Stable dry powder composition comprising biologically active microorganisms and/or bioactive materials and methods of making

Abstract

The present invention relates to embedding live or dead microorganisms and/or bioactive materials in a protective dry formulation matrix, wherein the formulation includes 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, and cooling the solution to a temperature above its freezing temperature. 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.

1 . A composition comprising (i) a bioactive microorganism or material as fresh, frozen or dry powder, (ii) at least two stabilizer agents, and (iii) at least two protective agents, wherein the composition is suitable for a liquid state drying and stabilizing the bioactive microorganism or material. 
     
     
         2 . The composition of  claim 1 , wherein total solids range from about 30 weight percent to about 70 weight percent. 
     
     
         3 . The composition of  claim 1 , wherein the bioactive microorganism or material is selected from a cell, a microbe, a virus, a culture, a probiotic, a yeast, an algae, a protein, a recombinant protein, an enzyme, a peptide, a hormone, a vaccine, a drug, a vitamin, a mineral, a microbiocide, a fungicide, a herbicide, an insecticide or a spermicide. 
     
     
         4 . The composition according to  claim 1 , wherein the stabilizer agent is a polysaccharide and or an oligosaccharide. 
     
     
         5 . The composition of  claim 4 , wherein the polysaccharides is selected from 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 combinations thereof. 
     
     
         6 . The composition of  claim 4 , wherein the oligosaccharide is selected from inulin, maltodextrins, dextrans and combinations thereof. 
     
     
         7 . The composition of  claim 4 , wherein the stabilizers are present in an amount ranging from about 1 weight percent to about 20 weight percent. 
     
     
         8 . The composition according to  claim 1 , wherein the protective agents are readily soluble in a solution and do not thicken or polymerize upon contact with water. 
     
     
         9 . The composition according to  claim 1 , wherein the protective agents are: proteins, such as human and bovine serum albumin, egg albumen, gelatin, immunoglobulins, isolated soya protein, wheat protein, skim milk powder, caseinate, whey protein, pea protein and any protein hydrolysates; carbohydrates including, monosaccharides (galactose, D-mannose, sorbose, etc.), disaccharides (lactose, trehalose, sucrose, etc.), cyclodextrins; 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 trihydric or higher sugar alcohols, e.g. glycerin, erythritol, glycerol, arabitol, xylitol, sorbitol, and mannitol; propylene glycol; polyethylene glycol; pluronics; surfactants; and combinations thereof. 
     
     
         10 . The composition of  claim 9 , wherein the protective agents are present in an amount ranging from about 1 weight percent to about 80 weight percent. 
     
     
         11 . The composition of  claim 1 , wherein the total amount of the protective agents is between 20 and 70 weight percent. 
     
     
         12 . A method for preparing a stable dry powder composition of  claim 1 , such method comprising: (i) combining under vacuum a bioactive microorganism or material in the form of fresh, frozen or dry powder, dry stabilizer agents and dry protective agents in an aqueous solvent; (ii) cooling the mixture of step (i) to a temperature above its freezing temperature; (iii) primary drying of the cooled mixture by evaporation, under vacuum, at a temperature above its freezing temperature; (iv) secondary drying of the mixture at a temperature of 20° C. or more for a time sufficient to reduce the water activity of the mixture to Aw−0.3 or less. 
     
     
         13 . The method of  claim 12 , wherein the mixture is cooled before drying to a temperature above its freezing temperature. 
     
     
         14 . The method of  claim 12 , wherein the drying of the mixture is done by evaporation. 
     
     
         15 . The method of  claim 12 , wherein the temperature of the mixture during the primary drying step is above its freezing temperature. 
     
     
         16 . The method of  claim 12 , wherein the secondary drying step is started when the temperature of the mixture is increased by at least 10° C. above its initial drying temperature. 
     
     
         17 . The method of  claim 12 , wherein the evaporation rate of the remaining solvent during the secondary drying step is accelerated by increasing the temperature and/or vacuum pressure. 
     
     
         18 . The method of  claim 12 , wherein the mixture is dried for a time sufficient to reduce the formulation to a water activity of Aw−0.3 or less. 
     
     
         19 . The method of  claim 12 , wherein the dried mixture is cut, crushed, milled or respectively pulverized into a free flowing powder. 
     
     
         20 . The method of  claim 19 , wherein particle size of the dried mixture is less than about 1000 μm. 
     
     
         21 . The method of  claim 12 , further comprising administering the composition to a mammal as a reconstituted liquid or as a ground powder and as a food or feed product. 
     
     
         22 . The composition of  claim 1 , wherein the protecting agents are dissolved in the solution and wherein the composition has a viscosity from about 10,000 cP to about 450,000 cP. 
     
     
         23 . (canceled) 
     
     
         24 . (canceled) 
     
     
         25 . (canceled) 
     
     
         26 . (canceled) 
     
     
         27 . (canceled) 
     
     
         28 . (canceled) 
     
     
         29 . (canceled) 
     
     
         30 . The method of  claim 12 , wherein the protecting agents are dissolved in the aqueous solution and the viscosity of the solution is from about 10,000 cP to about 450,000 cP to form a homogenous mixture and wherein vacuum pressure of step (iv) is less than 0.2 Torr for a time sufficient to reduce the water activity of the mixture to Aw−0.3 or less to form a dry mixture. 
     
     
         31 . The method according to  claim 30 , wherein the dried mixture is cut, crushed, milled or respectively pulverized into a free flowing powder. 
     
     
         32 . The method according to  claim 30 , wherein particle size of the dried mixture is less than about 1000 μm. 
     
     
         33 . The method according to  claim 30 , wherein the bioactive microorganism or material is selected from a cell, a microbe, a virus, a culture, a probiotic, a yeast, an algae, a protein, a recombinant protein, an enzyme, a peptide, a hormone, a vaccine, a drug, a vitamin, a mineral, a microbiocide, a fungicide, a herbicide, an insecticide or a spermicide. 
     
     
         34 . The method according to  claim 30 , wherein the dry stabilizer agents comprise a polysaccharide and an oligosaccharide. 
     
     
         35 . The method according to  claim 34 , wherein the polysaccharide is selected from 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 combinations thereof. 
     
     
         36 . The method according to  claim 34 , wherein the oligosaccharide is selected from inulin, maltodextrins, dextrans and combinations thereof. 
     
     
         37 . The method according to  claim 30 , wherein the protective agents do not thicken or polymerize upon contact with water. 
     
     
         38 . The method according to  claim 30 , wherein the protective agent is selected from a group consisting of human serum, bovine serum albumin, egg albumen, gelatin, immunoglobulins, isolated soya protein, wheat protein, skim milk powder, caseinate, whey protein, pea protein, a protein hydrolysate, galactose, D-mannose, sorbose, lactose, trehalose, sucrose, cyclodextrins, lysine, monosodium glutamate, glycine, alanine, arginine, histidine, tryptophan, tyrosine, leucine, phenylalanine, betaine, magnesium sulfate, glycerin, erythritol, glycerol, arabitol, xylitol, sorbitol, mannitol; propylene glycol; polyethylene glycol; pluronics; surfactants; and combinations thereof. 
     
     
         39 . The method of  claim 30 , wherein the bioactive microorganism or material is added to the aqueous solution after the protecting agents are dissolved. 
     
     
         40 . A method of feeding an animal a bioactive microorganism or material, the method comprising:
 administering a composition according to  claim 22 .