WO2010138522A2 - Stable dry powder composition comprising biologically active microorganisms and/or bioactive materials and methods of making - Google Patents
Stable dry powder composition comprising biologically active microorganisms and/or bioactive materials and methods of making Download PDFInfo
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- WO2010138522A2 WO2010138522A2 PCT/US2010/036098 US2010036098W WO2010138522A2 WO 2010138522 A2 WO2010138522 A2 WO 2010138522A2 US 2010036098 W US2010036098 W US 2010036098W WO 2010138522 A2 WO2010138522 A2 WO 2010138522A2
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/174—Vitamins
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/189—Enzymes
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K40/00—Shaping or working-up of animal feeding-stuffs
- A23K40/30—Shaping or working-up of animal feeding-stuffs by encapsulating; by coating
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/40—Feeding-stuffs specially adapted for particular animals for carnivorous animals, e.g. cats or dogs
- A23K50/42—Dry feed
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/06—Enzymes
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/15—Vitamins
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/40—Complete food formulations for specific consumer groups or specific purposes, e.g. infant formula
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
- A61K47/38—Cellulose; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
- A61K9/143—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with inorganic compounds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
- A61K9/146—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic macromolecular compounds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
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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
- 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
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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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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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
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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
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- 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
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
- Y02A40/818—Alternative feeds for fish, e.g. in aquacultures
Definitions
- the present invention is in the field of protection of bioactive microorganism and/or materials in high temperature and humid conditions.
- the invention relates to embedding live microorganisms and/or bioactive materials in a protective dry formulation matrix.
- Bioactive microorganisms such as live or dead bacteria and viruses, or bioactive materials, such as proteins, vitamins, minerals, hormones and cells are generally unstable when stored under conditions of high temperature and humidity.
- many commercially available probiotic bacteria such as lactobacillus rhamnosus can loose more than one log of viability in less than two weeks when stored in ambient atmosphere at room temperature (approximately 25°C).
- a common process to dry and protect these bioactive microorganisms after harvesting from a culture vessel (e.g., fermentor) is to drop a concentrated solution of the living cells into liquid nitrogen then store the frozen beads in -80 0 C refrigeration for later freeze drying or shipment to other locations. Freeze-drying has been a dominant method for drying sensitive bioactive material.
- the bioactive microorganism or materials is commonly mixed in a solution or suspension of protective agents, frozen, and then dehydrated by sublimation under full vacuum.
- the low temperatures of the freeze-drying process decrease the degradation reactions of the bioactive and minimize the loss of activity in the final dry form.
- the requirement for sub-zero temperatures is energy intensive, and the low surface area to volume ratios of the frozen material necessitates the use of long drying time (up to several days per batch cycle).
- the slow drying of the freeze-drying process also facilitates the formation of ice crystals that can damage or denature a sensitive bioactive.
- bioactive microorganism or materials such as viruses, bacteria, and cells that possess a cell wall or lipid membrane, pose significant challenges to the freeze-drying process.
- cryoprotective agents are highly soluble chemicals that are added to a formulation to protect cell membranes and intracellular proteins during freezing and to enhance stability during storage.
- Common stabilizers for live bacteria and viruses include sugars such as sucrose, glycerol, or sorbitol, at high concentrations with the cellular material or bioactive (Morgan et al., 2006; Capela et al., 2006).
- sugars such as sucrose, glycerol, or sorbitol
- Such protective agents may not penetrate adequately into the cell to protect active components within the intracellular volume. Therefore, a significant challenge remains to develop an optimal drying process and formulation that minimizes drying losses while achieving adequate storage stability of the dried material.
- the drying of the protective solution also involved a stepwise process of concentration under a moderate vacuum before application of a strong vacuum to cause frothy boiling of the remaining water to form dry stable foam.
- Busson and Schroeder US. Pat. No. 6,534,087 have proposed a drying process in a liquid state formulation for insensitive bioactives using a vacuum oven without boiling, by applying very mild vacuum pressure above 30 Torr. After achieving a certain level of drying without boiling the material, heat was applied at above 20 0 C and dried material was harvested after only a few hours.
- This type of drying process in which the bioactive solution is maintained in a liquid state during the entire drying process, has the advantage of faster drying due to convection of the liquid during boiling and the increased surface area presented by the foaming surfaces.
- boiling and foaming require input of a significant amount of heat to provide the necessary eruption of the solution.
- Such a drying process is not well adapted to drying of sensitive biologicals, such as viable viruses, cells or bacteria because the applied heat accelerates enzymatic processes (e.g., proteolysis), and chemical processes (e.g., oxidation and free radical attacks), which can destroy the activity or viability of the biological material.
- the drying process described above is also limited in its ability to be scaled to a large industrial process.
- the avoidance of freezing requires the process to be conducted at lower vacuum level (>7 Torr) than in conventional freeze drying or spray freeze drying process cycles.
- the most significant disadvantage of the above processes is the inability to control and limit the expansion of the foam within the vessel, tray or vial.
- the uncontrollable eruption and often-excessive foam formation makes it practically impossible to develop an industrial scale process.
- the eruption and foaming nature of the boiling step results in a portion of material being splattered on the walls of the vessel and into the drying chamber.
- Bronshtein U.S.
- Patent Nos.: 6,884,866, 6,306,345) has proposed special chambers and a controlled temperature/pressure application protocol that reduces overheating to an acceptable level.
- Another approach to contain the eruption and excessive foaming is described in US. Pat. App. No.: 2008/0229609, in which the bioactive solution is enclosed in a container or a bag covered with breathable membranes.
- bioactive microorganisms such as live or dead viruses, bacteria and cells
- Protective formulations and mild drying processes are required to provide adequate drying without exposure to high temperatures.
- a composition is needed that can protect such biologicals in storage under high temperature and humid conditions.
- the present invention as described below, provides a solution to all of these challenges.
- the present invention includes compositions and methods for preserving bioactive materials, such as peptides, proteins, hormones, vitamins, minerals, drugs, microbiocides, fungicides, herbicides, insecticides, spermicides, nucleic acids, antibodies, vaccines, and/or bioactive microorganism such as bacteria (probiotic or otherwise), viruses and/or cell suspensions, in storage.
- bioactive materials such as peptides, proteins, hormones, vitamins, minerals, drugs, microbiocides, fungicides, herbicides, insecticides, spermicides, nucleic acids, antibodies, vaccines, and/or bioactive microorganism such as bacteria (probiotic or otherwise), viruses and/or cell suspensions, in storage.
- the drying methods provide a process of controllable expansion of a formulation comprising the bioactive microorganism or material, a formulation stabilizer agent, and a protective agent.
- the formulation is prepared by dispersing all the solid components in a solution, with or without a vacuum.
- the solution is cooled to a temperature above its freezing temperature and dried under vacuum into a dry composition, which exhibits an unexpectedly high stability.
- the methods include a primary drying step of the formulation at a desired temperature and time period, and an accelerated secondary drying step under maximum vacuum and elevated temperature, to achieve a final desirable water activity of the dry material.
- the formulation comprises sufficient amounts of formulation stabilizer agents, in which the microorganisms are embedded.
- suitable formulation stabilizer agent include, but are not limited to, cellulose acetate phthalate (CAP), carboxy-methyl-cellulose, pectin, sodium alginate, salts of alginic acid, hydroxyl propyl methyl cellulose (HPMC), methyl cellulose, carrageenan, guar gum, gum acacia, xanthan gum, locust bean gum, chitosan and chitosan derivatives, collagen, polyglycolic acid, starches and modified starches, cyclodextrins and oligosaccharides (inulin, maltodextrins, dextrans, etc.); and combinations thereof.
- CAP cellulose acetate phthalate
- HPMC hydroxyl propyl methyl cellulose
- HPMC hydroxyl propyl methyl cellulose
- carrageenan guar gum
- gum acacia xanthan gum
- the preferred formulation stabilizer agent is sodium alginate.
- the formulation comprises, in percent by weight of total dry matter, 0.1-10%, preferably 1-6%, more preferably 2-4% of formulation stabilizer agent.
- the formulation stabilizer comprises a mixture of sodium alginate and oligosaccharides in a weight ratio of 1 :1-10, more preferably 1 :1-5 of sodium alginate/oligosaccharides.
- the formulation stabilizer is cross-linked with divalent metals ions to form a firm hydro gel.
- the formulation comprises significant amounts of protecting agents, in which the microorganisms are embedded.
- suitable protecting agent include but not limited to proteins such as human and bovine serum albumin, egg albumen, gelatin, immunoglobulin, isolated soya protein, wheat protein, skim milk powder, caseinate, whey protein and any protein hydrolysates; carbohydrates including monosaccharides (e.g., galactose, D-mannose, sorbose, etc.), disaccharides (e.g., lactose, trehalose, sucrose, etc.), an amino acid such as lysine, monosodium glutamate, glycine, alanine, arginine or histidine, as well as hydrophobic amino acids (tryptophan, tyrosine, leucine, phenylalanine, etc.); a methylamine such as betaine; an excipient salt such as magnesium sulfate; a polyol such as trihydr
- glycerin erythritol, glycerol, arabitol, xylitol, sorbitol, and mannitol
- propylene glycol polyethylene glycol
- pluronics surfactants; and combinations thereof.
- the protecting agent comprises a mixture of a disaccharide, a protein, and a protein hydrolysate.
- the preferred protecting agent is a mixture of trehalose, soy protein isolate or whey protein and their hydro lysates.
- the formulation comprises, in percent by weight of total dry matter, 10-90%, of trehalose, 0.1-30% soy protein isolate or whey proteins and 0.1-30% soy or whey protein hydrolysate.
- trehalose Preferably 20-80% of trehalose, 0.1-20% soy protein isolate or whey proteins and 1-20% soy or whey protein hydrolysate, more preferably 40-80% of trehalose, 0.1-20% soy protein isolate or whey proteins and 1-20% soy or whey protein hydrolysate.
- the method of the invention typically includes blending with or without a vacuum, concentrated solution or dry powder of bioactive microorganism (e.g., live or dead vaccines, bacteria, algae, viruses and/or cell suspensions) or a bioactive material (e.g., peptides, proteins, hormones, vitamins, minerals, drugs, microbiocides, fungicides, herbicides, insecticides, spermicides, nucleic acids, antibodies, vaccines), a stabilizer agent, and a protective agent into a homogeneous formulation, cooling the formulation to a temperature above its freezing temperature, and drying under vacuum at a shelf temperature above 20 0 C.
- bioactive microorganism e.g., live or dead vaccines, bacteria, algae, viruses and/or cell suspensions
- a bioactive material e.g., peptides, proteins, hormones, vitamins, minerals, drugs, microbiocides, fungicides, herbicides, insecticides, spermicides, nucleic acids, antibodies, vaccines
- the drying process can involve a primary vacuum drying at a shelf temperature of 20 0 C or above, followed by an accelerating secondary drying of the formulation under maximum vacuum and elevated temperature for a time sufficient to reduce the water activity of the dried formulation to 0.3 Aw or less.
- the bioactive microorganism or material is in a dry stabilized form and is further dry blended with the dry stabilizer agents and protective agents. This dry blend is then added to water and mixed under the appropriate vacuum and agitation to give a homogeneous slurry of the desired density.
- the bioactive microorganism or material is in the form of a concentrated solution or paste. The solution is mixed with all the other formulation ingredients before adding to water.
- the bioactive microorganism or material is in the form of dry powder.
- the dry powder is mixed with all the other formulation ingredients before adding to water.
- the dry bioactive microorganism or material is mixed with just a portion of the formulation ingredients, and this mixture is added to the pre-formed slurry, prepared from the addition of the other formulation ingredients to water.
- the bioactive microorganism is mixed under vacuum in a solution including a formulation stabilizer agent and a protective agent.
- the bioactive microorganism comprises live bacteria (e.g., probiotic bacteria).
- suitable microorganisms include, but are not limited to, yeasts such as Saccharomyces, Debaromyces, Candida, Pichia and Torulopsis, moulds such as Aspergillus, Rhizopus, Mucor, Penicillium and Torulopsis and bacteria such as the genera Bifidobacterium, Clostridium, Fusobacterium, Melissococcus, Propionibacterium, Streptococcus, Enterococcus, Lactococcus, Kocuriaw, Staphylococcus, Peptostrepococcus, Bacillus, Pediococcus, Micrococcus, Leuconostoc, Weiss ella, Aerococcus, Oenococcus and Lactobacillus.
- yeasts such as Saccharomyces, Debaromyces, Candida, Pichia and Torulopsis
- moulds such as Aspergillus, Rhizopus, Mucor, Penicillium and Torulopsis
- bacteria such as the
- probiotic microorganisms would be represented by the following species and include all culture biotypes within those species: Aspergillus niger, A. oryzae, Bacillus coagulans, B. lentus, B. licheniformis, B. mesentericus, B. pumilus, B. subtilis, B. natto, Bacteroides amylophilus, Bac. capillosus, Bac. ruminocola, Bac. suis, Bifidobacterium adolescentis, B. animalis, B. breve, B. bifidum, B. infantis, B. lactis, B. longum, B. pseudolongum, B.
- thermophilum Candida pintolepesii, Clostridium butyricum, Enterococcus cremoris, E. diacety lactis, E faecium, E. intermedius, E. lactis, E. muntdi, E. thermophilus, Escherichia coli, Kluyveromyces fragilis, Lactobacillus acidophilus, L. alimentarius, L. amylovorus, L. crispatus, L. brevis, L. case 4 L. curvatus, L. cellobiosus, L. delbrueckii ss. bulgaricus, L farciminis, L. fermentum, L. gasseri, L. helveticus, L.
- lactis L. plantarum, L. johnsonii, L. reuteri, L. rhamnosus, L. sakei, L. salivarius, Leuconostoc mesenteroides, P. cereviseae ( damnosus ), Pediococcus acidilactici, P. pentosaceus, Propionibacterium freudenreichii, Prop, shertnanii, Saccharomyces cereviseae, Staphylococcus carnosus, Staph, xylosus, Streptococcus infantarius, Strep, salivarius ss. thermophilus, Strep. Thermophilus and Strep, lactis.
- the formulation is mixed under vacuum at room temperature (e.g., from 20 0 C to 30 0 C). After mixing to homogeneity, the formulation is then cooled to a temperature above the freezing temperature of the formulation. Typically, the formulation is cooled to between -10 0 C to +10 0 C, more preferably the formulation is cooled to between -5°C and +5°C. In a preferred embodiment, the cooled formulation is then transferred to a drying chamber where heating is applied (20 0 C or more) while controlling an initial vacuum pressure at a level to maintain the original pre-cooling temperature. Typically, the desirable vacuum pressure is below 7 Torr but no less than 3 Torr.
- the secondary drying comprises removal of water at a pressure of less than 1 Torr, and in an especially preferred embodiment to less than 0.2 Torr.
- the wet formulation can be in the form of viscous slurry or hydrogel particles ranging from 0.05 to 10 mm.
- the dried formulation can be used directly as a flake, or ground into a powder with an average particle size from about 10 ⁇ m to about 1000 ⁇ m.
- the formulation can be administrated directly to an animal, including human, as a concentrated powder, as a reconstituted liquid, (e.g., beverage), or it can be incorporated either in flake or powder form into an existing food or feed product.
- Figure 1 shows the stability trend of the probiotic bacteria, L. rhamnosus, which was subjected to storage at 40 0 C and 33% relative humidity.
- Figure 2 shows the process temperatures and cumulative viability loss for a formulation process ending with Aw of 0.28 secondary drying step.
- Figure 3 shows the effect of different formulation stabilizers on storage stability.
- Figure 4 shows the effect of alginate viscosity on the formulation expansion under vacuum.
- Figure 5 shows the effect of different combinations of stabilizer agents on bacteria viability.
- Figure 6 shows the effect of the formulation density on expansion rate under vacuum.
- Figure 7 shows the effect of the formulation pre-cooling temperature on expansion under vacuum.
- Figure 8 shows the effect of the vacuum pressure on formulation temperature during primary drying step.
- Figure 9 shows the effect of the vacuum pressure on drying rate of the formulation.
- Figure 10 shows the stability of the probiotic bacteria, L. acidophilus dried with the formulation and method of the invention under storage at 37°C and 33% relative humidity.
- Figure 11 shows a flow chart of the method of production stable dry formulation from hydrogel formulation according to the invention.
- Ambient room temperatures or conditions are those at any given time in a given environment. Typically, ambient room temperature is 22-25°C, ambient atmospheric pressure, and ambient humidity are readily measured and will vary depending on the time of year, weather and climactic conditions, altitude, etc.
- Degassing refers to the release of a gas from solution in a liquid when the partial pressure of the gas is greater than the applied pressure. This is not boiling, and can often occur at pressures above a pressure that would boil a solution.
- bottled carbonated soft drinks contained a high partial pressure of CO 2 . Removing the bottle cap reduces the partial pressure and the drink bubbles vigorously (it degasses, but does not boil).
- Boiling refers to the rapid phase transition from liquid to gas that takes place when the temperature of a liquid is above its boiling temperature.
- the boiling temperature is the temperature at which the vapor pressure of a liquid is equal to the applied pressure. Boiling can be particularly vigorous when heat is added to a liquid that is already at its boiling point.
- Relative Humidity or “RH” in the context of storage stability refers to the amount of water vapor in the air at a given temperature. Relative humidity is usually less than that required to saturate the air and expressed in percent of saturation humidity.
- Primary drying refers to the drying that takes place from the time of initial vacuum application to the point where secondary drying starts. Typically, the bulk of primary drying takes place by extensive evaporation, while the product temperature remained significantly lower than the temperatures of the heat source.
- Secondary drying refers to a drying step that takes place at temperatures above freezing temperatures of the formulation and near the temperature of the heat source.
- a secondary drying step reduces the water activity of the formulation to an Aw of 0.3 or less.
- Bioactive microorganism or “biologically active microorganism or formulation” refers to live or dead microorganism preparations, which are in such a form as to permit the biological activity of the microorganism to be unequivocally effective.
- Live microorganism as dry powder refers to a bacterial biomass in which at least 10% W/W is live bacteria.
- Delivery microorganism as dry powder refers to a bacterial biomass in which at least 99.999% is dead bacteria.
- Bioactive material refers to preparations, which are in such a form as to permit the biological activity of the bioactive ingredients to be unequivocally effective.
- bioactive materials include but not limited to peptides, proteins, hormones, vitamins, minerals, drugs, microbiocides, fungicides, herbicides, insecticides, spermicides, nucleic acids, antibodies, and vaccines.
- Stabilizer or Stabilizing agent refers to compounds or materials that are added to the formulation to increase the viscosity of the wet formulation or to form a hydrogel.
- suitable stabilizer agent include but are not limited to polysaccharides, such as, cellulose acetate phthalate (CAP), carboxy-methyl- cellulose, pectin, sodium alginate, salts of alginic acid, hydroxyl propyl methyl cellulose (HPMC), methyl cellulose, carrageenan, guar gum, gum acacia, xanthan gum, locust bean gum, chitosan and chitosan derivatives, collagen, polyglycolic acid, starches and modified starches, cyclodextrins and oligosaccharides (inulin, maltodextrins, raffinose, dextrans, etc.) and combinations thereof.
- CAP cellulose acetate phthalate
- HPMC hydroxyl propyl methyl cellulose
- Protecting agent or “protective agent” or “protectant” generally refers to compounds or materials that are added to ensure or increase the stability of the bioactive material during the drying process and afterwards, or for long-term storage stability of the dry powder product. Suitable protectants are generally readily soluble in a solution and do not thicken or polymerize upon contact with water.
- Suitable protectants include, but are not limited to, proteins such as human and bovine serum albumin, whey protein, soy protein, caseinate, gelatin, immunoglobulins, carbohydrates including monosaccharides (galactose, D-mannose, sorbose, etc.), disaccharides (lactose, trehalose, sucrose, etc.), an amino acid such as monosodium glutamate, lysine, glycine, alanine, arginine or histidine, as well as hydrophobic amino acids (tryptophan, tyrosine, leucine, phenylalanine, etc.); a methylamine such as betaine; an excipient salt such as magnesium sulfate; a polyol such as trihydric or higher sugar alcohols (e.g., glycerin, erythritol, glycerol, arabitol, xylitol, sorbitol, and manni
- proteins
- a “stable" formulation or composition is one in which the bioactive microorganism or material therein essentially retains its viability, and/or biological activity upon storage. Stability can be measured at a selected temperature and humidity conditions for a selected time period. Trend analysis can be used to estimate an expected shelf life before a material has actually been in storage for that time period. For live bacteria, for example, stability is defined as the time it takes to loose 1 log of CFU/g dry formulation under predefined conditions of temperature, humidity and time period.
- Viability with regard to bacteria, refers to the ability to form a colony (CFU or Colony Forming Unit) on a nutrient media appropriate for the growth of the bacteria. Viability, with regard to viruses, refers to the ability to infect and reproduce in a suitable host cell, resulting in the formation of a plaque on a lawn of host cells.
- compositions and methods of the present invention solves the problem of providing a cost effective and industrially scalable drying processes for producing a dry formulation containing bioactive microorganisms or materials, such as live or dead vaccines, bacteria, algae viruses and/or cell suspensions, peptides, proteins, hormones, vitamins, minerals, drugs, microbiocides, fungicides, herbicides, insecticides, spermicides, nucleic acids, antibodies, vaccines with a significantly extended lifetime in the dry state.
- bioactive microorganisms or materials such as live or dead vaccines, bacteria, algae viruses and/or cell suspensions, peptides, proteins, hormones, vitamins, minerals, drugs, microbiocides, fungicides, herbicides, insecticides, spermicides, nucleic acids, antibodies, vaccines with a significantly extended lifetime in the dry state.
- the invention provides a formulation comprising a bioactive microorganism or material with a stabilizer agent and a protecting agent in a solution, cooling said formulation to a temperature above its freezing temperature, and stabilizing the formulation by removing the moisture under a regimen of reduced pressure while supplying heat to the composition.
- the present invention provides a formulation and an industrially scalable drying process that minimizes losses during the drying and protects the bioactive microorganism under harsh storage conditions thereafter.
- the present invention includes formulation compositions of a bioactive microorganism or material, a stabilizer agent and a protecting agent in a viscous solution.
- the formulations of the invention were found to be inherently different in their physical structure and function from non-viscous or concentrated formulations that were dried without pre-cooling.
- formulations of the prior art were initially "foamed" to facilitate effective drying.
- the foaming step generally resulted in an extensive boiling and eruption of the solution that is an unavoidable consequence of the vacuum drying in a liquid state and as a result, only a very low loading capacity of material in a vial or a vessel can be achieved (see for example US. Pat. No.
- compositions and drying methods of the present invention allow only a limited and controlled expansion of the formulation thereby enabling much higher loading of material per drying area and, as a result, can be easily scaled up to the production of large quantities of material.
- the bioactive microorganism of the invention is probiotic bacteria.
- the formulation is prepared according to the compositions and methods of the invention including obtaining live probiotic bacteria in concentrated solution, paste, frozen beads or dry powder from. Mixing the probiotic bacteria under vacuum with a stabilizer agent and a protecting agent, cooling the viscous formulation to a temperature above its freezing temperature, applying sufficient vacuum pressure to maintain that pre- cooling temperature and supplying a heat source of 20 0 C and above to facilitate water removal. Maintaining the pre-cooled temperature of the formulation can be by conduction of heat away from the formulation, and/or by loss of latent heat due to water evaporation.
- a secondary drying step is applied, at higher vacuum up to 0.1 Torr and at elevated temperature up to 70 0 C, to provide a final composition with water activity with an Aw of 0.3 or less.
- Such a composition can remain stable in storage conditions of 40 0 C and 33%RH for 60 days or more (see Figure 1).
- the specified processes of the invention have shown to result in the unexpected ability of the cells to retain their viability beyond that of established drying processes.
- the initial viability loss through the entire drying process according to the present invention was only 0.3 logs (see Figure 2).
- compositions to be mixed with the preferred microorganism or material for the preparation of dry powder compositions according to the invention includes a stabilizer agent and protective agent.
- Such constituents, when mixed with the preferred bioactive microorganisms or material, can be processed according to methods of the invention to provide large quantities of stable dry compositions for storage and administration of said microorganisms.
- the formulation stabilizers can include a mixture of a polysaccharide and an oligosaccharide.
- alginate is superior to other polysaccharides such as pectin and gum acacia in reducing the drying losses of sensitive biologicals such as probiotics (Figure 3). It was also preferred because of its hydrogel forming characteristics with non-toxic metals at mild temperatures. Alginate was also found to effectively stabilize the formulation under vacuum, by providing appropriate viscosity to the formulation and allowing a controlled expansion of the formulation at a particular viscosity (Figure 4).
- At least one of the formulation stabilizer agents is preferably a gum that can form a firm hydrogel by cross-linking with metal ions.
- Protective agents of the invention can include various proteins, peptides, sugars, sugar alcohols and amino acids.
- the protective agent is preferably one that does not crystallize and/or destabilize the biologically active material in the formulation at freezing temperatures (e.g., -20 0 C). It can be beneficial to include two or more different protective agents to inhibit the formation of crystals and stabilize the dried bioactive material formulation in storage conditions for long time periods.
- the wet formulations can include a substantial amount of total solids (constituents minus the solvent, such as water).
- a major portion of the total solids can consist of the bioactive material, the stabilizer agent and the protective agent.
- the bioactive material can be present in the formulation in a concentration ranging from about 2-50 weight percent, the stabilizer agent from about 1-20 weight percent, and the protective agent from about 20-80 weight percent.
- the stabilizer agent can be present in the formulation in a concentration ranging from about 0.5-10 weight percent, and the protective agent from about 10-40 weight percent.
- the wet formulation should have solids content between about 5% and 80%; more preferably between about 30% to 60%.
- the viscosity of formulations of the invention are typically greater than 1000 centipoises (cP); more preferably, greater than 10,000 cP and less than 450,000; and most preferably greater than 30,000 cP and less than 100,000 cP.
- the viscosity of formulations of the invention can be as high as 450,000 cP, provided that the protective agents are completely dissolved in the solution.
- Highly viscous and homogenous slurries containing substantial amount of total solids can be achieved at elevated temperature, depending on the thermo and osmo-sensitivity of the bioactive material.
- live cells formulations containing 30-60% of total solids can be mixed at elevated temperature of about 35-40°C and the mixing is carried out until all the protective agents are completely dissolved.
- Methods for preparing stable dry formulations for the preservation of bioactive microorganisms include, obtaining a live culture of a specific microorganism in a concentrated solution, paste, frozen beads or dry powder from (stabilized or otherwise). Preparation of a formulation by mixing, under vacuum, the bioactive microorganism or material with a stabilizer agent and a protecting agent in a solution, cooling the formulation to a temperature of no more than 10 0 C above its freezing temperature, and drying the formulation by evaporating the moisture under reduced pressure while supplying heat to the formulation.
- a formulation comprising a bioactive microorganism or material, a formulation stabilizer agent, and a protecting agent are mixed to homogeneity, under mild vacuum of about 10-50 Torr, in a solution.
- Figure 6 shows the effect of different densities of the formulation on its expansion under vacuum.
- the introduction of air during mixing of the formulation constituents in a solution results in excessive and un-controllable foaming even at relatively high vacuum pressure.
- the mixing under vacuum step according to the invention addresses this problem by eliminating the introduction of air or gas into the formulation solution, thereby eliminating excessive and uncontrolled foaming of the solution.
- FIG. 7 shows the effect of pre-cooling of the formulation solution on its expansion under vacuum pressure. It was surprisingly and unexpectedly found that boiling can be effectively eliminated even under a relatively higher vacuum pressure and formulation expansion is better controlled when the solution temperature is reduced to no more than 10 0 C above its freezing temperature. As can be seen from Figure 7, a vacuum pressure of 3 Torr can be applied without excessive foaming provided that the formulation is cooled to +5°C and preferably to - 3°C.
- Figure 8 shows the effect of the applied vacuum pressure on the temperature of the formulation solution.
- the formulation temperature increased to over 6°C and will continued to rapidly increase toward the shelf or chamber temperature.
- the solution will continue foaming and further expanding.
- This embodiment is distinguished from the prior art discussed above (see for example US. Pat. No. 6,534,087, where the applied vacuum pressure is between 3-7 Torr and even higher), in which a stronger vacuum pressure is applied ( ⁇ 3 Torr) while controlling the expansion of the formulation.
- Another embodiment of the invention provides methods to prepare hydrogel formulation compositions for preservation of bioactive microorganisms or materials.
- a formulation containing a probiotic bacteria in a dry powder form, a stabilizer agent and a protective agent are mixed in a solution, cross-linked to a hydrogel by adding metal ions or divalent cations and then dried under low vacuum and temperature as described above.
- the pre-cooled temperature of the formulation can be maintained by conduction of heat away from the formulation, and/or by loss of latent heat due to water evaporation.
- the formulation includes a concentrated fresh or frozen or dry culture of live probiotic bacteria in a solution of 1 to 2.5% sodium alginate (preferably 1.5% sodium alginate), 1% to about 5% inulin (preferably 2.5% inulin), 20% to 60% trehalose (preferably40% trehalose) and 3% to 15% casein hydrolysate (preferably 8% casein hydrolysate).
- the formulation is mixed under vacuum at a temperature slightly above the room temperature (typically between 25°C -37°C) until all the components are completely dissolved.
- all the ingredients are dissolved in the solution at elevated temperature, then the slurry is cooled down to a temperature between 0 0 C to -5°C and a dry powder of live microorganism is mixed in until all the components are completely dissolved.
- a small amount of trehalose can be added to the dry powder (typically a mixture containing equal portions of dry powder and trehalose is sufficient.
- the formulation slurry is spread on trays at loading capacity of about 200 g/sq ft and trays are placed on shelves in a freeze drier.
- the shelf temperature is adjusted to 0 to -5 0 C (preferably -2°C) and the slurry allowed to cool to that temperature.
- Vacuum pressure is then applied at 1 to 5 Torr (preferably 3 Torr) and shelf temperature increased to 20° to 45 0 C (preferably 30 0 C) for conductive heat transfer.
- the formulation temperature remained at about the temperature 0 to -5 0 C during the primary evaporation step to prevent the sample from freezing.
- Secondary drying step at maximum vacuum of O.lTorr and shelf temperature of 40 0 C is started when product temperature reached about +10 0 C. The entire drying process proceeds for about 4 hours at which time the product is harvested and water activity is at Aw - 0.3 or less.
- the loaded trays are pre-cooled to - 2°C in a cold room then immediately loaded in a vacuum oven drier for radiant heat transfer.
- the primary and secondary drying steps are then applied as described above for conductive heat transfer.
- Formulations of the invention can include fresh, frozen or dry live microorganisms formulated into a solution or suspension containing a formulation stabilizer agent and a protective agent.
- the formulation stabilizer and/or high concentration of protective agent can be dissolved into a heated aqueous solution with agitation before cooling and mixing with the bioactive microorganisms.
- the microorganisms such as cultured virus or bacterium, can be concentrated and separated from culture media by centrifugation or filtration, then directly mixed into the formulation of the present invention, or added with conventional cryoprotectants dropped into liquid nitrogen and the small frozen beads stored at -80C until mixed into the formulation.
- the frozen beads can be freeze dried, milled into a fine powder, packed in air tight bags and stored refrigerated until mixed in the formulation of the invention.
- the totality of the water in the formulation is provided in the liquid of the concentrated live organism and the live organism suspension is maintained at a temperature slightly above room temperature.
- the dry components of the formulation stabilizer agent and the protective agent are blended together and then slowly added to the warm suspension of the live organism.
- the formulation suspension is gently agitated under mild vacuum in a planetary mixer until all components are fully dispersed and uniform slurry is obtained.
- the bioactive microorganism is in the dry powder form and is premixed dry with formulation ingredients before the resulting dry mixture is added to water at a temperature slightly above room temperature.
- the bioactive microorganism or material can provide any bioactivity, such as enzymatic activity, induction of immune responses, cellular multiplication, infection, inhibition of cell growth, stimulation of cell growth, therapeutic effects, pharmacologic effects, antimicrobial effects, and/or the like.
- the bioactive microorganism or material can be nonliving cells or liposomes useful as vaccines or delivery vehicles for therapeutic agents.
- Bioactive microorganism of the invention can be live viruses and live attenuated viruses and/or the like.
- Formulation stabilizers provide structural stability to the formulation and/or physical and chemical protective benefits to the bioactive microorganisms.
- the stabilizers can provide thickening viscosity to the formulation and better control over its expansion properties under vacuum pressure and increased structural strength to the dried formulation compositions of the invention.
- the protective agents can include a variety of proteins, protein hydrolysates, sugars, sugar alcohols and amino acids.
- sugars such as sucrose or trehalose can physically surround the bioactive material to promote retention of molecular structure throughout the drying process and impart structural rigidity to the amorphous matrix in the dry state.
- the protective agent can replace water of hydration lost during drying, to prevent damage to cell membranes and denaturation of enzymes.
- Other functions of the protective agents can include protecting the bioactive material from exposure to damaging light, oxygen, oxidative agents and moisture. Most protective agents can be readily dissolved in a solution in amounts ranging from about 0.1 weight percent to about 60 weight percent.
- Formulations of the invention can be pre-cooled before applying vacuum pressure of the drying process, to provide benefits such as a further thickening of the formulation slurry, a better control over the expansion of formulations under low vacuum pressure, stabilization of bioactive microorganism or material, and/or enhancing the penetration of formulation constituents through cell membranes.
- Cooling can be applied by any appropriate technique known in the art. For example, cooling can be by contact and conduction with cold surfaces, loss of latent heat, and/or the like.
- formulations are held in vessels or spread on metal trays and place in contact with a controlled temperature surface or a chamber where they equilibrate to the controlled temperature.
- the formulations of the invention can be pre-cooled to a temperature above its freezing temperature (e.g., between -5°C and +5°C).
- Typical processes for preservation of bioactive microorganisms such as, live or attenuated organisms include a combination of freezing and vacuum conditions that can result in membrane damage and denaturation of cell constituents.
- the prior art teaches the use of higher vacuum pressures (e.g., less than 100 Torr), addition of specific cryoprotective agents, concentrating steps to obtain thick solutions (syrup), and/or higher initial temperatures to prevent freezing.
- the use of formulations and process parameters of the present invention overcome these limitations and allow for higher loading capacity per drying area that significantly improves industrial output.
- the formulation in the present invention is dried by evaporation. Removal of solvent (moisture) from the gaseous environment around the formulation can be driven by condensation or desiccation. Evaporation of solvent from the formulation can provide accelerated primary drying of the formulation under low vacuum pressure. The controlled expansion of the formulation accelerates the primary drying of the formulation by rapid transfer of solvent out of the formulation. The controlled expansion of the formulation is achieved by gentle degassing (not boiling) of the remaining dissolved gases when the drying vacuum is applied. Since it is desirable not to boil or excessively foam the formulation because the cavitations and shear forces associated with bubble formation during boiling and/or the formulation may spill out from containment or have a negative impact on the bioactive microorganism.
- the formulation structure is stabilized.
- the heat supplied in the drying chamber compensates for the loss of latent heat caused by evaporation of solvent and the formulation temperature is maintained within 10 0 C above its freezing temperature.
- the rate of evaporation of solvent slows and the formulation temperature begins to increase due to superfluous supply of heat in the drying chamber. This point indicates the end of the primary drying step in this invention.
- the protective agents in solution become concentrated and thicker until it stops flowing as a liquid.
- the amorphous and/or glassy formulation preserves a stable formulation structure.
- Secondary drying of the structurally stable formulation removes the remaining entrapped or bound moisture and provides a composition that is stable in storage for an extended period of time at ambient temperatures. Secondary drying involves the application of elevated temperatures and a strong vacuum for several hours to days. In preferred embodiments the time period necessary to complete the secondary drying step is double the time of the primary drying step. Preferably, the water activity of the formulation at the end of the secondary drying step is less than an Aw of 0.3.
- the drying temperature can range from about room temperature to about 70 0 C.
- a typical secondary drying process for many bioactive microorganisms can include raising the temperature from about 30 0 C to about 40 0 C, and holding from about 30 minutes to about 24 hours (preferably from about 30 minutes to about 4 hours), to provide a stable dried formulation composition with water activity of less than an Aw of 0.3.
- the drying temperature is slowly raised from primary drying conditions at a rate that can further preserve the activity of live biologicals such as live microorganisms.
- a strong vacuum can be provided in the secondary drying process to accelerate the rate of water removal to lower residual moisture levels.
- the vacuum during the secondary drying can be less than 1 Torr and, preferably, less than about 0.2 Torr.
- the drying methods of the invention result in a biologically active microorganism or bioactive material that is encased within an amorphous glassy matrix, thereby preventing the unfolding of proteins and significantly slowing molecular interactions or cross-reactivity, due to greatly reduced mobility of the compound and other molecules within the amorphous glassy composition.
- the amorphous solid is at a temperature below its glass transition temperature and the residual moisture remains relatively low (i.e., below Aw of 0.3), the labile bioactive microorganism can remain relatively stable. It should be noted that achieving a glassy state is not a prerequisite for long term stability as some bioactive microorganisms or materials may fare better in a more crystalline state.
- the dried formulation can be used en bloc, cut into desired shapes and sizes, or crushed and milled into a free flowing powder that provides easy downstream processing like wet or dry agglomeration, granulation, tabletting, compaction, pelletization or any other kind of delivery process.
- Processes for crushing, milling, grinding or pulverizing are well known in the art. For example, a hammer mill, an impact mill, a jet mill, a pin mill, a Wiley mill, or similar milling device can be used.
- the preferred particle size is less than about 1000 ⁇ m and preferably less than 500 ⁇ m.
- compositions and methods described herein preserve the biological activity of the encased biologically active microorganism or bioactive materials.
- the compositions are tested for stability by subjecting them at elevated temperature (e.g., 40 0 C) and high humidity (e.g. 33%RH) and measuring the biological activity of the formulations.
- elevated temperature e.g. 40 0 C
- high humidity e.g. 33%RH
- results of these studies demonstrate that the bacteria formulated in these formulations are stable for at least 60 days (see Figure 1). Stability is defined as time for one log CFU/g potency loss.
- Such formulations are stable even when high concentrations of the biologically active material are used.
- these formulations are advantageous in that they may be shipped and stored at temperatures at or above room temperature for long periods of time.
- Lactobacillus Acidophilus 100 g frozen concentrate from a lab fermentation harvest
- Protein hydrolysate premix (10Og, Table 1) was slowly added to the thawed slurry of probiotic bacteria in a jacketed dual planetary mixer (DPM, lqt, Ross Engineering, Inc. Worcester, GA,). Mixing was carried out under mild vacuum (25 Torr) at 40 RPM and 37 0 C for 10 minutes.
- the homogenous slurry was evenly spread on a tray at a loading capacity of 200 g/sq ft and the tray placed on a shelf in a freeze drier (Model 25 SRC, Virtis, Gardiner, NY).
- Figure 10 shows the storage stability of the dry formulation under accelerated storage conditions of 37°C and 33%RH. After four weeks at these storage conditions, the viability loss of the probiotic bacteria stabilized in the formulation of the invention was only 0.8 logs.
- Concentrated probiotic slurry was prepared according to Example 2 but using the whey protein premix of Table 1. To this slurry, 0.5 g of dibasic calcium phosphate was added, followed by 0.5 g of gluconolactone. The slurry was allowed to harden at room temperature over the next 2 hours to form a solid hydrogel. The firm gel was sliced to thin and long threads, using a commercially available slicer/shredder. The thin threads were loaded on a tray at a loading capacity of 200g/sq ft and placed in a freeze drier for drying as described in Example 2. Four hours after establishing maximum vacuum of 0.1 Torr, the dried product was taken out of the freeze drier.
- FIG. 11 present a flow chart of the method of production stable dry formulation from a hydrogel formulation according to the invention.
- a commercially available pelleted pet food for dogs is dried in a convection oven to a water activity of 0.1, and then coated with the stable probiotic dry formulation prepared as described in Example 3.
- the dry pellets are sprayed with about 5% of fat-based moisture barrier (a mixture of 40% chicken fat, 40% cocoa butter and 20% beeswax), mixed in a drum tumbler with the dry powder formulation (usually 0.1-0.5% of total pet food that provides a dosage of 10. sup.8 CFU/g), and finally sprayed with additional coat of the fat-based moisture barrier.
- the total amount of coating is about 15% (of the pet food). Coating time is about 30 min.
- Pelleted feed for fish according to the present invention was prepared with a mixture of several probiotics.
- a stable dry probiotic formulation containing a mixture of L, rhamnosus, L, acidophilus and Bifidobacterium lactis was prepared as described in Example 2.
- a commercially available starter feed for salmon (Zeigler Bros., Gardners, PA) was first dried in a convection oven to a water activity of 0.1, and then coated with the probiotics formulation in a drum tumbler.
- the pellets (100 g) were first sprayed with about 5% by weight of fat-based moisture barrier (a mixture of 40% fish oil, 40% cocoa butter and 20% beeswax), then mixed with 1 g of the stable dry probiotic formulation (to attain a dosage of 10 7 cfu/g feed), and finally sprayed with additional coat of the fat-based moisture barrier.
- the total amount of coating was about 10% of the fish feed.
- Example 2 is prepared according to Example 2 followed by a sieving into two particle size groups (above 50 ⁇ m and below 150 ⁇ m).
- An infant formula is prepared by mixing 99 g of Nutramigen (Mead Johnson; Evansville, IL) with 0.1 g of the small size particles (below 50 ⁇ m). The final product contains about 10 cfu of Lactobacillus GG per 100 g infant formula.
- the tubes are sonicated for 10 min at 45°C, followed by a brief centrifugation at 5,000 rpm for 10 min. Aliquots of the clear DMSO/NaOH/SDS/Citrate solution are taken into wells of a microplate and analyzed for protein content using the Bradford assay method.
- the storage stability of the stable enzyme formulation is significantly higher than a dry enzyme without the formulation of the present invention.
- Pelleted bait for specifically targeted invasive species is prepared containing a pesticide.
- the whey protein premix of Table 1 is added to 200 gm of water.
- To this solution is added 90 gm of rotenone and 0.5 gm of calcium phosphate dibasic, followed by 0.5 gm of gluconolactone.
- the slurry is allowed to harden at room temperature over 2 hours.
- the firm gel is sliced to thin and long threads through a slicer/shredder.
- the thin threads are loaded on a tray and placed in a vacuum oven dryer. Drying is stopped after achieving a water activity of 0.10.
- the dry formulation is ground to the appropriate size distribution for the bait size specification for the specific species targeted.
- a protected soluble granular formulation of a pesticide that would otherwise be subject to decomposition by other ingredients in a formulation during storage is prepared by the process of the present invention.
- the soy protein premix of Table 1 is added to 200 g of water.
- To this solution is added 80 g of a dry formulation of a sensitive formulated pesticide.
- the slurry is transferred to a vacuum oven dryer and dried to a water activity of 0.1.
- the dry formulation is milled to the desired size and packaged.
- a protected insoluble granular formulation of a pesticide that would otherwise be subject to decomposition by other ingredients in a formulation during storage is prepared with the formulation and the method of the present invention.
- the soy protein premix of Table 1 is added to 200 g water.
- To this solution is added 90 g of a dry formulation of a sensitive pesticide and 0.5 g of calcium phosphate dibasic, followed by 0.55 g of gluconolactone.
- the slurry is allowed to harden at room temperature over 2 hours, and then sliced to thin, long threads through a slicer/shredder.
- the thin threads are loaded on trays and dried in a vacuum oven dryer to reach a water activity of 0.1.
- the dry formulation is further milled to the desired size distribution and packaged.
- Example 1 100 g of the protein hydro lisate premix of Example 1 (table 1). This dry mixture is slowly added to 100 gm of deionized water at 35° C in a jacketed dual planetary mixer, and mixed for 10 minutes at 40 rpm. The homogeneous slurry is evenly spread on a tray at a loading capacity of 100 gm/ sq ft, and the tray is placed on a shelf in a freeze dryer (Model 25 SRC, Virtis, Gardiner, NY). The shelf temperature is set at 5° C to cool the slurry. Vacuum is applied to reduce the pressure to 3 Torr, at which time the shelf temperature is raised to 3O 0 C.
- EXAMPLE 13 [00131] Twenty (20) grams of dry Lactobacillus Rhamnosus GG is mixed with 100 g whey protein premix Example 1. This dry mixture is slowly added to 100 gm of deionized water at 35° C in a jacketed dual planetary mixer, and mixed for 10 minutes at 40 rpm. The homogeneous slurry is evenly spread on a tray at a loading capacity of 100 gm/ sq ft, and the tray is placed on a shelf in a freeze dryer (Model 25 SRC, Virtis, Gardiner, NY). The shelf temperature is set at 5 0 C to cool the slurry.
- Vacuum is applied to reduce the pressure to 3 Torr, at which time the shelf temperature is raised to 30° C. After 2 hours the pressure is reduced further to 150 milliTorr with the shelf temperature still held at 3O 0 C. Drying is continued for an additional 3 hours at which point the product temperature has risen to within 2 0 C of the shelf temperature. The dried product is then removed from the freeze dryer and the water activity of the dry formulation at this point is measured by a Hygropalm Awl instrument. Viability losses during formulation, preparation and drying processes are measured and recorded.
- the homogeneous slurry is evenly spread on a tray at a loading capacity of 100 gm/ sq ft, and the tray is placed on a shelf in a freeze dryer (Model 25 SRC, Virtis, Gardiner, NY).
- the shelf temperature is set at 5 0 C to cool the slurry. Vacuum is applied to reduce the pressure to 3 Torr, at which time the shelf temperature is raised to 3O 0 C. After 2 hours the pressure is reduced further to 150 milliTorr with the shelf temperature still held at 3O 0 C. Drying is continued for an additional 3 hours at which point the product temperature has risen to within 2 0 C of the shelf temperature.
- the dried product is removed from the freeze dryer and the water activity of the dry formulation at this point is measured using a Hygropalm Awl instrument. Viability losses during formulation, preparation and drying processes are measured and recorded. Storage stability testing of the dry formulation is conducted under accelerated storage conditions of 32 0 C and 20 % RH. Results for the trial where the dryer is maintained at 3O 0 C and compared to those where the dryer is maintained at 5O 0 C are shown in Table below.
- the homogeneous slurry is evenly spread on a tray at a loading capacity of 100 gm/ sq ft, and the tray is placed on a shelf in a freeze dryer (Model 25 SRC, Virtis, Gardiner, NY).
- EXAMPLE 17 [00142] The same parameters as Example #1, except the mixing done in the
- Ross mixer is under 25 inches of vacuum to give a slurry density of 1.2 gm/cc.
- EXAMPLE 19 Lactobacillus Rhamnosus GG (LGG)OnQ hundred (100) grams of unthawed, frozen concentrate and lOOg of protein hydro lysate premix were added to a jacketed dual planetary mixer (DPM, lpt, Ross Engineering, Inc., Worcester, GA). This process can also be done by thawing the frozen concentrate first. Mixing was carried out at 40 RPM and 37 0 C for 10 minutes. The homogeneous slurry was measured for viscosity (Brookfield viscometer, Model # LVDVEl 15, Brookfield Engineering Laboratories, Inc.), and then evenly spread on a tray at a loading capacity of 100g/sq ft.
- Viscosity Brookfield viscometer, Model # LVDVEl 15, Brookfield Engineering Laboratories, Inc.
- Viscosity Parameters for high viscosity ranges were: 300g sample in a 40OmL Pyrex beaker, 33-37C, Spindle #64, 1.0 RPM speed, operated without a guard-leg. The tray was then loaded into a -4 0 C refrigerator for cooling for 30min. After cooling, the drying began using a freeze drier (Model 25 SRC, Virtis, Gardiner, NY) with a shelf temperature set at 3O 0 C throughout, and 2800mTorr of pressure for at least 2.5 hours. After at least 2.5 hours, the pressure was decreased to lOOmTorr for at least another 2.5 hours. This experiment was repeated with two different batches of LGG fermentate, and included washing of one batch with 3% DMV and reconstitution with de-ionized water prior to adding the hydrolysate premix.
- a freeze drier Model 25 SRC, Virtis, Gardiner, NY
- Viscosity Parameters for medium viscosity ranges were: 300g sample in a 40OmL Pyrex beaker, 33-37 0 C, Spindle #64, 5.0 RPM speed, operated without a guard-leg.
- LGG Batch # 4 1.39x 10 +11 0.18 58,100 Washed LGG Batch
- Lactobacillus Rhamnosus GG (LGG) One hundred (100) grams of frozen concentrate was thawed at 37 0 C and added to a jacketed dual planetary mixer (DPM, lpt, Ross Engineering, Inc., Worcester, GA). To it, lOOg of protein hydrolysate premix was added. Unthawed frozen concentrate may also be used. Mixing was carried out at 40 RPM and 37 0 C for 10 minutes, and then the slurry was evenly spread onto trays at a loading capacity of 100g/sq ft. The trays were then loaded into a -4 0 C refrigerator for cooling for 30min.
- the drying begins using a freeze drier (Model 25 SRC, Virtis, Gardiner, NY) with a shelf temperature set at 3O 0 C throughout, and 2800mTorr of pressure for at least 2.5 hours. After at least 2.5 hours, the pressure was decreased to lOOmTorr for at least another 2.5 hours. This same process was applied to 1Og of dried (powdered) LGG material, which was mixed into lOOg of protein hydrolysate. This dry mixture was then slowly added to 9Og of de -ionized water in the jacketed dual planetary mixer.
- a freeze drier Model 25 SRC, Virtis, Gardiner, NY
- Vacuum is applied to reduce the pressure to 3 Torr, at which time the shelf temperature is raised to 6O 0 C. After 1 hour the pressure is reduced further to 150 milliTorr with the shelf temperature still held at 6O 0 C. Drying is continued for an additional 1 hour at which point the product temperature had risen to within 2 0 C of the shelf temperature. The dried product is removed from the freeze dryer.
- the dry sample is accurately weighed ( ⁇ 100 mg) in a microcentrifuge tube and 200 ⁇ g of dimethyl sulfoxide (DMSO) is added. The formulation is dissolved in the DMSO buffer by vortexing.
- DMSO dimethyl sulfoxide
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CN114854651B (en) * | 2022-07-06 | 2022-09-30 | 广东顺德鼎一生物科技有限公司 | Micrococcus compositus preparation and preparation method and application thereof |
CN114854651A (en) * | 2022-07-06 | 2022-08-05 | 广东顺德鼎一生物科技有限公司 | Micrococcus compositus preparation and preparation method and application thereof |
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EP2435554B1 (en) | 2017-07-26 |
SG176253A1 (en) | 2011-12-29 |
US20220081471A1 (en) | 2022-03-17 |
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CA2763074C (en) | 2021-02-23 |
DK2435554T3 (en) | 2017-10-30 |
US20190194259A1 (en) | 2019-06-27 |
KR20120017460A (en) | 2012-02-28 |
CN106987525B (en) | 2020-10-27 |
JP2012527898A (en) | 2012-11-12 |
ES2643148T3 (en) | 2017-11-21 |
BRPI1013809B1 (en) | 2021-01-19 |
RU2011151788A (en) | 2013-07-10 |
EP2435554A4 (en) | 2012-11-07 |
CN106987525A (en) | 2017-07-28 |
CA2763074A1 (en) | 2010-12-02 |
CN102459568A (en) | 2012-05-16 |
EP2435554A2 (en) | 2012-04-04 |
MY157343A (en) | 2016-05-31 |
PL2435554T3 (en) | 2018-01-31 |
JP5841527B2 (en) | 2016-01-13 |
US11214597B2 (en) | 2022-01-04 |
RU2567668C2 (en) | 2015-11-10 |
BRPI1013809A2 (en) | 2016-11-01 |
NZ597053A (en) | 2014-02-28 |
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