticle contains about 10 to about 10 9 microorganisms.
  • the composition is in the form of a powder. In some embodiments, the composition is in the form of a liquid suspension of microparticles.
  • the disclosure provides a method for treating or preventing dag formation in animals, the method comprising: applying to the floor surface of an animal pen comprising animal pen material a composition comprising a dormant enzyme-producing microorganism encapsulated by a coating, wherein the coating is configured to release the microorganism upon hydration, and wherein the microorganism is activated by hydration to produce one or more enzymes; mixing the composition with the animal pen material to form a mixture; and hydrating portions of the mixture over a period of time, wherein the enzymes produced by the activated microorganisms degrade elements of the pen material that adhere to animal skins or hides when animals contact the pen material, thereby treating or preventing dag formation.
  • the mixing is effected by at least one animal traversing across the animal pen and/or rainfall.
  • the hydration is provided by rainfall.
  • the period of time corresponds to a season comprising multiple wet/dry cycles. In some embodiments, the period of time is at least about 100 days. In some embodiments, the period of time is at least about one year.
  • the pen material comprises one or more of soil, manure, undigested silage, or biomass.
  • the enzyme-producing microorganism is a prokaryotic organism, a eukaryotic organism, or a combination thereof.
  • the prokaryotic organism is selected from the group consisting of Bacillis subtilis, non-pathogenic Escherichia coli, actinomycetes, Archaea, and combinations thereof.
  • the eukaryotic organism is selected from the group consisting of fungi, algae, protozoa, and combinations thereof.
  • the one or more enzymes is one or more selected from the group consisting of hemicellulases, cellulases, amylases, glucanases, papain, pectinases, xylanases, proteases ⁇ e.g., keratinases, trypsin), nucleases, lipases, laccases ⁇ e.g., ligninases), peroxidases ⁇ e.g., lignin peroxidases), hydrolases, oxidoreductases, and lyases.
  • the coating comprises one or more materials that degrade upon hydration.
  • the material that degrades upon hydration is one or more selected from the group consisting of soil, biomass, manure, alginate, ethyl cellulose, polyvinyl alcohol (PVA), chitosan, and gelatin.
  • the coating further comprises one or more biodegradable binders selected from the group consisting of polylactic acid (PLA), polyvinyl alcohol (PVA), starch, natural resins, lignin, and other bioadhesives such as proteins ⁇ e.g., casein).
  • PVA polylactic acid
  • PVA polyvinyl alcohol
  • starch natural resins
  • lignin lignin
  • other bioadhesives such as proteins ⁇ e.g., casein.
  • the thickness of the coating material ranges from about 10 nm to about 1 mm. In some embodiments, the thickness of the coating material ranges from about 10 nm to about 100 nm. In some embodiments, the thickness of the coating material ranges from about 100 nm to 200 nm. In some embodiments, the thickness of the coating material ranges from about 200 nm to about 300 nm.
  • the thickness of the coating material ranges from about 300 nm to about 400 nm. In some embodiments, the thickness of the coating material ranges from about 400 nm to about 500 nm. In some embodiments, the thickness of the coating material ranges from about 500 nm to about 600 nm. In some embodiments, the thickness of the coating material ranges from about 600 nm to about 700 nm. In some embodiments, the thickness of the coating material ranges from about 700 nm to about 800 nm. In some embodiments, the thickness of the coating material ranges from about 800 nm to about 900 nm. In some embodiments, the thickness of the coating material ranges from about 900 nm to about 1 mm.
  • the coating material is crosslinked with a crosslinking agent selected from the group consisting of aldehydes (e.g., formaldehyde), alcohols (e.g., ethanol, methanol), and oxidizing agents (e.g., potassium permanganate), or a combination thereof.
  • a crosslinking agent selected from the group consisting of aldehydes (e.g., formaldehyde), alcohols (e.g., ethanol, methanol), and oxidizing agents (e.g., potassium permanganate), or a combination thereof.
  • the microorganism is present in a dehydrated state.
  • the composition is in the form of
  • each microparticle contains about 10 to about 10 9 microorganisms.
  • the method further comprises contacting the animal with the pen material comprising enzymes produced by the activated microorganisms, thereby treating or preventing dag formation.
  • the composition is in the form of a powder. In some embodiments, the method comprises dusting the animal with the powder. [0032] In some embodiments of the method, the composition is in the form of a liquid suspension of microparticles. In some embodiments, the method comprises spraying the animal with the liquid suspension of microparticles.
  • Figure 1 is an illustrative overview of a system in which the compositions of the present technology comprising microcapsules produce enzymatically active microorganisms to prevent dag formation.
  • Figure 2 is an illustrative example of release and activation profiles for
  • compositions and methods related to treating and preventing dag formation, and related disorders or conditions in animals, over the course of a relatively long period of time e.g., over the course of a season comprising multiple wet/dry cycles.
  • Figure 1 provides an illustrative overview of the methods and compositions described herein.
  • compositions and methods of the present technology comprise the use of a steady release encapsulated microbiome (e.g., microcapsules) 102 comprising enzyme-producing microorganisms 104 that degrade dag precursors or binding elements (e.g., complex sugars, proteins, fat, lignin) 106 to reduce the ability of animal pen material (e.g., dung, urine, mud, biomass) 108 to adhere to an animal' s coat 110.
  • animal pen material e.g., dung, urine, mud, biomass
  • the pen material that does adhere to the animal' s coat does so only weakly due to the degradation of binding elements 106 in the pen material 108 by enzymes 112 produced by the microorganism and is easily brushed or washed away.
  • compositions and methods of the present technology provide for the steady release of the enzymes 112 over the course of a relatively long period of time to ensure that the catalytic activity of the enzymes is applied throughout the time period.
  • the steady release of the enzymes 112 is accomplished by microencapsulating dormant enzyme- producing microorganisms 104 in a coating material that degrades when hydrated 114 to release and activate the microorganisms 104 in a controlled manner throughout the period of time, such as throughout a season comprising multiple wet/dry cycles.
  • the desired enzymatic activity can be directed to the animal's coat 110 as the animal will contact the activated microorganism through normal activity (e.g., mixing the animal pen or feedlot material and lying in the material).
  • the activation of the microorganisms is triggered by the very event that typically causes dag formation, e.g., rainfall 118, which will hydrate the encapsulant and release and activate at least a portion of the microorganisms.
  • the animals coming into contact with the animal pen or feedlot floor material 108 comprising the activated microorganisms 120 will ensure that the enzymatic activity is transferred to the location at which the dag needs to be prevented, i.e., the animal's coat 110.
  • Enzymes 112 produced by the activated microorganisms 120 degrade elements in the pen material that would adhere to an animal's coat and may also contact the animal when the animal comes into contact with the pen material, thereby reducing the adhesion of dag material to the animal's coat.
  • the enzymes may react with elements of the animal's hide to reduce the ability of the animal pen or feedlot material to bind to the animal's coat 110.
  • An example of such an enzyme is keratinase.
  • the encapsulation coating comprises elements of the animal pen or feedlot itself, such as soil, biomass, and manure.
  • the present technology provides for the direct application of the encapsulated enzyme-producing microorganisms to the feedlot or animal pen through either their direct application to the animal or to the feedlot or animal pen floor.
  • not all of the microcapsules of the present technology release all of their contents at once. Rather, as illustrated by Figure 1, a subset of the microcapsules may release their contents upon hydration 124, while a separate subset of microcapsules 102 will remain to be activated by hydration ⁇ e.g., during a subsequence wet period or rainfall).
  • animal pen and “feedlot” are used interchangeably and include any enclosure in which animals are kept. The terms also include a lot or building or a combination of lots and buildings intended for confined feeding, breeding, raising, or holding animals where manure may accumulate.
  • biomass may include lignin, lignocellulose, cellulose, or combinations thereof.
  • Biomass can be derived from a variety of sources, such as waste organic matter (e.g. , from compostable sources) or from numerous types of plants, such as miscanthus, switchgrass, hemp, corn, poplar, willow, sorghum, sugarcane, bamboo, or trees such as eucalyptus or palm.
  • the term "breech region,” refers to the area around the vulva and/or anus of the animal that may collect urine and/or feces.
  • the term "dormant,” with reference to the enzyme-producing microorganisms of the present technology, refers to a state in which the enzymatic and/or metabolic activities of the microorganism are suspended (e.g., by dehydration or in a spore state), and in which the microorganism may be activated or re-animated (e.g. , by hydration or oxygenation).
  • the microorganisms are in a spore state (e.g. , dormant) and are encapsulated according to the present technology.
  • the spores are dehydrated, e.g., by freeze drying, and encapsulated according to the present technology.
  • coating refers to any compound, natural or synthesized, that coats or encapsulates the microorganisms to contribute to a delay of the release of the microorganism until the coating is hydrated.
  • Exemplary, non-limiting coatings may include the same materials present in the animal pen or feedlot, such as soil (e.g. , clay), biomass, and manure.
  • the encapsulation materials include, but are not limited to, alginate, ethyl cellulose, polyvinyl alcohol (PVA), chitosan, and gelatin.
  • biodegradable binders such as polylactic acid (PL A), polyvinyl alcohol (PVA), starch (e.g., dextrin), natural resins, lignin, or other bioadhesives, such as proteins (e.g., casein) and crosslinking agents may be added to the coating.
  • Exemplary, non-limiting crosslinking agents may include aldehydes (e.g., formaldehyde), alcohols, (e.g., ethanol, methanol), and oxidizing agents (e.g., potassium permanganate), or combinations thereof.
  • aldehydes e.g., formaldehyde
  • alcohols e.g., ethanol, methanol
  • oxidizing agents e.g., potassium permanganate
  • the amount and/or thickness of either the coating material and/or crosslinking of the coating material can be varied.
  • larvastrike refers to an infestation by larvae or maggots of flies.
  • microorganism means naturally-occurring and/or genetically-modified prokaryotic and eukaryotic microbial species, spores, or progenitors thereof from the domains Archaea, Bacteria, and Eukarya, the latter including yeast and filamentous fungi, protozoa, algae, and higher Protista.
  • the terms “treating” or “treatment” or “alleviation” refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted condition.
  • a subject is successfully “treated” for dag formation if, after contacting an effective amount of the microencapsulated enzyme-producing microorganisms according to the methods described herein, the subject shows observable and/or measurable reduction of dags.
  • the various modes of treatment or prevention of dag formation as described are intended to mean “substantial,” which includes total but also less than total treatment or prevention, and wherein some biologically or medically relevant result is achieved.
  • prevention or “preventing” of a disorder or condition refers to a compound that reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample.
  • compositions comprising dormant enzyme-producing microorganisms that are encapsulated in a material configured to degrade when hydrated and to provide activated enzyme-producing microorganisms over a relatively long period of time (e.g., over the course of an entire season comprising multiple wet/dry cycles) to prevent or treat the formation of dags in animals during that period of time.
  • the encapsulating material may comprise the same materials present in the animal pen or feedlot (e.g., soil, biomass, and manure).
  • the microorganisms are capable of producing enzymes for several days or weeks, as long as they remain hydrated (e.g., during a wet period).
  • the enzymatic activity of the microorganisms subsides until the next wet period activates a new subset of the encapsulated microorganisms.
  • compositions comprising one or more enzyme-producing microorganisms.
  • microorganisms already present in the animal pen or feedlot material are suitable candidates. Additionally or alternatively, in some embodiments, selection of microorganisms that produce a significant amount of the appropriate enzymes is a consideration.
  • the criteria for selecting the microorganisms of the present technology may include: the ability of the microorganism to express the appropriate catalytic enzyme;
  • biosafety for the animals and workers in contact with the animal pen and feedlot material minimal impact on the environment; ease of production; and ability to be dormant for extended periods of time and robustly activate.
  • the microorganisms of the present technology may include naturally-occurring and/or genetically-modified prokaryotic and eukaryotic microbial species from the domains Archaea, Bacteria, and Eukarya, or combinations thereof.
  • Prokaryotic organisms include, but are not limited to bacteria such as, Bacillis subtilis, non-pathogenic Escherichia coli, actinomycetes, and Archaea.
  • Eukaryotic organisms include, but are not limited to, fungi, algae, and protozoa.
  • microorganisms of the present technology include one or more microorganisms selected from the group consisting of Acetobacter aceti, Achlya, Achnanthes, Acinetobacter calcoaceticus, Actinosphaerium, Aerococcus, Aerococcus salmonicida, Aeromonas punctate, Agrobacterium radiobacter, Alcaligenes eutrophus, Alcaligenes faecalis, Alcaligenes viscolactis, Alicyclobacillus acidocaldarius,
  • Alicyclobacillus acidoterrestris Alicyclobacillus cycloheptanicus, Alloyces, Amoeba chaos (Pelomyxa), Amoeba proteus, Ampelomyces quisqualis, Amphidinium, Anabaena, Anacystis, Ankistrodesmus, Antracobia, Aquaspirillum, Aquaspirillum itersonii, Aquaspirillum polymorphism, Aquaspirillum serpens, Aquaspirillum sinuosum, Arcella, Armillariella mellea, Arthobacter globiformis, Arthobacter oligospora, Arthrobotrys conoides, Aspergillus, Aspergillus flavus, Aureobasidium pullulans, Azospirillum, Azotobacter, Azotobacter chrooccum, Azotobacter vinelandii, Bacillus aerophilus, Bacillus amyloliquefaciens,
  • Candida oleophila Candida saitoana, Careria, Caulerpa, Caulobacter, Cellumonas, Chilomonas, Chlamydomonas, Chlorella, Chromobacteria, Citrobacter, Cladophora, Clavicorona pyxidata, Clonostachys rosea f. catenulate, Closterium,
  • Corynebacterium xerosis Cosmarium, Crithidia fasciulata, Cryphonectria parasitica, Cryptococcus albidus, Cyanophora, Cyclotella, Cylindrospermum, Dactylaris, Derbesia, desmids, Desulfovibrio, Deuteromycetes, Dictyostelium, Dictyota, Didinium, Difflugia, Dunaliella, Ectocarpus, Edwardsiella, Egregia, Enterobacter, Enterococcus faecium, Enteromorpha, Epulopiscium spp., Eremosphaera, Erwinia, Erwinia dissolvens, Escherichia coli, Eudorina, Euglena, Euplotes, Fischerella, Flavobacterium, Fritschiella, Fucus,
  • Leuconstoc mesenteroides Lucibacterium spp., Lyngbya, Lysinibacillus boronitolerans, Lysinibacillus boronitolerans, Lysinibacillus boronitolerans, Macrocystis, Merismopedia, Mesotaenium, Metabacterium polyspora, Methanomonas methylovora, Metschnikowia fructicola, Micrasterias, Microbacterium testaceum, Micrococcus luteus, Micrococcus mutans, Micrococcus roseus, Micrococcus salivarius, Micrococcus stereothermophilus, Microdochium dimerum, Microspora, Mougeotia, Mucor rouxii, Mycobacterium phlei, Mycobacterium smegmatis, Myxogastria, Navicula, Neisseria flava, Neisseria sicca,
  • Nereocystis Netrium, Neurospora crassa, Nitella, Nitrobacter, Nitrosomonas, Nostoc, Ochromonas, Oedogonium, Oscillatoria, Paenibacillus amylolyticus, Paenibacillus barcinonensis, Paenibacillus glycanilyticus, Paenibacillus lautus, Paenibacillus peoriae, Paenibacillus polymyxa, Paenibacillus taichonnesis, Paenibacillus xylanexedens,
  • Pandorina Paramecium, Pediastrum, Pediococcus, Penicillium camemberti, Penicillium roqueforti, Peranema, Peridinium, Phacus, Phlebiopsis gigantean, Photobacterium,
  • Pseudozyma flocculosa Pyrsonympha, Pythium oligandrum, Rhizobium, Rhodococcus rhodochrous, Rhodospirillum rubrum, Rhodotorula rubrum, Rhyzopus, Ruminococcus, Saccharomyces cerevisiae, Saccharomyces uvarum, Saccharomycoides ludwigii, Salmonella choleraesuis, Saprolegnia, Sarcina aurantiaca, Sarcina flava, Sarcina lutea, Scenedesmus, Schizophyllum, Schizosaccharomyces octosporus, Sclerotium rolfsii, Scytonema,
  • Selanastrum Selanastrum, Selenomonas, Serratia marcescens, Serratia liquefaciens, Solibacillus silvestris, Sordaria flmicola, Spirillum serpens, Spirillum volutans, Spirogyra, Spirostomum,
  • Spirostomum volutans Spirulina, Sporosarcina globispora, Sporosarcina psychrophila, Sporosarcina ureae, Staphylococcus saprophyticus, Staphylococcus aureus, Staphylococcus epidermidis, Staurastrum, Stentor, Stigeoclonium, Streptococcus antibioticus, Streptococcus cremoris, Streptococcus diacetilactis, Streptococcus durans, Streptococcus faecalis,
  • Streptococcus thermophiles Streptococcus venezuelae, Streptomyces albus, Succinomonas, Sulfolobus, Synedra, Synura, Talaromyces flavus, Taxomyces andreanae, Tetrahymena, Thalassiosira, Thermomyces lanuginosus, Thermoplasma, Thermothrix, Thiobacillus, Thiobacillus thioparus, Tolypothrix, Trachelomonas, Tribonema, Trichoderma asperellum, Trichoderma atroviride, Trichoderma harzianum, Trichoderma polysporum, Trichoderma stromaticum, Trichoderma virens, Trichoderma viride, Trichonympha, Tritrichomonas augusta, Trypanosoma lewisi, Trypanosoma ranarum, Ulocladium oudemansii, Ulothrix
  • the one or more microorganisms selected are typically non-pathogenic to the animals, and are present in animal pens or in the environment of animal pens. Additionally or alternatively, in some embodiments, the one or more microorganisms selected are typically non-pathogenic to the animals and are present naturally in or on the animal, e.g., in the animal gastrointestinal tract, on the animal's coat, etc. In some embodiments, the microorganisms are genetically modified to express one or more enzymes (e.g., as described in section C, below).
  • the microorganisms of the present technology are dormant until activated by hydration to produce enzymes that degrade elements of animal pen material that otherwise adhere to hair of animal skin or hides thereby preventing dag formation or treating dag formation by weakening the structure of pen material that may adhere to the hair of animal skin or hides.
  • the capsules in which they are contained may be dehydrated. Cryo-preservation, freeze-drying or lyophilization, foam drying, spray drying, and fluidized bed drying are standard means for preserving microorganisms. These methodologies for dehydration suspend enzymatic activities of the microorganisms such as the activities of proteases, hydrolases, and nucleases. Other conventional techniques for rendering microorganisms dormant may also be used.
  • the microorganisms are rendered dormant by spore formation.
  • Spore formation can be induced several ways, such as by incubating the microorganisms in a sporulation media.
  • Bacillus spores can be formed by transferring cultures of the bacilli to sporulation medium.
  • Another dormant form of microorganisms is what is known as viable but nonculturable (VBNC), which can be induced by exposure to stress such as adverse nutrient, temperature, osmotic, oxygen, or light conditions.
  • VBNC viable but nonculturable
  • Another form of dormancy demonstrated in eukaryotes is the induction of a metabolic hibernation state, which can be achieved, for example, by exposure to anoxia or reversibly removing the ability of the cell to utilize oxygen such as by exposure to hydrogen sulfides.
  • a metabolic hibernation state which can be achieved, for example, by exposure to anoxia or reversibly removing the ability of the cell to utilize oxygen such as by exposure to hydrogen sulfides.
  • this hydration will also act as the trigger for the re-animation or activation of the microorganisms and their subsequent production of enzymes.
  • the enzymes produced by the activated microorganisms of the present technology target dag precursor elements (e.g., complex sugars, proteins, fats, lignin) present in animal pen or feedlot materials (e.g., soil, manure, undigested silage, and other biomass present in the pen or feedlot) for degradation to break down the elements that may adhere the pen or feedlot material to an animal' s coat.
  • dag precursor elements e.g., complex sugars, proteins, fats, lignin
  • animal pen or feedlot materials e.g., soil, manure, undigested silage, and other biomass present in the pen or feedlot
  • the enzymes e.g., keratinases
  • the enzymes produced by microorganisms of the present technology to prevent and treat dag formation include, but are not limited to, hemicellulases, cellulases, amylases, glucanases, papain, pectinases, xylanases, proteases (e.g., keratinases, trypsin), nucleases, lipases, laccases (e.g., ligninases), peroxidases (e.g., lignin peroxidases), hydrolases, oxidoreductases, and lyases.
  • the enzymes may be applied to the animal pen or feedlot at pH values close to their activity maxima (e.g., pH 4-9).
  • the dag-degrading enzymes are derived from naturally- occurring microorganisms. Additionally or alternatively, in some embodiments, genetically- modified microorganisms engineered to produce dag-degrading enzymes may be used in the compositions of the present technology.
  • the genetically-modified microorganisms are engineered to include an exogenous nucleic acid encoding one or more enzymes selected from the group consisting of hemicellulases, cellulases, amylases, glucanases, papain, pectinases, xylanases, proteases (e.g., keratinases, trypsin), nucleases, lipases, laccases (e.g., ligninases), peroxidases (e.g., lignin peroxidases), hydrolases, oxidoreductases, and lyases.
  • one or more enzymes selected from the group consisting of hemicellulases, cellulases, amylases, glucanases, papain, pectinases, xylanases, proteases (e.g., keratinases, trypsin), nucleases, lipases, laccases (e.
  • One aspect of the present technology is the ability to deliver the active enzymes from microorganisms to prevent or treat dag formation in animals during wet periods, which alternate throughout the year and may extend over one hundred days.
  • the present technology provides compositions and methods for releasing enzyme-producing microorganisms not all at once, but over the course of a season, which may comprise several wet/dry cycles.
  • the present technology provides encapsulated enzyme-producing microorganisms.
  • Figure 2 provides an illustrative example of the release and activation profiles of the encapsulated microorganisms of the present technology over the course of several wet/dry cycles.
  • the encapsulating material comprises materials present in the animal pen or feedlot, including, but not limited to, soil, biomass, and manure.
  • the encapsulation materials include, but are not limited to, alginate, ethyl cellulose, polyvinyl alcohol (PVA), chitosan, and gelatin.
  • PVA polyvinyl alcohol
  • the use of such materials not only provides a cost effective means of producing the encapsulating material, but also provides an encapsulation material that is environmentally and biologically compatible with the animal pen and feedlot production.
  • the encapsulation materials can be easily integrated into the pen and feedlot to minimize any potential harm to the animals or environment that may otherwise result from introducing foreign materials into the animal pen or feedlot.
  • the encapsulation material may also include a biodegradable binder including, but not limited to, polylactic acid (PLA), polyvinyl alcohol (PVA), starch (e.g., dextrin), natural resins, lignin, or other bioadhesives, such as proteins (e.g., casein).
  • Crosslinking agents may also be added to the encapsulation material.
  • Exemplary, non-limiting crosslinking agents may include aldehydes (e.g., formaldehyde), alcohols, (e.g., ethanol, methanol), and oxidizing agents (e.g., potassium permanganate), or combinations thereof.
  • the encapsulation material is configured to degrade and release activated microorganisms when hydrated, such as during a wet period of rainfall. Accordingly, the activated microorganisms are released at a time that is critical for dag formation and are also released at a location that is effective for preventing or treating dag formation, e.g., in the animal pen or feedlot material with which the animal comes into contact or directly on the animal.
  • the enzyme-producing microorganisms disclosed herein may be coated or encapsulated to ensure controlled release over a relatively long period of time.
  • the period of time corresponds to a season comprising multiple wet/dry cycles.
  • the period of time is at least about 100 days. In some embodiments, the period of time is at least about one year.
  • amount/thickness of the encapsulation material and/or crosslinking of the encapsulant can be varied.
  • crosslinking agents include, but are not limited to aldehydes (e.g., formaldehyde), alcohols (e.g., ethanol or methanol), and oxidizing agents (e.g., potassium permanganate).
  • Coating thickness may be increased by adding additional layers of the encapsulant and/or increasing the thickness of the encapsulant.
  • the animal pen and feedlot conditions such as the amount of rainfall and/or pH may also affect the rate of degradation of the encapsulation material.
  • compositions comprising a plurality of capsules with variable encapsulant thicknesses and/or crosslinking amounts may be delivered to the animal pen or feedlot.
  • the encapsulant of the present technology may be formed into microcapsules or nanocapsules.
  • the nanocapsules may have an average diameter of about 100 nm to 1000 nm, 200 nm to 900 nm, 300 nm to 800 nm, 400 nm to 700 nm, 500 nm to 600 nm.
  • the microcapsules may have an average diameter of about 1 ⁇ to 20 ⁇ , 20 ⁇ to 50 ⁇ , 50 ⁇ to 100 ⁇ , 100 ⁇ to 200 ⁇ , 200 ⁇ to 300 ⁇ , 300 ⁇ to 400 ⁇ , 400 ⁇ to 500 ⁇ , 500 ⁇ to 600 ⁇ , 600 ⁇ to 700 ⁇ , 700 ⁇ to 800 ⁇ , 800 ⁇ to 900 ⁇ , or 900 ⁇ to 1000 ⁇ .
  • each microcapsule or nanocapsule contains about 10- 10 9 microorganisms per capsule.
  • the composition is present in the form of a powder.
  • the composition is in the form of a liquid suspension of microcapsules or nanocapsules. In some embodiments of the liquid
  • the microcapsules or nanocapsules are formulated to degrade over an extended period of time.
  • the liquid formulation may comprise a plurality of capsules with variable encapsulant thicknesses and/or crosslinking amounts.
  • the compositions are formulated as a soil conditioner that can be added to the flooring of an animal pen or feedlot.
  • adhering agents are added to the encapsulation material to ensure that the encapsulated material associates with animal coats.
  • adhering agents include, but are not limited to, starch (e.g., dextrin), natural resins, lignin, or other bioadhesives such as proteins (e.g., casein).
  • Indicators such as a natural dye, may be added to the formulation (e.g., in the microencapsulation itself, or in the shell material), in order to provide a visual identification of the presence of the material in the soil. Additionally, a dye may be released by the microorganisms upon activation, to demonstrate their presence and activity, especially on the hide of the animal. Similarly, at least a portion of the microorganisms may produce a volatile organic compound upon active metabolism, which will provide an olfactory cue as to their effectiveness.
  • the animals coming into contact with the pen or feedlot floor material comprising the microorganisms of the present technology will provide for the ability to mix the compositions with the pen or feedlot material and transfer the enzymatic activity of the microorganisms to the animal's coat to prevent and/or treat dag formation.
  • Multiple routes may be taken to introduce the encapsulated microorganisms of the present technology to the animal pen or feedlot.
  • compositions of the present technology can be added directly to the animal pen or feedlot by dusting or spraying the compositions onto the pen or feedlot flooring.
  • the compositions can be combined with the material already present in the pen or feedlot (e.g., soil, manure, biomass).
  • the compositions can be mixed with the pen or feedlot material as animals traverse the pen and/or by conventional means including plowing, tilling, raking, or shoveling, for example. In this manner, different portions of the encapsulated compositions are brought to the surface of the animal pen or feedlot where they may be exposed to hydration and subsequent activation.
  • the compositions may be applied directly to the animals themselves.
  • the compositions of the present technology may be sprayed or dusted directly onto the animals.
  • the compositions may be applied as a spray or dusting as the animals move through a gangway or are otherwise introduced into the feedlot or pen.
  • the encapsulated compositions are mixed with the pen material over a course of time, e.g., over the course of several wet/dry cycles.
  • Figure 2 provides an example of the release and activation profiles for the encapsulated microorganisms of the present technology.
  • a portion of the capsules of the present technology will degrade in response to hydration and release activated microorganisms.
  • the activity of the microorganisms 206 will decrease as the source of hydration (e.g., rainfall) is removed.
  • microorganisms During the next wet period 202, another portion of microorganisms will be released and activated. As the wet/dry cycles continue, the cumulative amount of microorganisms released from the encapsulant in the pen material 208 increases, and as illustrated by Figure 2, the enzymes are released at a time when they are needed to prevent and treat dag formation (e.g., during the wet period).
  • LB broth lysogeny broth
  • the cells are then centrifuged at 10,000g for 30 minutes to remove the media, and then are re-suspended in a cryopreservation media containing 1% Dimethyl sulfoxide (DMSO) and 1% glycerol in Phosphate Buffered Saline (PBS).
  • DMSO Dimethyl sulfoxide
  • PBS Phosphate Buffered Saline
  • This mixture is then frozen to -100 °C, and placed under vacuum in a lyophilizer for 8 hours.
  • the freeze dried material is then combined with a homogenized solution of clay, modified starch, and cellulose acetate to form an emulsion and then injected into a spray drier with an inlet temperature of 120 °C and an outlet temperature of 65 °C.
  • This material can be stored at room temperature for extended periods of time before use.
  • the encapsulated microorganisms are applied onto a feedlot pen floor and turned into the bedding material. This involves covering a majority of the area of the feedlot pen with the preparation from the 100L initial culture. The material is then tilled into the pen bedding typically with a tiller attached to a tractor. The animals are introduced to the pen as normal. The encapsulated material is then re-applied prior to the next wet season or when the feedlot pen material is scraped.

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Abstract

La présente invention concerne de nouvelles compositions et de nouveaux procédés pour traiter ou prévenir la formation de mèches de laine coagulées (daggins). Les compositions et procédés de la présente invention concernent des microorganismes microencapsulés biodégradables qui restent dormants jusqu'à leur activation par hydratation pour produire des enzymes qui dégradent des éléments du matériau d'enclos d'animaux qui sinon adhèrent aux poils de la peau ou du cuir des animaux, empêchant ainsi la formation de daggins ou affaiblissant la structure du matériau d'enclos pouvant adhérer aux poils de peau ou de cuir d'animaux. L'enrobage des microcapsules se dégrade et libère des micro-organismes activés lorsqu'il est hydraté, sinon les micro-organismes encapsulés restent dormants. La condition de libération spécifique de micro-organismes activés fournit la capacité de délivrer l'activité enzymatique des microorganismes au matériau d'enclos et/ou aux poils de peau ou cuir d'animaux au moment approprié, par exemple pendant une saison humide.
PCT/US2016/061143 2015-11-18 2016-11-09 Méthodes et compositions destinées au traitement et à la prévention de la formation de daggins WO2017087234A1 (fr)

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CN108004174A (zh) * 2017-12-25 2018-05-08 江苏世邦生物工程科技有限公司 治理土壤污染的复合微生物菌剂及其制备方法和应用
CN109355218A (zh) * 2018-10-18 2019-02-19 东北农业大学 用于畜禽粪便堆肥快速升温除味的多功能发酵复合菌剂及其制备方法与应用
WO2019035849A1 (fr) * 2017-08-18 2019-02-21 Invention Development Management Company Méthodes et compositions pour atténuer la formation de daggins
WO2019068133A1 (fr) * 2017-10-06 2019-04-11 Meat & Livestock Australia Ltd Élimination de dépôts organiques
CN109769807A (zh) * 2018-11-14 2019-05-21 浙江农林大学 一种具有双分子结构的缓释灭藻微胶囊及其制备方法
CN110734885A (zh) * 2019-12-06 2020-01-31 安徽瑞驰兰德生物科技有限公司 一种用于发酵海杂鱼的复合微生物菌剂及酶解鱼蛋白氨基酸水溶肥的制备方法
WO2020089872A1 (fr) * 2018-11-03 2020-05-07 Gopalkrishna Shetty Mahesh Composition et procédé pour augmenter les capacités d'une population d'agents biologiques et traitement de déchets associé
KR102255930B1 (ko) * 2019-12-03 2021-05-26 국립낙동강생물자원관 담수에서 분리한 리그닌 분해 활성을 가지는 플레비옵시스 크라사 nnibrfg4544 균주 및 이의 용도
CN114938753A (zh) * 2022-06-07 2022-08-26 广西红树林研究中心 一种防治红树林浒苔危害的方法

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WO2019035849A1 (fr) * 2017-08-18 2019-02-21 Invention Development Management Company Méthodes et compositions pour atténuer la formation de daggins
WO2019068133A1 (fr) * 2017-10-06 2019-04-11 Meat & Livestock Australia Ltd Élimination de dépôts organiques
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CN108004174A (zh) * 2017-12-25 2018-05-08 江苏世邦生物工程科技有限公司 治理土壤污染的复合微生物菌剂及其制备方法和应用
CN109355218A (zh) * 2018-10-18 2019-02-19 东北农业大学 用于畜禽粪便堆肥快速升温除味的多功能发酵复合菌剂及其制备方法与应用
WO2020089872A1 (fr) * 2018-11-03 2020-05-07 Gopalkrishna Shetty Mahesh Composition et procédé pour augmenter les capacités d'une population d'agents biologiques et traitement de déchets associé
CN109769807A (zh) * 2018-11-14 2019-05-21 浙江农林大学 一种具有双分子结构的缓释灭藻微胶囊及其制备方法
KR102255930B1 (ko) * 2019-12-03 2021-05-26 국립낙동강생물자원관 담수에서 분리한 리그닌 분해 활성을 가지는 플레비옵시스 크라사 nnibrfg4544 균주 및 이의 용도
CN110734885A (zh) * 2019-12-06 2020-01-31 安徽瑞驰兰德生物科技有限公司 一种用于发酵海杂鱼的复合微生物菌剂及酶解鱼蛋白氨基酸水溶肥的制备方法
CN110734885B (zh) * 2019-12-06 2021-07-06 安徽瑞驰兰德生物科技有限公司 一种用于发酵海杂鱼的复合微生物菌剂及酶解鱼蛋白氨基酸水溶肥的制备方法
CN114938753A (zh) * 2022-06-07 2022-08-26 广西红树林研究中心 一种防治红树林浒苔危害的方法
CN114938753B (zh) * 2022-06-07 2023-06-30 广西红树林研究中心 一种防治红树林浒苔危害的方法

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