• 专利附录
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    • 专利摘要:
    "dry glassy composition comprising a bioactive material". the present invention relates to formulations and methods for stabilizing and protecting biological materials during use and harsh storage conditions, wherein the formulations refer to encrusted and biological bioactive materials, including live bacteria, in a protective matrix.
    公开号:BR112012018839B1
    申请号:R112012018839
    申请日:2011-01-28
    公开日:2020-04-14
    发明作者:Moti Harel
    申请人:Advanced Bionutrition Corp；
    IPC主号:
    专利说明:

    "DRY GLASS COMPOSITION UNDERSTANDING A BIOACTIVE MATERIAL"
    CROSS REFERENCE TO RELATED ORDER
    This application claims priority for US Provisional Application No. 61 / 299,315 filed with the United States Patent and Trademark Office on January 28, 2010, the content of which is hereby incorporated by reference into this document for all purposes.
    BACKGROUND OF THE INVENTION
    Field of invention
    The present invention relates to the stabilization and protection of biological materials during conditions of use and hard storage, and more particularly, the invention relates to the fouling of bioactive and biological materials including live bacteria in a protective formulation of an amorphous glassy matrix.
    Related Technique
    Freeze drying has traditionally been the most common method of preserving sensitive biological substances such as live or dead bacteria and viruses and proteins, while other methods such as spray drying, fluid spray drying and drying are generally not suitable. The high drying temperatures used in these methods result in significant damage to the bioactive material on its own. In addition, the material may not be sufficiently dried to meet specific residual moisture or water activity requirements for product stability, and thus an additional drying step by other means may be required. A conventional freeze-drying process typically involves freezing the solution containing the bioactive material, and lyophilizing the frozen biomaterial under a complete vacuum while remaining frozen. The low temperatures of the freeze drying process decrease the degradation reaction of the bioactive material and minimize the loss of activity in the final dry form. The freeze-drying process often results in significant loss of activity and damage to bioactive material
    2/34 due to the formation of ice crystals during the slow drying process. In addition, the freezing step itself, if not done correctly, can denature or inactivate the bioactive material. The damage caused by the formation of an ice crystal structure can be overcome, to a certain degree, by the addition of cryoprotective agents to the bioactive solution (Morgan el al., 2006). Such protective agents are highly soluble chemicals that are added to a formulation to protect cell membranes and proteins during freezing and to increase stability during storage. Common stabilizers for live bacteria and viruses include higher sugars such as sucrose, glycerol, or sorbitol, at high concentrations with cell or bioactive material (Morgan el al., 2006; Capela et al., 2006). However, such protective agents may not adequately penetrate the cell to protect active components within the intracellular volume that can lead to instability after freezing the dry substances. For this reason, membranous biomaterials such as viruses, bacteria, and cells do not survive well in the freeze-drying process. Therefore, a significant challenge remains to develop an optimal drying process and formulation that minimizes drying losses while achieving adequate dry material storage stability.
    Some of the problems associated with freeze drying have been solved using a combination of certain formulations and vacuum drying in a glassy state, particularly sugar glasses (U.S. Pat. 6,190,701). Dry stabilized bioactive materials are protected in a glassy matrix against hostile environments such as high temperatures and humidity. Generally, glass formation stabilization is initiated by concentrating the sugar solution containing a bioactive molecule to form supersaturated syrup. Removing additional water progressively solidifies the syrup, which eventually becomes a glass of solid sugar with a low residual water content. Chemical diffusion is negligible in glass and therefore chemical reactions virtually cease. Since denaturation or membrane damage are chemical changes, they cannot occur in the glass and the bioactive material is stabilized and protected. Many glasses fail to stabilize because they react with the bioactive material during storage. Obvious problems occur
    3/34 with the reduction of sugars, which can form good physical glasses, but then their aldehyde groups attack amino groups in the bioactive in a typical Maillard reaction, while non-reactive sugars give stable products, which do not require refrigeration at all .
    Since sugars are inherently hygroscopic, removal of water and final drying of the supersaturated syrup becomes extremely difficult. This inconvenience was first addressed by (Annear 1962) who developed a formulation containing bacteria in a solution of sugars and amino acids and a vacuum drying process that involves boiling and foaming the concentrated syrup. Roser el al. (US. Pat 6,964,771) have revealed a similar concept of foaming drying which includes a concentration step by evaporating the solvent mass followed by boiling and foaming the concentrated syrup under vacuum. To mitigate the damage by oxidation and denaturation that can occur during the boiling stage, Bronshtein (US Patents 5,766,520, 7,153,472) introduced an improved protective formula containing carbohydrates and surfactants. The drying of the protective solution also involved a step-by-step concentration process under a moderate vacuum before applying a strong vacuum to cause foamy boiling of the remaining water to form a stable dry foam. To bypass the boiling stage, Busson and Schroeder (US Pat. No. 6,534,087) introduced a process of liquid drying a formulation suitable for sensitive bioactive materials and using a vacuum oven under very moderate vacuum pressure above 30 Torr. After reaching a certain level of drying without boiling the material, heat was applied to above 20 ° C and the dried material was collected after only a few hours.
    This type of drying process, in which the bioactive solution is kept in a liquid state throughout the drying process, has the advantage of drying faster due to the evaporation of the liquid during boiling and the increased surface area presented by the foaming surfaces. . However, boiling and foaming requires the entry of a significant amount of heat to provide the necessary solution eruption. Such drying process is not well suited for drying sensitive biologicals, such as viable viruses, cells or
    4/34 bacteria because, the applied heat accelerates enzymatic degradation (for example, proteolysis), and chemical oxidation (for example, oxidation and free radical attacks), which can destroy the activity or viability of biological material.
    The drying process described above is also limited in its ability to scale to a larger industrial process. Freezing avoidance requires that the process be conducted at a lower vacuum level (> 7 TORR) than in conventional freeze-drying or freeze-drying cycles. The most significant disadvantage of the above processes is the inability to control and limit the expansion of the foam within the container, tray or vial. The uncontrollable eruption and foaming too often makes it virtually impossible to develop an industrial scale process. The eruption and foam nature of the boiling step result in a portion of material being plaited on the walls of the container and in the drying chamber. To smooth out the eruption during boiling, Bronshtein (US Patent 6,884,866, 6,306,345) proposed special chambers and a controlled temperature / pressure application protocol that reduces overheating to an acceptable level. Another approach to containing the rash and excessive foaming is described in Pat. US No. 2008/0229609, in which the bioactive solution is contained in a container or bag covered with breathable membranes. Again, these protocols are difficult to implement at an industrial level, require special equipment and are difficult to reliably reproduce with different formulations.
    The dry foam process, as is known in the art, is not particularly well suited for the conservation of membranous biological materials, such as liposomes, viruses or viable cells and bacteria. Lipid membranes often prevent the penetration of protective agents into closed volumes or prevent adequate removal of water from the staged volume. Without adequate penetration of protective agents, enzymatic processes, such as proteolysis, and chemical processes, such as oxidation and free radical attacks, can destroy the activity or viability of the membranous biological material. Hyposmotic fluids that remain within closed membrane volumes can promote the instability of biological material. Truong-le, Vu (US Pat. 7,381,425) describes a drying process by
    5/34 freezing suitable for membranous bioactive. The compositions of the invention include a polyol and a bioactive membrane material. The drying process begins by cooling the formulation to a temperature of about a phase transition temperature of the lipid membranes, reducing the pressure on the formulation to form a stable foam, freezing the foam, and then sublimating the water from the frozen foam. to provide a lyophilized dry foam composition. Secondary drying conditions can be used to further dry the foam.
    A need remains for a suitable protective formulation that can be dried in a glassy state without boiling and excessive foaming. There is a particular need for an economical formulation and scalable drying process that is also suitable for applications outside the pharmaceutical industry such as the food and agriculture industries. Protective formulations and gentle drying processes are required to provide adequate drying without exposure to high temperatures. A composition is necessary which can protect such biological materials in storage under high temperature and humidity conditions. The present invention provides a solution to all of these challenges as described below. The dehydration process of the present invention is very smooth and does not expose the active agent to boiling or foaming and is therefore advantageous over conventional freeze drying and foam drying techniques that would subject the sample to one or both of these stresses.
    SUMMARY OF THE INVENTION
    The present invention includes compositions and drying methods for conserving sensitive bioactive materials, such as peptides, proteins, enzymes, hormones, vitamins, carotenoids, minerals, drugs, antibiotics, microbiocides, fungicides, herbicides, insecticides, spermicides, nucleic acids, antibodies, vaccines, bacteria (probiotic or otherwise), viruses and / or cell suspensions in storage. Drying methods provide a process for drying a formulation comprising bioactive materials, a matrix forming agent, and a glass forming agent. The formulation is prepared by dispersing all the
    6/34 solid components and bioactive materials in one solution. The solution is quickly frozen by means known in the art such as liquid nitrogen or dry ice to form an amorphous composition in small beads, strands or droplets. The frozen particles can be stored in a freezer (between -30 ° C and - 80 ° C) until drying or immediately placed in trays in a frozen amorphous state for drying liquid in a conventional freeze dryer. The drying method is initiated by a step of structure stabilization and short purging of the frozen particles under a vacuum pressure of less than <2000 mTORR followed by a primary drying step under more than> 2000 mTORR of vacuum pressure and at a desired temperature. During the secondary and final drying step of the amorphous vitreous material, a complete vacuum pressure and high temperature are applied, to achieve a desirable final water activity of the dry material.
    In one embodiment, the formulation comprises sufficient amounts of matrix forming agents, in which the bioactive material is encrusted. Examples of a suitable matrix agent include, but are not limited to, cellulose acetate phthalate (CAP), carboxymethyl cellulose, pectin, sodium alginate, alginic acid salts, hydroxyl propyl methyl cellulose (HPMC), methyl cellulose , carrageenan, guar gum, acacia gum, xanthan gum, locust bean gum, chitosan and chitosan derivatives, collagen, polyglycolic acid, modified starches, cyclodextrins and oligosaccharides (inulin, maltodextrins, dextrans, etc.); and combinations thereof. In a particular embodiment, the preferred matrix forming agent is sodium alginate. Preferably, the formulation comprises, in weight percent of total dry matter, 0.1-20% and more preferably 1-12%.
    In a further embodiment, the matrix forming agent comprises a mixture of sodium alginate and oligosaccharides in a weight ratio of 1: 1-10, more preferably 1: 1-5 sodium alginate / oligosaccharides.
    In yet another embodiment of the present invention, the matrix forming agent is cross-linked with divalent metal ions to form a firm hydrogel. The crosslinked hydrogel formulation is formed by atomizing or extruding the paste in a bath containing divalent metal ion solution or adding divalent metal ions directly into the paste and allowing the formulation to harden and form a
    7/34 hydrogel. The hydrogel formulation is then quickly frozen and dried according to the drying methods of the invention.
    In yet another embodiment, the formulation comprises significant amounts of glass-forming agents, in which the microorganisms are encrusted. Examples of a suitable agent include, but are not limited to, proteins such as egg albumen, egg white, gelatin, immunoglobulin, isolated soy protein, wheat protein, pea protein, cottonseed protein, skimmed milk powder , caseinate, whey protein and any hydrolyzed protein; carbohydrates including monosaccharides (eg, galactose, D-mannose, sorbose, etc.), disaccharides (eg, lactose, trehalose, sucrose, etc.), an amino acid such as lysine, 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 trihydric or higher sugar alcohols, (for example, glycerin, erythritol, glycerol, arabitol, xylitol, sorbitol, and mannitol); propylene glycol; polyethylene glycol; pluronic; surfactants; and combinations thereof.
    In a preferred embodiment; the glass forming agent comprises a mixture of a disaccharide and a hydrolyzed protein. In a particular embodiment, the preferred glass-forming agent is a mixture of trehalose and hydrolyzed protein. Preferably, the formulation comprises, in weight percent of total dry matter, 10-90% trehalose and 0.1-30% hydrolyzed protein, more preferably 20-80% trehalose and 0.1-20% hydrolyzed protein , and more preferably 40-80% trehalose and 0.1-20% hydrolyzed protein.
    The method of the invention typically includes mixing bioactive materials (for example, peptides, proteins, enzymes, hormones, vitamins, carotenoids, minerals, drugs, antibiotics, microbiocides, fungicides, herbicides, insecticides, spermicides, nucleic acids, antibodies, vaccines) , bacteria, viruses and / or cell suspensions), at least one matrix forming agent, and at least two glass forming agents in a homogeneous paste, quickly freeze the paste by atomization, dripping or extrusion in liquid nitrogen bath. Collect the beads, microcounts, threads or droplets from the liquid nitrogen bath and dry in a
    8/34 liquid state in a freeze dryer, or alternatively storing them in a freezer (between -30 ° C and -80 ° C) until drying.
    In a variation of the present invention, the amount of matrix forming agent in the formulation is adjusted to achieve a desired formulation viscosity and density that allows for efficient primary drying while avoiding the excessive boiling and foaming that typically occur during primary liquid drying step. A desired density of the liquid formulation can be achieved by any means known in the art, for example, tapping or gas injection such as air, nitrogen, carbon dioxide, argon etc. Preferably, nitrogen is injected into the viscous slurry formulation under mixing to form a stable creamy or porous slurry prior to the rapid freezing step.
    According to the invention, the drying process involves three main steps; 1. Structure stabilization step and short purging of frozen particles under a vacuum pressure of less than <2000 mTORR, 2. Primary liquid drying step under vacuum pressure of more than> 2000 mTORR and at a desired temperature, 3 Secondary and final drying step of the vitreous material under full vacuum pressure and elevated temperature for a time sufficient to reduce the water activity of the dry formulation to 0.3 Aw or less.
    In preferred embodiments of the drying methods, the bioactive material is mixed in a solution including a matrix forming agent and a glass forming agent. In a particular embodiment, the bioactive material comprises live bacteria (for example, probiotic bacteria). Examples of suitable microorganisms include, but are not limited to, yeasts such as Saccharomyces, Debaromyces, Candida, Pichia and Torulopsis, molds such as Aspergillus, Rhizopus, Mucor, Penicillium and Torulopsis and bacteria such as the genera Bifidobacterium, Clostridium, Fusobacterium, Melboccus , Propionibacterium, Streptococcus, Enterococcus, Lactococcus, Kocuriaw, Staphilococcus, Peptostrepococcus, Bacillus, Pediococcus, Micrococcus, Leuconostoc, Weissella, Aerococcus, Oenococcus and Lactobacillus. Specific examples of suitable 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.
    9/34 licheniformis, B. mesentericus, B. pumilus, B. suhtilis, B. natto, Bacteroides amilophilus, Bac. capillosus, Bac. ruminocola, Bac. suis, Bifidobacterium adolescentis, B. animalis, B. breve, B. bifidum, B. infantil, B. lactis, B. longum, B. pseudoIongum, B. thermophilum, Candida pintolepesii, Clostridium butyricum, Enterococcus cremoris, E. diacetilactis, E faecium, E. intermedius, E. lactis, E. muntdi, E. thermophilus, Escherichia coli, Kluyveromyces fragilis, Lactobacillus acidophilus, L. alimentias, L. amilovorus, L. crispatus, L. brevis, L. case 4 L. curvatus, L. cellobiosus, L. delbrueckii ss. bulgaricus, L farciminis, L. ferment, L. gasseri, L. helveticus, L. lactis, L. plantarum, L. johnsonii, L. reuteri, L. rhamnosus, L. sakei, L. salivarias, Leuconostoc mesenteroides, P. cereviseae (damnosus), Pediococcus acidilactici, P. pentosaceus, Propionibacterium freudenreichii, Prop, shermanii, Saccharomyces cereviseae, Staphilococcus carnosus, Staph, xilosus, Streptococcus infantarius, Strep, salivary ss. thermophilus, Strep. Thermophilus and Strep, lactis.
    In preferred methods, the formulation is mixed at room temperature or slightly heated to assist solubilization of the materials in viscous solution (for example, from 20 ° C to 40 ° C). After mixing until homogeneous, the viscous paste is then quickly frozen by atomization, dripping, or extrusion in liquid nitrogen. The frozen particles are collected from the liquid nitrogen bath and are either immediately dried or alternatively stored in a freezer for later drying. Typically, the paste containing the bioactive is frozen quickly at -30 ° C to -180 ° C, more preferably the formulation is frozen quickly in liquid nitrogen.
    In a preferred embodiment, the frozen particles are quickly dried immediately or otherwise stored in a freezer, preferably at -80 ° C, until drying. The frozen particles are then loaded onto trays and immediately transferred to a vacuum drying chamber where they are dried in accordance with the present invention. Preferably, drying is initiated by subjecting the frozen particles under vacuum pressure between 0 and 2000 mTORR. The frozen particles are degassed and their structure and volume are allowed to develop and stabilize for a short period of time. Typically, the desirable time period to subject the frozen particle to high vacuum pressure is no longer
    10/34 of 30 minutes, more preferably the time period is between 1 and 20 min. After the initial short degassing and stabilization of the frozen particle structure, the vacuum is adjusted to between 2000 and 10,000 mTORR and heat applied to thaw the particles at a temperature above their freezing point. Typically, the vacuum is adjusted to between 2000 and 4000 mTORR and the particle temperature is increased to between -5 ° C and +5 ° C. Under these preferred primary drying conditions the frozen particles are quickly thawed and loosely retain their original shape while accelerated dehydration begins. Subsequent secondary drying is established after removing about 60-90% of the free water, a maximum vacuum pressure is applied and heat that provides temperature to the formulation is raised to from 30 ° C to 60 ° C. To maximize the stability of the final product, the formulation is preferably scca long enough to reduce the water activity of the formulation to Aw ~ 0.3 or less. In a preferred embodiment of the invention, secondary drying comprises removing water bound to a pressure of less than 1000 mTORR.
    The dry formulation can be used directly as a flake, or ground into a powder and sieved to an average particle size from approximately 10 pm to approximately 1000 pm. The formulation can be administered directly to an animal, including man, as a concentrated powder, as a reconstituted liquid, (for example, drink), or it can be incorporated as a flake or powder into an existing food or animal feed product .
    BRIEF DESCRIPTION OF THE FIGURES
    Figure 1. Visual and microscopic observations of different dry compositions containing various matrices and glass-forming agents as a frozen solid bead according to the method of the present invention.
    Figure 2. The effect of the L. rhamnosus culture form as fresh, frozen beads or dry powder cultures on your initial UFC counts in a dry composition.
    Figure 3. The freezing temperature effect of a composition containing L. rhamnosus as solid beads frozen in liquid nitrogen or
    11/34 freezer at -80 ° C and as a non-frozen viscous paste at +4 ° C in the initial bacterial CFU counts in the dry composition. The results show only the freezing temperature effect of the paste with no additional purging steps before drying.
    Figure 4. The effect of freezing temperature of a composition containing Bifidobacterium animalis Bbl2 as solid beads frozen in liquid nitrogen and as a viscous non-frozen paste at +4 ° C in the initial CFU bacterial counts in the dry composition. The results show only the freezing temperature effect of the paste with no additional purging steps before drying.
    Figure 5. The effect of vacuum purge duration of frozen solid beads on initial CF counts of L rhamnosus in a dry composition.
    Figure 6. Drying profile in a lyophilizer of the composition according to the method of the invention.
    Figure 7. Process and drying losses of L. rhamnosus in compositions and drying methods of the invention.
    Figure 8. Trends in stability of dry probiotic bacteria, composition of L. rhamnosus in storage at 40 ° C and 33% relative humidity.
    DETAILED DESCRIPTION OF THE INVENTION
    DEFINITIONS
    And to be understood that the terminology used in this document is for the purpose of describing particular modalities only, and is not intended to be limiting.
    As used in this specification and the appended claims, the singular forms one, one and a / o include references to the plural unless the content clearly dictates otherwise. Thus, for example, reference to a protein includes a single protein or a combination of two or more proteins; reference to enzyme, vitamin, bacterium, etc., includes singular or mixtures of several, and the like.
    Bioactive material, bioactive composition, or bioactive formulation refers to preparations, which are in such a way as to allow the biological activity of the bioactive ingredients to be unequivocally effective.
    12/34
    Matrix forming agent refers to compounds or materials that are added to the formulation to increase the viscosity and or density of the wet formulation or to form a hydrogel. Examples of a suitable matrix forming agent include, but are not limited to, water soluble cellulose derivatives such as methylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, and hypromellose; alginates, galactomannan, gellan gum, tragacanth, including any derivatives thereof, cellulose acetate phthalate (CAP), carboxymethyl cellulose, pectin, sodium alginate, alginic acid salts, hydroxyl propyl methyl cellulose (HPMC), methyl cellulose, carrageenan, guar gum, acacia gum, xanthan gum, locust bean gum, chitosan and chitosan derivatives, collagen, polyglycolic acid, modified starches, cyclodextrins and oligosaccharides (inulin, maltodextrins, dextrans, etc.) and combinations thereof.
    Glass forming agent or sucking glass forming agent generally refers to compounds or materials that are readily soluble in a solution and do not thicken or polymerize after contact with water. These agents are added to ensure or increase the stability of the bioactive material during the drying process and thereafter, or for long-term storage stability of the dry powder product. Useful glass forming agents can be monomeric, oligomeric or polymeric.
    According to one of the preferred embodiments, the glass forming agent is a saccharide. A saccharide, or carbohydrate, is defined as a compound predominantly composed of carbon, hydrogen, and oxygen. Useful saccharides include reducing and non-reducing sugars and sugar alcohols, oligosaccharides, water-soluble polysaccharides and derivatives thereof. Preferred saccharides according to the invention include glucose, fructose, lactose, sucrose, trehalose, maltose, cellobiose, galactose, maltotriose, raffinose, dextrin, dextran, inulin, mannitol, sorbitol, xylitol. Particularly preferred saccharides are glucose and trehalose.
    Other useful glass-forming agents can be selected from other chemical classes, such as water-soluble amino acids, peptides or proteins and hydrolyzed protein. For example, lysine, glycine, alanine, arginine or histidine, as well as hydrophobic amino acids (tryptophan, tyrosine, leucine, phenylalanine, etc.); a methylamine such as betaine. Useful proteins include gelatin,
    13/34 egg albumin, egg white, whey protein, caseinate, immunoglobulins, soy protein, pea protein, cottonseed protein or other food, dairy or vegetable proteins.
    Hydrolysed proteins generally refer to proteins from animal, dairy or plant sources that have been degraded by enzymatic hydrolysis or digestion into shorter peptide fragments and / or amino acids. Useful hydrolyzed proteins are those that undergo an extensive hydrolysis process that reduces the molecular weight of 99% of native proteins to less than 50,000 Dalton, preferable to less than 10,000 Dalton.
    Ambient ambient conditions or temperatures are those at any given time in a given environment. Typically, ambient ambient temperature is 2225 ° C, ambient atmospheric pressure, and ambient humidity are readily measured and will vary depending on the time of year, climate and climatic conditions, altitude, etc.
    Purge or gasification in the context of the present invention refers to the release of a gas from a solid formulation or liquid formulation in which the partial pressure of the gas is greater than the applied pressure. This is not the boiling of solution in liquid form, and can often occur at pressures above a pressure that would boil a solution.
    Boiling refers to the rapid phase transition from liquid to gas that occurs 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 pressure applied. Boiling can be particularly vigorous when heat is added to a liquid that is already at its boiling point.
    Water activity or ’’ Aw ’’, in the context of dry formulation compositions, refers to the availability of water and represents the energy status of water in a system. It is defined as the water vapor pressure above a sample divided by that of pure water at the same temperature. Pure distilled water has a water activity of exactly one or Aw - 1.0.
    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 as a percentage of
    14/34 saturation humidity.
    Dry and variations thereof refer to a physical state that is freeze-dried, dehydrated or anhydrous, i.e., liquid substantially lacking. Drying includes, for example, spray drying, fluid bed drying, lyophilization, and vacuum drying.
    Freeze drying or Freeze drying refers to the preparation of a composition in the dry form by rapid freezing and dehydration in the frozen state (sometimes referred to as sublimation). This process can take place under vacuum at a pressure sufficient to keep the product frozen, preferably below approximately <2000 mTORR.
    Primary drying or Liquid drying, with respect to the processes described in this document, refers to drying by dehydration that occurs from the time of defrosting the frozen particles to the point where secondary drying begins. Typically, the primary drying mass occurs by extensive evaporation, while the product temperature remained significantly below the temperatures of the heat source. This process can take place under vacuum at a pressure sufficient to keep the product thawed, preferably above approximately> 2000 mTORR.
    Secondary drying, with respect to the processes described in this document, refers to a drying step that occurs at temperatures above the freezing temperatures of the formulation and close to the temperature of the heat source. This process can take place under vacuum at a pressure sufficient to reduce the water activity of a formulation, preferably less than approximately <1000 mTORR. In a typical formulation drying process, a secondary drying step reduces the water activity of the formulation to an Aw of 0.3 or less.
    Foaming refers to a procedure for biologicals sensitive to boiling drying under vacuum under conditions where the biologicals retain activity or viability for extended periods at ambient and higher temperatures. The specific procedure for the formation of a mechanically stable porous structure proceeds in two stages; (1) Foam - Primary and boiling drying under vacuum (2) Drying / Stability Glazing, and are disclosed in US Patent No.
    15/34
    5,766,520 to Bronshtein.
    A stable formulation or composition is one in which the biologically active material in it essentially retains its physical stability, chemical stability, and / or biological activity after storage. Stability can be measured at a selected temperature and humidity conditions for a selected period of time. Trend analysis can be used to estimate an expected useful life before a material has actually been in storage for that period of time. For live bacteria, for example, stability is defined as the time it takes to lose 1 log CFU / g of dry formulation under predefined conditions of temperature, humidity and time period.
    Viability with respect to bacteria, refers to the ability to form a colony (UFC or Colony Forming Unit) in an appropriate nutrient medium for the growth of bacteria. Viability, with respect to viruses, refers to the ability to infect and reproduce in a suitable host cell, resulting in the formation of a plaque in a layer of host cells.
    The compositions and methods of the present invention solve the problem of providing an economical and industrially scalable drying process for formulations containing sensitive bioactive materials, such as peptides, proteins, enzymes, hormones, vitamins, carotenoids, minerals, drugs, antibiotics, microbiocides, fungicides , herbicides, insecticides, spermicides, nucleic acids, antibodies, vaccines, bacteria, viruses and / or cell suspensions, with a significantly extended life in the dry state.
    The invention provides a composition comprising a bioactive material and matrix and glass forming agents in a mixing solution and a drying method comprising rapidly freezing said composition in liquid nitrogen to form a solid amorphous structure in the form of droplets, threads, beads or microcounts and purge the frozen particles under high vacuum followed by stabilizing the bioactive material in a sugar glass formation by lyophilization or evaporation of moisture under a reduced pressure regime while providing heat to the composition.
    Most of the loss of viability of microorganisms during the processes
    16/34 drying can be attributed to a combination of ice crystal formation, high osmotic and oxidative stresses, shear forces and energy release during bubble cavitations associated with boiling ”and foaming of the solution under low and high drying pressure temperature. The present invention avoids such negative effects and provides drying compositions and methods with minimal loss and which results in a bioactive material protected in a sugar glass matrix under harsh storage and handling conditions thereafter.
    COMPOSITIONS OF THE INVENTION
    The present invention includes compositions of a bioactive material, a matrix forming agent and glass forming agents in a viscous solution. It has been found that the formulations of the invention are inherently different in their physical structure and function from non-viscous or concentrated formulations that have been dried with or without rapid freezing and purging. For example, prior art formulations were initially foamed ”by boiling to facilitate effective drying. The foaming step generally resulted in extensive boiling and eruption of the solution which is an inevitable consequence of drying in a liquid state and as a result, only a very low load capacity of material in a vial or container can be achieved (see , for example, US Pat. No. 6,534,087, where the thickness of the final foamed product is less than 2 mm).
    The drying compositions and methods of the present invention prevent boiling and extensive foaming of the formulation thereby enabling much greater material loading per drying area and, as a result, can easily be scaled up to the production of large quantities of material without the use of specifically designed containers, trays or equipment.
    Probiotic bacteria have been shown to benefit particularly from the formulations and drying methods of the present invention. The formulation is prepared according to the compositions and methods of the invention including mixing fresh, frozen or dry cultures of probiotic bacteria with at least one matrix forming agent and at least two glass forming agents, quickly freezing the viscous formulation in nitrogen liquid to form a structure
    17/34 amorphous of frozen solid droplets, threads or beads. For primary drying, sufficient vacuum pressure is applied to purge and stabilize the frozen particle structure and then the frozen particles are lyophilized or evaporated under reduced vacuum pressure and increased temperature above the freezing temperature of the formulation. Keeping the temperature of the formulation above the freezing point can be achieved by conducting heat away from the formulation, and / or by the loss of latent heat due to water evaporation. To complete the drying process and further reduce the water activity of the formulation, a secondary drying step can be applied, the vacuum pressure higher than 1000 mTORR and lower and the temperature elevated up to 70 ° C, to provide a final composition with water activity with an Aw of 0.3 or less. Such a composition can remain stable under storage conditions of 40 ° C and 33% RH for 30 days or more, as shown in Figure 8.
    Preparation of the compositions
    The materials, to be mixed in a solution with the preferred bioactive for the preparation of dry powder compositions according to the invention, include at least one matrix forming agent and at least two glass forming agents. Such materials, when mixed with the preferred bioactive material, form beads, microcounts, strands or droplets in liquid nitrogen and can be effectively lyophilized or dehydrated in an amorphous glassy state according to the methods of the invention and to provide large quantities of stable dry compositions for storage and administration of said bioactive material (see Figures 1 A & B for visual and microscopic observations and water activity (Aw) of different formulations after drying). The matrix forming agent provides structural stability to the formulation, improved drying profile and / or physical and chemical protective benefits for bioactive materials. The matrix forming agent also provides thickening of the viscosity to the formulation and better control over the properties of the formulation under vacuum pressure and increased structural resistance to the dry formulation compositions of the invention (see Figure 1 B- Images 4, 4b, 4c for glassy structure and dryness of that particular formulation). The matrix forming agent includes a mixture of polysaccharides and oligosaccharides. The preferred polysaccharides,
    18/34 particularly for living organisms, are water-soluble gums, because of their distinctive characteristic to form viscous gel at moderate temperatures. Gums at a certain concentration also effectively stabilize the formulation and facilitate the formation of an amorphous glassy structure and improve the vacuum drying profile 5 (see Figure IA - images 3a, 3b, 3c, 4, and Figure IB - 4c and Figure 6) .
    Notably, seeing the images in Figure 1A in combination with the results shown below in Table 1, it is evident that samples 3b, 3c, 4, 5, and 6 were all sufficiently dry to provide some porosity in the amorphous glass structures.
    Table 1
    Visual Inspection of Various Dry Compositions 1 2 3rd 3b 3c 4 [5 6Dryness Not Dry Not Dry Not Scca Dry iSeca Dry ; Scca Scca ........ Porosity None None None Gift Gift Gift None Partial A W 0.847 .0,923 0.916 0.216 0.183 0.376 Ό.17Ι 0.112 Glass Structure None None None Partial = Partial Gift Partial Partial
    Glass forming agents of the invention can include various sugars, non-reducing sugars, sugar alcohols, amino acids, proteins, hydrolyzed proteins and peptides. The glass-forming compound is preferably one that does not crystallize and / or destabilize the biologically active material in the formulation at freezing temperatures (for example, below -20 ° C). For example, bioactive material can be physically embedded in amorphous sugar glass structures such as sucrose or trehalose to promote molecular structure retention throughout the drying process and impart structural rigidity to the amorphous matrix in the dry state. The glass-forming agent replaces hydrating water lost during drying, to prevent damage to cell membranes and denaturation of enzymes (see review by Crowe et al., 1998). Other functions of the glass-forming agent may include protecting bioactive material from exposure to harmful light, oxygen, oxidizing agents and moisture. More glass-forming agents need to be readily dissolved in a solution in amounts ranging from approximately 0.1 weight percent
    19/34 up to approximately 80 weight percent. It is beneficial to include two or more different glass-forming agents to inhibit crystal formation and improve the stability of the dry material bioactive formulation under extended storage conditions (see the effect of mixing sugars and proteins in Figure IA images 4, 5 and 6).
    Pre-dried formulations include a substantial amount of total solids (constituents minus the solvent, such as water). A larger portion of total solids consists of bioactive material, matrix forming agents and glass forming agents. For example, the bioactive material is present in the formulation at a concentration ranging from approximately 5-60 weight percent, the matrix forming agent approximately 1-20 weight percent, and the glass forming agent approximately 5-80 weight percent. In another example, the matrix forming agent may be present in the formulation at a concentration ranging from approximately 0.5-10 weight percent, and the glass forming agent from approximately 10-50 weight percent. Preferably, the wet formulation should have a solids content between approximately 5% and 80%; more preferably between 30% and 60%. The viscosity of formulations of the invention is typically greater than 1000 centipoises (cP); more preferably, greater than 5,000 cP; and more preferably greater than 10,000 cP. The density of the formulations of the invention is preferably between 0.9 and 1.2 g / ml.
    METHODS OF PREPARING STABLE DRY FORMULATIONS
    Methods for preparing stable dry formulations containing bioactive materials include; (1) preparing a formulation by mixing the bioactive material with matrix and glass-forming agents in a solution, (2) quickly freezing the formulation to form solid frozen particles, (3) subjecting the frozen particle to high vacuum pressure for a short time to purge the particles and stabilize their structure, (4) remove water by lyophilization and / or evaporation of moisture under reduced pressure while providing heat to the formulation at a temperature above the freezing temperature of the formulation, preferably above -10 ° C, (5) additionally reduce the water activity of the formulation to less than 0.3 Aw under
    20/34 complete vacuum and high temperature.
    In one embodiment, for example, the formulations of the invention include a bioactive material formulated in a solution or suspension containing a matrix and glass forming agents. The matrix forming agent and / or high concentration of glass forming agents are dissolved and disinfected in a hot aqueous solution with agitation before cooling and mixing with the bioactive material. Bioactive material, such as viruses or cultured bacteria, is concentrated and separated from the culture media by centrifugation or filtration before resuspension in the formulation.
    In one embodiment of the present invention, all of the water in the formulation is provided in the concentrated living organism liquid and the living organism suspension is maintained at a temperature slightly above room temperature. The dry components are mixed together and then slowly added to the warm suspension (25 ° C to 40 ° C) of the living organism. The formulation suspension is gently stirred in a planetary mixer until all components are completely dispersed and a uniform paste is obtained.
    The viscous solution is then quickly frozen by atomization, dripping or extrusion in a bath of liquid nitrogen to form small solid droplets strands or beads. The frozen solid particles can be stored in a freezer at -30 ° C to -80 ° C until drying or immediately placed in trays and lyophilized or dried according to the methods of the invention. The frozen solid particle is purged for a short time, typically between 1 and 20 minutes, under sufficient vacuum (for example, below <2000 mTORR). The particles generally remain in a frozen solid form at a temperature below -20 ° C during the purge step. After the initial purge step the vacuum pressure is increased to between 2000 and 10,000 mTORR and heat can be provided allowing the formulation temperature to rapidly rise to between -5 ° C and +5 ° C and the particles begin to thaw. Once the formulation temperature reaches the desired temperature, the heat is adjusted to maintain that temperature and the primary drying step is progressed. In this stage the formulation is already thawed and accelerated evaporation of water occurs without any boiling or foaming.
    Typical methods in the prior art involve extensive foaming and / or
    21/34 splashing and violent boiling that can be harmful to sensitive biologicals and make it difficult to increase the industrial scale to high load capacity (see, for example, US Pat. No. 6,534,087, where the applied vacuum pressure has as result of violent boiling and foaming), while the present compositions and methods prevent any excessive boiling or foaming of the formulation at the same time that it achieves a significantly faster drying rate and enables a high load capacity of the formulation. In addition, complete and efficient degassing of viscous slurries is difficult and may require an extended period of time. These obstacles have all been solved in the present invention using a suitable composition that allows an effective primary drying of liquid that forms an amorphous glassy formation without any boiling and excessive foaming. Surprisingly and importantly this was achieved mainly by quickly freezing a suitable composition and introducing a short purging step before the start of the primary liquid drying step. The loading of solid frozen particles in a tray as opposed to paste or viscous syrup allows for much greater loading capacity per drying area in trays than was permitted according to the prior art. After the primary liquid drying stage is completed, the stabilized amorphous glass formulation is maintained at elevated secondary drying temperatures (between 20 ° C and 70 ° C) and vacuum pressures of less than 1000 mTORR to further reduce water activity formulation in a very short time.
    Another embodiment of the invention provides methods for preparing hydrogel formulation compositions for conserving bioactive materials. For example, a formulation containing a bioactive material and matrix and glass-forming agents are mixed in a solution and cross-linked to form particles of firm hydrogel by atomization or extrusion in a bath containing divalent metal ions or by adding divalent metal ions directly to the paste and crushing the hydrogel plate hardened to small threads or pieces. The hydrogel particles are then dried according to the drying methods of the invention as described above.
    In a particular embodiment of the invention, for example, the formulation includes live probiotic bacteria in a solution of 1-4% sodium alginate and 10-40%
    22/34 trehalose. Proteins and particularly hydrolyzed proteins, such as casein, whey, peas, soybeans or cottonseed are added to the formulation at 5-10% to increase the drying process and the formation of a stable amorphous glass formulation structure (see Figure IA, images 4, 5 and 6). The probiotic culture can be fresh, frozen or already dried in a dry powder form (see Figure 2 for CFU counts of different culture forms of probiotic bacteria in the dry formulation) The solution is mixed at a temperature slightly above room temperature (typically between 25 ° C -37 ° C) until all components are completely dissolved. The formulation paste is atomized, extruded or dripped in liquid nitrogen to form small droplets or beads which are then removed from the liquid nitrogen, packaged in bags and can be stored in a -80 ° C freezer until drying.
    A typical method of drying live probiotic bacteria includes; spread the frozen solid beads on trays in an even layer at a load capacity between 100-1000 g / sq ft and the trays are immediately placed in a freeze dryer. The vacuum pressure is then applied at approximately 1000 mTORR and depending on the size of the freeze dryer and type of heat source, the shelf temperature adjusted to +20 ° C or at a temperature sufficient to keep the particles at approximately -20 ° C. The frozen solid beads are left to purge for approximately 5-30 minutes and vacuum adjusted to between 2000 and 10,000 mTORR and increased heat transfer to raise the formulation temperature to between -5 ° C and +5 ° C. These temperature and vacuum pressure conditions are maintained during the primary liquid drying step which can last from a few hours to up to 24 hours depending on the tray load, preferably from approximately 3 to 10 hours. At some point during the primary drying process, the solvent evaporation rate slows down and the formulation temperature starts to increase due to the superfluous supply of heat in the drying chamber. This point indicates the end of the primary drying step. As the solvent is directed out of the formulation, the glass-forming agents in the solution become concentrated and thicker until they stop flowing like a liquid and form an amorphous and / or stable glassy structure.
    A secondary drying step is then followed by complete vacuum and
    23/34 formulation temperature between 30 ° C and 50 ° C. The purpose of the secondary drying step is to remove the remaining trapped or bound moisture and provide a composition that is stable in storage for an extended period of time at ambient temperatures. The secondary drying step can take several hours and its end point is when the formulation is completely dry and its water activity is less than 0.3 Aw.
    The drying methods of the invention result in a biologically active material that is enclosed within an amorphous glassy matrix, thereby preventing the unfolding of proteins and significantly slowing molecular interactions or cross-reactivity, due to the greatly reduced mobility of the compound and other molecules within of the amorphous glassy composition. As long as the amorphous solid is at a temperature below its glass transition temperature and the residual humidity remains relatively low (that is, below Aw of 0.3), the bioactive material remains relatively stable (see Figure 8). It should be noted that achieving a glassy state is not a prerequisite for long-term stability as some bioactive ingredients can do better in a more crystalline state.
    Dry Powder Preparation
    The dry formulation can be used en bloc, cut into desired shapes and sizes, or crushed and crushed into a free flowing powder that provides easy downstream processing such as wet or dry agglomeration, granulation, tabletting, compaction, pelletizing or blending. food or animal feed products or any other type of administration process. Crushing, crushing, grinding or spraying processes are well known in the art. For example, a hammer mill, an air mill, an impact mill, a jet mill, a pin mill, a Wiley mill, or similar grinding device can be used. The preferred particle size of the crushed particles is less than approximately 1000 pm and preferably less than 500 pm.
    The compositions and methods described in this document preserve the biological activity of the closed biologically active material. For example, compositions are tested for stability by subjecting them to temperature
    24/34 high (for example, 40 ° C) and high humidity (for example, 33% RH) and measuring the biological activity of the formulations. As an example for live probiotic bacteria, the results of these studies demonstrate that the bacteria formulated in these formulations are stable for at least 20 days (see Figure 8). Stability is defined as time by a UFC / g log of power loss. Such formulations are stable even when high concentrations of the biologically active material are used. Thus, these formulations are advantageous in that they can be shipped and stored at temperatures at or above room temperature for long periods of time.
    EXAMPLES
    The following examples are offered to illustrate, but not to limit the claimed invention.
    EXAMPLE 1
    Preparation of dry and stable probiotic substance
    Basic formulation g of trehalose (Cargill Minneapolis, MN) and 22 g of extensively hydrolyzed casein (M arc or, Carlstadt, NJ) were uniformly mixed with 3 g of sodium alginate (ISP Corp., Wayne, NJ) dry. Fresh concentrate of Lactobacillus acidophilus (100 ml containing at least 10% solids, straight from fermentation harvest) was added in a mixer and kept at 35 ° C. The dry mixture of gum, sugar and hydrolyzed protein was slowly added to the probiotic culture and the mixture was carried out at 35 ° C for 10 minutes. The viscous paste was then transferred to a container that has a perforated bottom and allowed to drip in a nitrogen-containing bath. The beads were then removed from the liquid nitrogen and immediately transferred to drying.
    Drying of frozen beads of the basic formulation
    The frozen beads were evenly spread on a tray at a load capacity of 100 g / sq ft and immediately placed on a shelf in a freeze dryer (Model 25 SRC, Virtis, Gardiner, NY). The vacuum pressure was then applied at 1000 mTORR and the frozen solid beads were left for
    25/34 purge for 10 minutes. Vacuum was then adjusted to 2700 mTORR and shelf temperature raised to +30 ° C. This vacuum temperature and pressure was maintained for 3 hours. A secondary drying step was then followed at full vacuum (150-200 mTORR) and shelf temperature raised to 30 ° C for an additional 2 hours. The formulation was completely dried and its water activity measured by a Hygropalm Awl instrument (Rotonic Instrument Corp., Huntington, NY.) At Aw = 0.23.
    EXAMPLE 2
    Stable dry composition containing probiotic bacteria Lactobacillus rhamnosus LGG.
    Lactobacillus rhamnosus LGG (500 g of frozen concentrate from a commercial source) was thawed at 37 ° C in a jacketed double planetary mixer (DPM, Iqt, Ross Engineering, Inc. Savannah, GA,). Two glass forming agents; trehalose (387 g, Cargill Minneapolis, MN) and extensively hydrolyzed casein (83 g, Marcor, Carlstadt, NJ) were homogeneously mixed in dry form with two matrix forming agents; sodium alginate (15 g, ISP Corp., Wayne, NJ) and instant inulin (25 g, Cargill Minneapolis, MN). The dry mixture was slowly added to the thawed probiotic bacteria and the mixture was carried out at 40 RPM and 37 ° C for 10 minutes. The viscosity of the paste was adjusted to 12,000 Cp by adding 50-200 ml of water. The paste was then transferred to a container that has a perforated bottom and allowed to drip into a container containing liquid nitrogen. The beads were then removed from the liquid nitrogen, placed in a sealed aluminum foil pouch and stored in a -80 ° C freezer for several weeks.
    For drying, the frozen beads were evenly spread on trays at a load capacity ranging from 100 to 500 g / sq ft and the trays placed on shelves in a freeze dryer (Model 25 SRC, Virtis, Gardiner, NY). The vacuum pressure was applied at 1000 mTorr and the shelf temperature adjusted to 120 ° C. The frozen solid beads were left to purge for a period of time ranging from 1 to 30 minutes. The purge step was followed by a primary drying step after adjusting the vacuum pressure to 2700 mTORR and shelf temperature raised to +30 ° C. These temperature and vacuum pressure were maintained for
    26/34 hours. A secondary drying step was then followed at full vacuum (150200 mTORR) and shelf temperature maintained at 30 ° C for an additional 4 hours. The formulation was completely dry and its water activity measured at 0.23 Aw. Figure 6 shows the drying profile of the probiotic formulation.
    The viability losses after freezing the paste at different temperatures (+4 ° C, - 80 ° C and -180 ° C) and after the drying process including preparation of frozen beads, and drying in a freeze dryer are shown in Figure 3, 4 and 7. Losses of viability throughout the process were generally less than <1 log depending on the type of bacterial culture (frozen or dry cultures) and the freezing temperature of the viscous paste. The results show that quickly freezing probiotic bacteria in liquid nitrogen (-180 ° C) was less harmful than freezing at -80 ° C.
    Figures 5 & 8 show the effect of various purging times ranging from 0 min (without purging) to 30 min on the initial probiotic bacteria counts in the dry composition and on storage stability under accelerated 40 ° C storage conditions and 33% RH. The results suggest that a longer purging time generally improves the initial bacterial count in the dry formulation, but has no effect on the storage stability of the probiotic formulation.
    EXAMPLE 3
    Trealose (752 g, Cargill Minneapolis, MN), extensively hydrolyzed pea protein (167 g, Marcor, Carlstadt, NJ), sodium alginate (30 g, ISP Corp., Wayne, NJ) and 50 g instant inulin, Cargill Minneapolis , MN) were homogeneously mixed in the dry form. The dry mixture was slowly added to 1000 ml of hot deionized water at 80 ° C in a jacketed double planetary mixer (DPM, Iqt, Ross Engineering, Inc. Savannah, GA,) and the mixture was carried out at 40 RPM for 10 minutes. The temperature mixture was reduced to 37 ° C and 100 g of dry powder of Lactobacillus rhamnosus LGG obtained from a commercial source was slowly added and the mixing continued for 20 minutes. The paste was then extruded through a needle with a 2 mm hole in a bath containing liquid nitrogen. The
    27/34 / strands / beads were then removed from the liquid nitrogen placed in a sealed aluminum foil pouch and stored in a freezer at -80 ° C for several weeks. For drying, the frozen threads / beads were evenly spread on trays at a load capacity ranging from 100 to 500 g / sq ft and the trays were placed on shelves in a freeze dryer (Model 25 SRC, Virtis, Gardiner, NY) and dried as described in Example 2. All formulations were satisfactorily retained within the tray and no splashing or foaming was observed at all loading levels. The formulation was completely dry even at the highest load capacity and water activity measured at 0.26 Aw and lowest for all samples.
    EXAMPLE 4
    Preparation of a hydrogel formulation containing probiotic bacteria Bifidobacterium lactis (Bbl2):
    Concentrated probiotic paste of Bifidobacterium lactis (Bbl2) is prepared according to Example 1. The basic formulation, 0.5 g of dibasic calcium phosphate is 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 into long, thin threads using a commercially available slicer / shredder. The fine strands are quickly frozen in liquid nitrogen and loaded into a tray at a load capacity of 700g / sq ft and placed in a freeze dryer for drying as described in Example 2. The water activity (Aw) of the formulation was 0, 05 (Measured by HygroPalm Awl, Rotonic Huntington, NY). The dry formulation was further ground to fine powder using standard hammer milling equipment and sieved through 50-250 micron screens.
    EXAMPLE 5
    Allergen-free composition containing probiotic bacteria Lactobacillus acidophilus.
    Trealose (752 g, Cargill Minneapolis, MN), pea protein extensively
    28/34 hydrolyzed (167g, Marcor, Carlstadt, NJ), sodium alginate (30 g, ISP Corp., Wayne, NJ) and 50 g instant inulin, Cargill Minneapolis, MN) were homogeneously mixed in the dry form. The dry mixture was sterilized by slowly adding 1000 ml hot deionized water at 80 ° C in a jacketed double planetary mixer (DPM, Iqt, Ross Engineering, Inc. Savannah, GA,) and the mixture was carried out at 40 RPM for 10 minutes until a smooth, clear paste is formed. The temperature mixture was reduced to 37 ° C and 1000 g of frozen beads containing Lactobacillus acidophilus obtained from a commercial source were slowly added and mixing continued for 10 minutes. The paste was then extruded through a needle with a 2 mm hole in a bath containing liquid nitrogen. The / wires / beads were then removed from the liquid nitrogen placed in a sealed aluminum foil pouch and stored in a -80 ° C freezer for several weeks. For drying, the frozen threads / beads were evenly spread on trays at a load capacity of 1000 g / sq ft and the trays placed on shelves in a freeze dryer (Model 25 SRC, Virtis, Gardiner, NY) and dried as described in Example 2. The initial CFU counts of probiotic bacteria in the dry composition was 10.53 logs / g, and loss of viability after 42 days storage under accelerated storage conditions of 40 ° C and 33% RH was 0.69 log CFU / g.
    EXAMPLE 6
    An infant formula containing the dry formulation of the present invention:
    A stable dry formulation containing Lactobacillus GG (Valio Corp, Finland) was prepared according to Example 2 followed by sieving into two groups of particle size (above 50 pm and below 150 pm). An infant formula was prepared by mixing 99.9 g of Nutramigen (Mead Johnson; Evansville, IL) with 0.1 g of the dry formulation particles in the size range between 50 pm and 150 pm). The final product contains approximately 10 8 cfu of Lactobacillus GG per 100 g infant formula.
    29/34
    EXAMPLE 7
    A probiotic supplement containing the stable dry formulation of the present invention:
    A dry, stable composition containing Lactobacillus acidophilus is formulated into oral dosage forms, such as tablets, oblong tablets, or capsules. Orange flavor tablets containing 99.9 g of a compression agent (dextrose) and 0.1 g of dry formulation particles in the size range between 50 pm and 150 pm are prepared by direct compression on a rotating machine using a round concave tool 1/2 ”standard. The final product contains approximately 10 8 cfu / unit dose. The hardness of the tablets is in the range of 8-10 kp and the disintegration times are approximately 20 seconds. The compressed tablets are packaged in 180 cc HDPE bottles of 100 tablets each and exposed to controlled temperature / humidity of 40 ° C / 33% RH. The product is subjected to microbiological stability test monthly over a period of 12 months or until a reduction in the test count below 1 x 10 6 / unit dose is observed.
    EXAMPLE 8
    A functional drink containing the stable dry formulation of the present invention:
    A dry mixture containing (% by weight) 71% sucrose, 14% maltodextrin, 10% inulin, 2% dextrose, 1% anhydrous citric acid, 0.3% acacia gum, 0.3% flavor, 0, 3% tricalcium phosphate and 0.1% particles of dry probiotic formulation (Z. acidophilus) in the size range between 50 pm and 250 pm is prepared. The final product contains approximately 10 9 cfu / unit dose (30 g dry mix). The product is packaged in small laminated aluminum bags (30 g unit dose / bag) to drink by shaking in 340 thousand water. The stability of probiotic bacteria in the dry beverage mixture is subjected to microbiological stability test monthly over a period of 12 months or until a reduction in the test count below 1 x 10 7 / unit dose is observed.
    30/34
    EXAMPLE 9
    Preparation of probiotic animal feed:
    A commercially available pelleted dog food for dogs is dried in a convection oven at a water activity of 0.1, and then coated with the stable dry probiotic formulation prepared as described in Example 3. The dry pellets are sprayed with approximately 5% fat-based moisture barrier (a mixture of 40% chicken fat, 40% cocoa butter and 20% beeswax), mixed in a tilting mixer with the dry powder formulation (usually 0.1 -0.5% of total animal feed that provides a dosage of 10 8 CFU / g), and finally sprayed with an additional coating of the fat-based moisture barrier. The total amount of coating is approximately 15% (of the animal feed). The coating time is approximately 30 min.
    EXAMPLE 10
    Preparation of fish food with several probiotic microorganisms:
    Pelleted fish food according to the present invention is prepared with a mixture of several probiotics. A dry stable probiotic formulation containing a mixture of L. rhamnosus, L. acidophilus and Bifidobacterium lactis is prepared as described in Example 1. A commercially available salmon starter (Zeigler Bros., Gardners, PA) is first dried in an oven of convection to a water activity of 0.1, and then coated with the formulation of probiotics in a tilting mixer. The pellets (1000 g) are first sprayed with approximately 5% by weight of a 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 achieve a dosage of 10 7 cfu / g of feed), and finally sprayed with additional fat-based moisture barrier coating. The total amount of coating is approximately 10% (of the fish feed).
    31/34
    EXAMPLE 11
    Stable dry powder containing an enzyme:
    A hydrogel formula containing 40 weight percent Savinase (Novozymes, Denmark) is prepared by mixing 600 g of the formulation described in Example 4 and 400 g of savinase in 1000 g of solution in water. The cut hydrogel formulation is quickly frozen in liquid nitrogen and dried in a vacuum oven at a formulation drying temperature of 50 ° C. For the determination of load and storage stability of the dry formula: a dry sample is weighed precisely (<100 mg) in a microcentrifuge tube. 200 μΐ of dimethyl sulfoxide (DMSO) is added. The formulation is dissolved in the DMSO buffer by vortexing. To this sample, 0.8 ml of a solution containing 0.05 N NaOFI, 0.5% SDS and 0.075 M citric acid (trisodium salt) is added. 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 in wells of a microplate and analyzed for protein content using the Bradford assay method. The storage stability of the stable enzyme formulation is significantly superior to a dry enzyme without the formulation of the present invention.
    EXAMPLE 12
    Stable dry powder containing vitamin A:
    A formulation containing 30 weight percent Vitamin A is prepared by mixing 320 g of instant inulin, 320 g of maltodextrin DE-1 (Tate & Lile, London, UK), 50 g of sodium carboxymethyl cellulose (Ashland Aquaion Functional Ingredients, Wilmington, DE ), 10 g of sodium ascorbate and 300 g of crystalline vitamin A (BASF Corp., Florham Park, NJ) in 1000 g of water. The wet formulation is spray-dried in a Mobile-Minor spray dryer (GEA Process Engineering Inc., Columbia MD) at inlet and outlet temperature of 180 ° C and 80 ° C, respectively or quickly frozen in liquid nitrogen, then spread in trays at a load capacity of 1000 g / sq ft and dried as described in Example 2. The vitamin-A composition is stable (> 80%) at 40 ° C and 75% RH for 3 months.
    32/34
    EXAMPLE 13
    Carotene preparation in a protected formulation that has improved bioavailability:
    A formulation that protects and improves the bioavailability of carotenes that would otherwise be subjected to oxidation by other ingredients in a feed during storage or after feeding an organism is prepared according to the formulation and method of the present invention. A formulation containing 6 g of water-soluble chitosan (LSK BioPartners, Inc. Salt Lake City, Utah) is dissolved in 200 g of water. To this solution is added 90 g of natural astaxanthin (Naturose ™, Cyanotech Corp., Kailua-Kona, HI) and the paste is atomized or extruded in a bath containing 5% sodium tripoli phosphate. The hydrogel or yarn microparticles are left to harden at room temperature over 4 hours. The particles are removed from the cross-linking bath, washed with water and mixed with a dry mixture of 90 g of sucrose and 10 g of extensively hydrolyzed casein. The sugar / protein loaded particles are quickly frozen and immediately placed in trays at 500 g / sq ft and lyophilized in a lyophilizer until water activity is reduced to less than 0.3. The dry formulation is crushed in addition to the desired size distribution and packaged.
    EXAMPLE 14
    Preparation of Invasive Species Baits
    The pelletized bait for specifically targeted invasive species is prepared in accordance with the present invention. 200 g of a formulation containing a pesticide as described in Example 1 are prepared and added to 200 gm of water. To this solution is added 90 gm of Rotenone and 0.5 gm of dibasic calcium phosphate, followed by 0.5 gm of gluconolactone. The paste is left to harden at room temperature over 2 hours. The firm gel is sliced into long, thin strands through a slicer / shredder. The fine wires are loaded into a tray and placed in a freeze dryer. Shelf temperature is adjusted to -30 ° C and the formulation allowed to freeze before a full vacuum is applied and the shelf temperature was raised to +60 ° C by overnight drying. The dry formulation is ground to the distribution of
    33/34 size appropriate for specifying bait size for specific target species.
    EXAMPLE 15
    Preparation of a protected pesticide in a water-insoluble formulation:
    A granular formulation protected from a pesticide that would otherwise be subjected to decomposition by other ingredients in a formulation during storage or after application to the environment is prepared with the formulation and method of the present invention. A formulation containing 6 g of pectin and 102 g of sucrose is added to 200 g of water. To this solution is added 90 g of a dry formulation of a sensitive pesticide and a mixture containing 1.5 g of dibasic calcium phosphate and 0.5 g of calcium chloride, followed by 0.85 g of gluconolactone. The paste is left to harden at room temperature over 4 hours, and then sliced into long, thin strands through a slicer / shredder. The fine wires are loaded in trays and dried in a freeze dryer to achieve 0.1 water activity. The dry formulation is crushed to the desired size distribution and packaged.
    EXAMPLE 16
    Preparation of a protected plant probiotic formulation:
    A biological control agent such as Rhizohacteria is prepared in a dry composition according to Example 4. The effectiveness of the dry composition of Rhizohacteria is evaluated on the growth of lettuce under gnotobiotic conditions. Doses of 100 mg of dry Rhizohacteria composition per plant are inoculated in jars with sand and planted with pre-germinated lettuce seedlings (24 h). A nutrient dose of 5 ml of sterile Hoagland's solution is applied to the plants in the jar. The jars are randomly arranged in a growth chamber maintained at 28 ° C with 12 h of photoperiod. During each 7-day interval after inoculation, plants and adhering sand are carefully removed from the jars. The roots are washed in sterile phosphate buffer (pH 7.0), and the root length measurement is recorded.
    34/34
    REFERENCES
    The following references and all references cited in this document are hereby incorporated by reference in this document for all purposes.
    US Patent References:
    6,190,701 Composition and method for stable injectable liquids, March 1999, Roser et al.
    6,964,771 Method for stably incorporating substances within dry, foamed glass matrices, September 1997, Roser et al.
    5,766,520 Preservation by formulation formation, June 1998, Bronshtein
    6,534,087 Process for preparing a pharmaceutical composition, June 2001, Busson and Schroeder.
    6,884,866 Bulk drying and the effects of inducing bubble nucleation, April 2005, Bronshtein.
    7,153,472 Preservation and formulation of bioactive materials for storage and delivery in hydrophobic carriers, December, 2006, Bronshtein
    20080229609 Preservation by Vaporization, June 2005, Bronshtein
    6,306,345 Industrial scale barrier technology for preservation of sensitive biological materials at ambient temperatures, October 2001, Bronshtein et al.
    7381425 Preservation of bioactive materials by freeze dried foam, September 2006, Truong-le, Vu.
    Other References:
    Morgan, C.A., Herman, N., White, P.A., Vesey, G. 2006. Preservation of microorganisms by drying; the review. J. Microbiol. Methods. 66 (2): 183-93.
    Capela, P., Hay, T.K.C „& Shah, N. P. 2006. Effect of cryoprotectants, prebiotics and micro encapsulation on survival of probiotic organisms in yoghurt and freeze-dried yoghurt. Food Research International, 39 (3) 203-211).
    Annear, 1962. The Preservation of Lepto spires by Drying From the Liquid State, J. Gen. Microbiol., 27: 341-343.
    Crowe, J.F., Carpenter, J.F. and Crowe, L.M. 1998. THE ROLE OF VITRIFICATION IN ANHYDROBIOSIS. Annu. Rev. Physiol. 60: 73-103.
    权利要求:
    Claims (15)
    [1]
    1. Method for preparing a dry powder composition without boiling or foaming, said method characterized by the fact that it comprises:
    a) combining a bioactive material, a matrix forming agent and two glass forming agents in an aqueous solvent to form a viscous paste;
    b) quickly freeze the paste in liquid nitrogen to form solid frozen particles, in the form of beads, droplets or threads;
    c) primary drying of the frozen particles by evaporation under a pressure greater than 267 Pa (2000 mTorr) and at a temperature between -5 ° C and + 5 ° C, thus maintaining the temperature of the particles above the freezing point of the frozen particles, whereby a dry formulation is formed; and
    d) secondary drying of the dry formulation under vacuum and at a temperature between 20 ° C and 70 ° C for a time sufficient to reduce the water activity of the dry formulation to 0.3 Aw or less, whereby the dry glassy composition is prepared .
    [2]
    A method of preparing the composition according to claim 1, said method characterized by the fact that it additionally comprises purging the frozen particles before the primary drying of the frozen particles.
    [3]
    3. Method according to claim 1, characterized by the fact that the total pressure on the particles frozen in the primary drying step is between 267 Pa and 533 Pa (2,000 mTORR and 4,000 mTORR).
    [4]
    4. Method according to claim 1, characterized by the fact that the total pressure on the particles frozen in the primary drying step is between 267 Pa and 1333 Pa (2,000 mTORR and 10,000 mTORR).
    [5]
    Method according to claim 1, characterized in that it further comprises cutting, crushing, grinding or spraying the composition in a free flowing powder.
    Petition 870190088715, of 09/09/2019, p. 4/7
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    [6]
    6. Method according to claim 5, characterized in that the particle size of the powder is less than 1000 μm.
    [7]
    Method according to claim 1, characterized in that it further comprises administering the composition to an animal or plant as a reconstituted liquid or as a crushed powder in a food or food product.
    [8]
    8. Method according to claim 1, characterized by the fact that it also comprises (a) mixing the composition with a component selected from the group consisting of infant formula, functional drinks and pet food; and (b) administering the mixture from step (a) to a human infant, human adult, animal or plant.
    [9]
    Dry, glassy composition prepared by the method of claim 1 without boiling or foaming, characterized by the fact that it comprises a bioactive material, at least one matrix-forming agent and at least two glass-forming agents.
    [10]
    10. The composition of claim 9, characterized in that the composition comprises total solids in the range of 30 weight percent to 70 weight percent.
    [11]
    11. The composition of claim 9, characterized by the fact that the bioactive material comprises a cell, a microbe, a virus, a cell culture, a bacterium, a probiotic bacterium, a soil and plant probiotic bacterium, a yeast, a protein , a recombinant protein, an enzyme, a peptide, a hormone, a vaccine, a drug, an antibiotic, a vitamin, a carotenoid, a mineral, a microbicide, a fungicide, a herbicide, an insecticide or a spermicide.
    Petition 870190088715, of 09/09/2019, p. 5/7
    3/3
    [12]
    12. Composition of claim 9, characterized by the fact that the matrix-forming agent is selected from the group consisting of: cellulose acetophthalate (CAP), carboxymethyl cellulose, pectin, sodium alginate, alginic acid salts, hydroxyl propyl methyl cellulose (HPMC), methylcellulose, carrageenan, guar gum, gum arabic, xanthan gum, locust bean gum, chitosan and chitosan derivatives, modified starches, cyclodextrins, inulin, maltodextrins, dextrans and their combinations.
    [13]
    The composition of claim 9, characterized in that the matrix forming agent is present in the composition in an amount ranging from 1% by weight to 20% by weight.
    [14]
    14. Composition according to claim 9, characterized by the fact that each of the glass-forming agents is selected from the group consisting of proteins, carbohydrates, amino acids, methylamine, polyol, propylene glycol, polyethylene glycol, surfactants, phospholipids and their combinations.
    [15]
    The composition of claim 9, characterized in that the glass-forming agents are present in the composition in an amount ranging from 1% by weight to 80% by weight.
    类似技术:
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    US9731020B2|2017-08-15|Dry glassy composition comprising a bioactive material
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    同族专利:
    公开号 | 公开日
    CA2785815C|2018-04-24|
    BR112012018839A2|2015-09-15|
    MX336076B|2016-01-07|
    CN102725393B|2015-12-02|
    RU2535869C2|2014-12-20|
    US8834951B2|2014-09-16|
    CN102725393A|2012-10-10|
    PL2529004T3|2017-12-29|
    AR080073A1|2012-03-14|
    CA2785815A1|2011-08-04|
    WO2011094469A2|2011-08-04|
    JP5886763B2|2016-03-16|
    ES2639397T3|2017-10-26|
    US20150031544A1|2015-01-29|
    EP2529004A2|2012-12-05|
    SG182317A1|2012-08-30|
    WO2011094469A3|2011-12-29|
    RU2012134269A|2014-03-10|
    JP2013517801A|2013-05-20|
    US20120322663A1|2012-12-20|
    DK2529004T3|2017-09-25|
    EP2529004A4|2013-10-23|
    NZ601017A|2014-07-25|
    MX2012008795A|2012-08-17|
    US9731020B2|2017-08-15|
    EP2529004B1|2017-06-07|
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    法律状态:
    2018-03-06| B06T| Formal requirements before examination|
    2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according art. 34 industrial property law|
    2019-07-09| B07A| Technical examination (opinion): publication of technical examination (opinion)|
    2020-02-11| B09A| Decision: intention to grant|
    2020-04-14| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 28/01/2011, OBSERVADAS AS CONDICOES LEGAIS. |
    优先权:
    申请号 | 申请日 | 专利标题
    US29931510P| true| 2010-01-28|2010-01-28|
    PCT/US2011/022821|WO2011094469A2|2010-01-28|2011-01-28|Dry glassy composition comprising a bioactive material|
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