Dry glassy composition comprising a bioactive material

The present invention relates to the preservation and preservation of biological materials under severe conditions of storage and use, and more particularly the invention relates to the deposition of bioactive materials and biological agents comprising live bacteria in a protective formulation with an amorphous vitreous matrix.

Related art [0002] Traditionally, lyophilization has been the most common method for fixing sensitive biological substances such as live or dead bacteria, viruses and proteins, as other methods such as spray drying, fluidized spray drying, and drying are generally not suitable. The high drying temperatures used in these methods result in significant damage to the bioactive materials themselves. In addition, they may not sufficiently dry the material to a suitable residual moisture or water activity required for product durability, and therefore an additional drying step may be required in another way. A standard freeze drying process involves freezing a solution containing a bioactive material, and lyophilizing the frozen biomaterial under a full vacuum during which it remains frozen. Low temperatures of the lyophilization process lower the degradation reaction of the bioactive material and minimize the loss of activity in the final dry form. Often, the lyophilization process results in significant loss of activity and damage to the bioactive material due to the formation of ice crystals during the slow drying process. In addition, the lyophilisation step itself, if not done correctly, can denature or inactivate the bioactive material. Damage due to the formation of ice crystal structures can be circumvented, to some extent, by the addition of cryoprotective agents to the bioactive solution (Morgan et al., 2006). Such preservatives are well-soluble chemicals,

EP 2 529 004B1

Typical fixatives for live bacteria and viruses include large sugars such as sucrose, glycerol or sorbitol, at high concentrations with a cellular or bioactive material (Morgan et al., 2006, Capela et al., 2006). However, such safeners may not penetrate the cell intensely to protect active elements in the intracellular space, which may lead to instability during the storage of freeze-dried substances. For this reason, membrane biomaterials like viruses, bacteria and cells will not survive the lyophilization process well. Therefore, it is a major challenge to develop an optimal drying process and formulation that will minimize losses during drying while achieving adequate durability when storing dry material.

[0003] Several problems associated with lifolization have been solved by using a combination of several formulations and vacuum drying in the vitreous state, in particular of sugar glasses (US Patent 6,190,701). Dry fixed bioactive materials are protected in a glassy matrix from unfriendly environmental conditions such as high temperatures and humidity. In general, glass fixation is initiated by concentrating a sugar solution containing a bioactive particle into a supersaturated syrup. Further water removal gradually cures the syrup, which eventually turns into solid sugar glass with a low residual water content. Chemical diffusion is negligible in the glass and therefore chemical reactions are practically non-existent. Because denaturation or membrane damage are chemical changes, they can not take place in the glass, thus the bioactive material is fixed and protected. Many glasses can not be fixed because they react with the bioactive material during storage. Obvious problems exist with reducing sugars that can form glasses with good physical properties, but then their aldehyde groups attack the amino groups in bioactive agents in a typical Maillard reaction, which is why non-reactive sugars give stable products that do not require refrigeration at all.

[0004] As sugars are inherently hygroscopic, the removal of water and the final drying of the supersaturated syrup becomes extremely difficult. This difficulty first

EP 2 529 004 B1 was once described by (Annear 1962), who developed a formulation containing bacteria in a sugar and amino acid solution and a vacuum drying process that included boiling and foam formation of a concentrated syrup. Roser et al., (US Patent 6,964,771) disclose a similar concept of drying by foam formation, which includes a concentration step by evaporation of most of the solvent followed by boiling and foaming of the concentrated syrup under vacuum. To ameliorate damage caused by oxidation and denaturation that may occur during the boiling step of Bronshtein (US Patents 5,766,520, 7,153,472) introduced an improved protective formulation containing carbohydrates and surfactants. The drying of the security solution also applies to the gradual concentration process under moderate vacuum, before applying a strong vacuum to induce foamy boiling of the remaining water to form a dry, stable foam. To circumvent the boiling step, Busson and Schroeder (U.S. Patent No. 6,534,087) introduced a liquid-phase drying process for a formulation suitable for sensitive bioactive materials and the use of a vacuum oven under a very mild vacuum pressure above 30 Torr. After reaching a certain level of drying without boiling the material, heating was applied at above 20 ° C and the dry material was collected after only a few hours. 087) introduced a liquid-phase drying process of a formulation suitable for sensitive bioactive materials and the use of a vacuum oven under a very mild vacuum pressure above 30 Torr. After reaching a certain level of drying without boiling the material, heating was applied at above 20 ° C and the dry material was collected after only a few hours. 087) introduced a liquid-phase drying process of a formulation suitable for sensitive bioactive materials and the use of a vacuum oven under a very mild vacuum pressure above 30 Torr. After reaching a certain level of drying without boiling the material, heating was applied at above 20 ° C and the dry material was collected after only a few hours.

[0005] This type of drying process, in which the bioactive solution is kept in the liquid state during the entire drying process, has the advantage of fast drying due to evaporation of the liquid during boiling and increasing the surface area presented by the foaming surfaces. However, boiling and foaming requires a significant amount of heat to provide the necessary solution discharge. This drying process is not well suited for drying sensitive biological agents such as live viruses, cells or bacteria because the heat used accelerates enzymatic degradation (e.g., proteolysis), and chemical oxidation (e.g., oxidation and attacks of free radicals) that can destroy activity or viability of biological materials.

[0006] The drying process described above is also limited in its ability to be scaled to large industrial processes.

EP 2 529 004B1

Avoiding freezing requires a process to be carried out at a lower vacuum level (> 7 TORR) than in conventional cycles of the freeze drying or spray drying process. The most significant disadvantage of the above processes is the inability to control and limit the growth of foam within the tank, tray or vial. Uncontrolled burst and sometimes excessive foam formation makes it virtually impossible to develop an industrial scale process. The ejection and foaming nature of the boiling step results in a portion of the material that is sprayed on the walls of the vessel and in the drying chamber. To mitigate the burst during boiling, Bronshtein (US Patents 6,884,866, 6,306,345) has proposed special chambers and a temperature / pressure control application protocol that reduce overheating to an acceptable level. Another approach including ejection and excessive foaming is described in US Patent Publication. No. 2008/0229609, in which the bioactive solution is enclosed in a reservoir or bag covered with membranes capable of breathing. Again, these protocols are difficult to implement at the industrial level, they require special equipment and are difficult to reproduce accurately with different formulations.

[0007] The foam drying process as known in the art is not particularly well suited for protecting membrane biological materials such as liposomes, viruses, living cells and bacteria. Lipid membranes often prevent penetration of security measures into enclosed spaces or prevent accurate removal of water from enclosed spaces. Without adequate penetration of safeners, enzymatic processes, such as proteolysis, and chemical processes, such as oxidation and free radical attacks, can destroy the activity or viability of membrane biological materials. Hypoosmotic fluids that remain inside the sealed space of the membrane may contribute to biological material instability. Truong-le, Vu (U.S. Patent 7,381, 425) describe a freeze drying process suitable for bioactive membrane particles. Compositions of the invention include polyol and membrane bioactive materials. The drying process begins by cooling the formulation to a temperature around the phase transition temperature of the lipid membranes, reducing the pressure acting on

The formulation is used to form a stable foam, freeze the foam, and then sublimate the water from the frozen foam to provide a lyophilized dry foam composition. The secondary drying conditions can be used to further dry the foam.

[0008] Among the other bioactive compositions disclosed in the prior art, GB-A-1232057 discloses a finely powdered composition containing skimmed-milk powder that is granulated to improve the ability to be reconstituted with aqueous liquids by retaining the fluidized bed composition and introducing steam contact with the bed in countercurrent to the gas holding the bed. The composition may include sugars, vitamins, minerals, proteins, fats, bulking agents such as methylcellulose, monosodium glutamate, and dyes. US-A-2004/241313 indicates a protein-based nutritional composition that contains proteins, erythritol, maltitol syrup, glycerol, inulin and maltodextrin, and a process for preparing a protein-based nutritional composition. US-A-5262187 relates to a low-fat dry mix, ready-to-use raw dough and baked composition consisting of a sweetened base component from cereal grains with a fat mimetic system of polydextrose, cellulose material, skim milk in a solid form or a replacement, emulsifier, edible modified starch, and a mixture of xanthan gum and guar gum or rubber from carob bean flour preferably with lecithin and a whey protein concentrate. The baked composition is delicate, fragile with a pleasant mouthfeel, but preferably contains one third of fewer calories than a similar full fat composition. US-A-2007/031534 relates to animal feed which contains amino acids or their salts, instead of raw protein materials that may become allergens, or which contain, additionally, amino acid (s), crude protein materials with low allergenicity,

[0009] There remains a need for a suitable protective formulation that can be dried in the vitreous state without boiling and

EP 2 529 004B1 excessive foaming. There is a particular need for a cost-effective formulation and a scalable drying process that is also suitable for use outside the pharmaceutical industry, such as the food and agricultural industry. Protective formulations and gentle drying processes are required to ensure adequate drying without exposure to high temperatures. A composition is required that can protect such biological agents during storage under conditions of high temperatures and humidity. The present invention provides a solution to all challenges as described below. The dehydrating process according to the present invention is very delicate and does not expose the active agent to boiling or foaming, and therefore it is advantageous compared to conventional lyophilisation and foam drying techniques,

SUMMARY OF THE INVENTION [0010] The present invention includes compositions and methods of drying protecting sensitive bioactive materials such as peptides, proteins, enzymes, hormones, vitamins, carotenoids, minerals, drugs, antibiotics, microbicides, fungicides, herbicides, insecticides, spermicides, nucleic acids, antibodies, vaccines, bacteria (probiotic or other), viruses and / or cell suspensions during storage. The drying methods provide a drying process for a formulation containing bioactive materials, a matrix forming agent and a glazing agent. The formulation is prepared by dispersing all solid ingredients and bioactive materials in solution. The solution is rapidly frozen with agents known in the art such as liquid nitrogen or dry ice to form an amorphous composition in the form of small beads, fibers or drops. Frozen particles can be stored in a freezer (between 30 ° C and - 80 ° C) until drying or placed directly on trays in a frozen amorphous state for drying the liquid in a standard lyophilizer. The drying method is initiated by a short blow and a step of fixing the structure of the frozen particles under a vacuum pressure lower than <2,000 mTORR, followed by a basic drying step under a vacuum pressure higher than> 2,000 mTORR and at the desired temperature. During Frozen particles can be stored in a freezer (between 30 ° C and - 80 ° C) until drying or placed directly on trays in a frozen amorphous state for drying the liquid in a standard lyophilizer. The drying method is initiated by a short blow and a step of fixing the structure of the frozen particles under a vacuum pressure lower than <2,000 mTORR, followed by a basic drying step under a vacuum pressure higher than> 2,000 mTORR and at the desired temperature. During Frozen particles can be stored in a freezer (between 30 ° C and - 80 ° C) until drying or placed directly on trays in a frozen amorphous state for drying the liquid in a standard lyophilizer. The drying method is initiated by a short blow and a step of fixing the structure of the frozen particles under a vacuum pressure lower than <2,000 mTORR, followed by a basic drying step under a vacuum pressure higher than> 2,000 mTORR and at the desired temperature. During 2000 mTORR and at the desired temperature. During 2000 mTORR and at the desired temperature. During

EP 2 529 004 B1 for the secondary and final step of glass drying the amorphous material, full vacuum pressure and elevated temperature are used to achieve the final desired water activity of the dry material.

[0011] In one embodiment, the formulation contains sufficient amounts of matrix forming agents in which the bioactive material is deposited. Examples of suitable matrix agents include, but are not limited to, cellulose acetate phthalate (CAP), carboxymethylcellulose, pectin, sodium alginate, salts of alginic acid, hydroxylpropylmethylcellulose (HPMC), methylcellulose, carrageenan, guar gum, gum arabic, xanthan gum, gum from bread flour midsummer, chitosan and chitosan derivatives, collagen, polyglycolic acid, starch and modified starches, cyclodextrins and oligosaccharides (inulin, maltodextrins, dextranes, etc.); and their combinations. In one particular embodiment of the invention, the preferred matrix-forming agent is sodium alginate. Preferably, the formulation contains, in percent by weight of the total dry substance, 0,

[0012] In an additional 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 [0013] In yet another embodiment of the present invention, the agent forming the matrix is ​​cross-linked with divalent metal ions to form a compact hydrogel. A cross-linked hydrogel formulation is formed by atomizing or extruding a slurry into a bath containing a solution of divalent metal ions or by adding divalent metal ions directly to the slurry and allowing the formulation to cure and form a hydrogel. The hydrogel formulation is then rapidly frozen and dried according to the drying method of the invention.

[0014] In yet another embodiment, the formulation contains significant amounts of a glass forming agent in which the microorganisms are embedded. Examples of suitable agents include, but are not limited to, proteins such as egg albumin, egg white, gelatin, immunoglobulins, isolated protein

Soybean protein, wheat protein, pea protein, cotton seed protein, skimmed milk powder, caseinate, whey protein and each hydrolyzed protein; carbohydrates including monosaccharides (e.g., galactose, D-mannose, sorbose, etc.), disaccharides (e.g., lactose, trehalose, sucrose, etc.), amino acids such as lysine, glutamate, glycine, alanine, arginine or histidine, as well as hydrophobic amino acids (tryptophan, tyrosine, leucine, phenylalanine, etc.), methylamine such as betaine; an auxiliary salt such as magnesium sulfate, a polyol such as trihydroxy or higher sugar alcohols (e.g., glycerol, erythritol, glycerol, arabitol, xylitol, sorbitol and mannitol); propylene glycol, polyethylene glycol; pluronics, surfactants and combinations thereof.

[0015] In one preferred embodiment, the sclerosing agent comprises a mixture of disaccharide and hydrolyzed protein. In a particular embodiment, the preferred glazing agent is a mixture of trehalose and hydrolyzed protein. Preferably, the formulation contains, in percent by weight of dry matter, 10-90%, trehalose and 0.1-30% hydrolysed protein, more preferably 20-80% trehalose and 0.1-20% hydrolysed protein and most preferably 40-80% trehalose and 0.1-20% hydrolysed protein.

The method of the invention typically comprises mixing in a solution of a bioactive material (e.g., 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 scorching agents to obtain a homogeneous suspension, quick freezing of the suspension by atomization, instilling or extrusion into a liquid nitrogen bath. Collecting, spheres, microspheres, fibers or drops from a bath with liquid nitrogen and drying in the liquid state in a freeze dryer or, alternatively, storing them in a freezer (between -30 ° C and -80 ° C) until drying.

[0017] In an embodiment of the present invention, the amount of matrix forming agent in

The formulation is adapted to achieve the desired viscosity and density of the formulation, which allows effective basic drying and prevents boiling and excessive foaming that typically occur during the basic drying step of the liquid. The desired density of the liquid formulation can be achieved by any means known in the art, e.g., compacting or injecting a gas such as air, nitrogen, carbon dioxide, argon etc. Preferably, nitrogen is injected into the viscous slurry formulation while mixing to form a stable porous and cream colored suspensions prior to the rapid freezing stage.

[0018] According to the invention, the drying process comprises three main steps; 1. Short purging and stage of fixing the structure of frozen particles under a vacuum pressure of less than <2,000 mTORR. 2. Stage of basic drying of liquids under vacuum pressure higher than> 2000 mTORR and at the desired temperature. 3. A step of secondary and final drying of the glassy 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.

[0019] In preferred embodiments of the drying processes, the bioactive material is mixed in a solution comprising a matrix-forming agent and a glass-forming agent. In a particular embodiment of the invention, the bioactive material contains live bacteria (e.g., 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 from such species as Bifidobacterium, Clostridium, Fusobacterium, Melissococcus, Propionibacterium, Streptococcus, Enterococcus, Lactococcus, Kocuriaw, Staphylococcus, Peptostrepococcus, Bacillus, Pediococcus, Micrococcus, Leuconostoc, Weissella, Aerococcus, Oenococcus and Lactobacillus.

EP 2 529 004B1

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. diacetylactis, 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, P. 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. cerevisis (damnosus), Pediococcus acidilactici, P. pentosaceus, Propionibacterium freudenreichii, Prop. shermanii, Saccharomyces cereviseae, Staphylococcus carnosus, Staph. xylosus, Streptococcus infantarius, Strep. salivarius ss. thermophilus, Strep. Thermophilus and Strep. lactis.

[0020] In preferred methods, the formulation is stirred at room temperature or slightly heated to help dissolve the materials in a viscous solution (e.g., from 20 ° C to 40 ° C). After mixing to homogeneity, the viscous slurry is then quickly frozen by atomization, dropping or injection into liquid nitrogen. Frozen particles are collected from the bath in liquid nitrogen and are directly dried or possibly stored in a freezer for subsequent drying. Typically, the suspension containing bioactive materials is rapidly frozen to a temperature between -30 ° C to -180 ° C, more preferably the formulation is rapidly frozen in liquid nitrogen.

[0021] In a preferred embodiment of the invention, the quick-frozen particles are directly dried or optionally stored in a freezer, preferably at -80 ° C, until drying. The frozen particles are then loaded into trays and directly transferred to the vacuum drying chamber, where they are dried in accordance with the present invention. Preferably, the drying is initiated by subjecting the frozen particles to a vacuum pressure between 0 and 2,000 mTORR. Frozen particles are degasified, and their structure and volume allow to develop and consolidate for a short period of time. Usually the desired time period of being frozen

The high vacuum pressure of the particles is not more than 30 minutes, more preferably, the time period is between 1 and 20 minutes. After a brief preliminary degassing and fixing of the structure of the frozen particles, the vacuum is set to between 2000 and 10000 mTORR and heat is provided to thaw the particles at a temperature above its freezing point. Typically, the vacuum is set between 2000 and 4000 mTORR and the particle temperature rises to between -5 ° C and + 5 ° C. In these preferred basic drying conditions, the frozen particles are quickly thawed and freely retain their original shape while accelerated dehydration begins. Then, after removing about 60-90% of free water, secondary drying is started, the maximum vacuum pressure is set and heat is supplied to the formulation to raise the temperature from 30 ° C to 60 ° C. To increase the stability of the final product, the formulation is preferably dried for a time sufficient to reduce the water activity of the formulation to 0.3 Aw or less. In a preferred embodiment, the secondary drying comprises removing the bound water at a pressure lower than 1000 mTORR. [0022] The dried formulation may be used directly as a flake, or milled in powder form, and sieved to an average particle size of from 10 Pm to about 1000 Pm. The formulation may be administered directly to animals, including humans, as a concentrated powder, as a reconstituted liquid (e.g. a beverage), or it may be contained in either a flake or powder form in existing food or feed products. To increase the stability of the final product, the formulation is preferably dried for a time sufficient to reduce the water activity of the formulation to 0.3 Aw or less. In a preferred embodiment, the secondary drying comprises removing the bound water at a pressure lower than 1000 mTORR. [0022] The dried formulation may be used directly as a flake, or milled in powder form, and sieved to an average particle size of from 10 Pm to about 1000 Pm. The formulation may be administered directly to animals, including humans, as a concentrated powder, as a reconstituted liquid (e.g. a beverage), or it may be contained in either a flake or powder form in existing food or feed products. To increase the stability of the final product, the formulation is preferably dried for a time sufficient to reduce the water activity of the formulation to 0.3 Aw or less. In a preferred embodiment, the secondary drying comprises removing the bound water at a pressure lower than 1000 mTORR. [0022] The dried formulation may be used directly as a flake, or milled in powder form, and sieved to an average particle size of from 10 Pm to about 1000 Pm. The formulation may be administered directly to animals, including humans, as a concentrated powder, as a reconstituted liquid (e.g. a beverage), or it may be contained in either a flake or powder form in existing food or feed products. the formulation is preferably dried for a time sufficient to reduce the water activity of the formulation to 0.3 Aw or less. In a preferred embodiment, the secondary drying comprises removing the bound water at a pressure lower than 1000 mTORR. [0022] The dried formulation may be used directly as a flake, or milled in powder form, and sieved to an average particle size of from 10 Pm to about 1000 Pm. The formulation may be administered directly to animals, including humans, as a concentrated powder, as a reconstituted liquid (e.g. a beverage), or it may be contained in either a flake or powder form in existing food or feed products. the formulation is preferably dried for a time sufficient to reduce the water activity of the formulation to 0.3 Aw or less. In a preferred embodiment, the secondary drying comprises removing the bound water at a pressure lower than 1000 mTORR. [0022] The dried formulation may be used directly as a flake, or milled in powder form, and sieved to an average particle size of from 10 Pm to about 1000 Pm. The formulation may be administered directly to animals, including humans, as a concentrated powder, as a reconstituted liquid (e.g. a beverage), or it may be contained in either a flake or powder form in existing food or feed products. [0022] The dried formulation may be used directly as a flake, or milled in powder form, and sieved to an average particle size of from 10 Pm to about 1000 Pm. The formulation may be administered directly to animals, including humans, as a concentrated powder, as a reconstituted liquid (e.g. a beverage), or it may be contained in either a flake or powder form in existing food or feed products. [0022] The dried formulation may be used directly as a flake, or milled in powder form, and sieved to an average particle size of from 10 Pm to about 1000 Pm. The formulation may be administered directly to animals, including humans, as a concentrated powder, as a reconstituted liquid (e.g. a beverage), or it may be contained in either a flake or powder form in existing food or feed products.

<a name="caption1"></a>BRIEF DESCRIPTION OF THE FIGURES [0023]

Figure 1. Visual and microscopic observations of various dry compositions comprising various matrix and glass forming agents in the form of frozen solid beads in accordance with the method of the present invention

Figure 2. Effect of an L. rhamnosus culture form appearing as fresh, frozen beads or dry powder on its initial JTK number in a dry composition.

Figure 3. Effect of freezing temperature of a composition containing L.

Rhamnosus in the form of frozen solid beads in liquid nitrogen or at -80 ° C in a freezer and in an undivided viscous slurry at + 4 ° C, for the initial bacterial amount of JTK in the dry composition. The results show only the effect of the freezing temperature on the slurry without the additional blowing step before drying.

Figure 4. Effect of freezing temperature of the composition comprising Bifidobacterium animalis Bb12 in the form of frozen solid beads in liquid nitrogen and in the form of an unfrozen viscous suspension at + 4 ° C, on the initial bacterial amount of JTK in the dry composition. The results show only the effect of the freezing temperature on the slurry without the additional blowing step before drying.

Figure 5. Influence of the duration of blowing under vacuum of frozen solid beads on the initial amount of JTK L. rhamnosus in the dry composition

Figure 6. Drying profile in a lyophilizer composition according to the method of the invention.

Figure 7. Process losses and drying of L. rhamnosus in the compositions and methods of drying according to the invention.

Figure 8. Trends in the stability of dry probiotic bacteria, L. rhamnosus composition during storage at 40 ° C and 33% relative humidity.

DETAILED DESCRIPTION OF THE INVENTION

DEFINITIONS [0024] It is to be understood that the terminology used in this document is intended to describe only specific embodiments.

[0025] As used in this specification and the appended claims, the single forms "a," "an" and "the" include plural references unless the content clearly indicates otherwise. Thus, for example, the reference to "protein" includes a single protein or a combination of two or more proteins, a reference to "enzyme", "vitamin", "bacterium", includes a single or a mixture of several, etc ..

EP 2 529 004 B1 [0026] "Bioactive material", "bioactive composition", or "bioactive formulation" refers to formulations that, being in this form, allow to be unambiguously effective biological activity of bioactive components.

[0027] A "matrix-forming agent" refers to ingredients or materials that, added to the formulation, increase the viscosity and / or density of the wet formulation or form a hydrogel. Examples of suitable matrix-forming agents include, but are not limited to, water-soluble cellulose derivatives such as methylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, and hypromellose; alginates, galactomannans, gellan gum, tragacanth, including any derivatives thereof, cellulose acetate phthalate (CAP), carboxymethylcellulose, pectin, sodium alginate, salts of alginate acid, hydroxypropylmethylcellulose (HPMC), methylcellulose, carrageenan, guar gum, gum arabic, xanthan gum , carob bean gum, chitosan and chitosan derivatives, collagen, polyglycolic acid, starch and modified starches, cyclodextrins and oligosaccharides (inulin,

[0028] "Glass-forming agent" or "sugar-glass forming agent" generally refers to compounds or materials that are readily soluble in solution and do not thicken or polymerise on contact with water. These agents are added to provide an increase in bioactive material durability during the drying process and later, or durability during long storage of the dry product. Useable sclerosing agents may be monomeric, oligomeric or polymeric.

[0029] According to one preferred embodiment, the sclerosing agent is a saccharide, a saccharide or a carbohydrate, is defined as a compound composed mainly of carbon, hydrogen and oxygen. Useful saccharides include reducing and non-reducing sugars and sugar alcohols, oligosaccharides, water-soluble polysaccharides and their derivatives. Preferred saccharides according to the invention include glucose, fructose, lactose, sucrose, trehalose, maltose, cellobiose, galactose, maltotriose, raffinose, dextrin, dextran, inulin, mannitol, sorbitol, xylitol. Glucose and trehalose are particularly preferred saccharides.

[0030] Other useful sclerosing agents may be selected from other chemical classes, such as water-soluble amino acids, peptides, proteins and hydrolysed proteins. For example, lysine, glycine, alanine, arginine or histidine, as well as hydrophobic amino acids (tryptophan, tyrosine, leucine, phenylalanine, etc.), methylamine such as betaine. Useful proteins include gelatin, egg albumin, egg white, whey protein, caseinate, immunoglobulins, soy protein, pea protein, cotton seed protein or other food, dairy or vegetable proteins.

[0031] "Hydrolysed proteins" generally refer to proteins from both animal and dairy or vegetable sources that have been broken down by enzymatic hydrolysis into shorter peptide fragments and / or amino acids. Useful hydrolysed proteins are those subjected to an accurate hydrolysis process that reduces 99% of the molecular weight of native proteins to less than 50,000 Daltons, preferably to less than 10,000 Daltons.

[0032] Peaceful "ambient" temperatures or conditions are conditions at a given moment in and in a given environment. Usually, the room ambient temperature is 22-25 ° C, atmospheric ambient pressure and ambient humidity are easily measured and can vary depending on the season, weather and climatic conditions, altitude, etc.

[0033] "Blowing" or "Degassing" in the context of the present invention relates to the release of gas from a solid or liquid formulation where the gas partial pressure is higher than the applied pressure. This is not boiling the solution in liquid form, and it can sometimes occur at a pressure above the pressure at which the solution can boil.

[0034] "Boiling" refers to the rapid phase transition from liquid to gas which takes place when the liquid temperature is above its boiling point. The boiling point is the temperature at which the vapor pressure of the liquid is the same as the pressure used. Boiling can be particularly vigorous when the heat is delivered to a liquid that has just reached the boiling point.

[0035] "Water activity" or "Aw" in the context of a dry formulation formulation relates to the availability of water and represents the state of the water energy in the system. This is defined as the water vapor pressure above the sample divided by pressure

EP 2 529 004 B1 water vapor over clean water at the same temperature. Pure distilled water has a water activity of exactly one or Aw = 1.0.

[0036] "Relative humidity" or "WW" in the context of storage stability refers to the amount of water vapor in the air at a maintained temperature. Relative humidity is sometimes less than required for saturation of air, expressed in percentage of saturation humidity.

[0037] "Dry" and its variants relate to the physical state, i.e. lyophilized, dehydrated, anhydrous, i.e. practically no liquid. Drying includes, for example, spray drying, fluidized bed drying, freeze drying and vacuum drying.

[0038] "Lyophilization" or "Freeze-drying" refers to the preparation of the composition in a dry form by rapid freezing and dehydration in a frozen state (sometimes referred to as sublimation). This process can be carried out under vacuum at a pressure sufficient to keep the frozen product, preferably less than about &lt;2,000 mTORR.

[0039] "Basic drying" or "Drying of liquids" refers to the processes described in this document, which relates to a dewatering drying that takes place from the thawing of the frozen particles to the point at which the secondary drying commences. Usually, all of the basic drying is carried out by thorough evaporation, while the product temperature remains significantly lower than the heat source temperature. This process can be carried out under vacuum at a pressure sufficient to maintain the thawed product, preferably higher than about&gt; 2,000 mTORR.

[0040] The "secondary drying" with reference to the described processes in this document relates to a drying step that takes place at temperatures above the freezing temperatures of the formulation and close to the temperature of the heat source. This process can be carried out under vacuum at a pressure suitable for reducing the water activity of the formulation, preferably less than about &lt;1000 mTORR. In the usual drying process of the formulation, the secondary drying step reduces the water activity of the formulation to Aw of 0.3 or less.

[0041] "Foam formation" refers to a procedure for drying sensitive biological particles by boiling in a vacuum under conditions in which the particles

The biological remains active or viable over a prolonged period of time at ambient and higher temperatures. The specific procedure for creating a mechanically stable porous structure proceeds in two stages; (1) Basic Drying-Foam and boiling in vacuum (2) Fixative Drying / Glazing and are disclosed in US Patent No. 5, 766, 520 Bronshtein.

[0042] A "stable" formulation or composition is one in which the biologically active material substantially retains in it physical stability, chemical stability, and / or biological activity during storage. The durability can be measured at the selected temperature and humidity conditions for a selected period of time. Trend analysis can be used to determine the expected expiration date before the material is actually stored for this period of time. For live bacteria, for example, durability is defined as the time at which it loses 1 log JTK / g dry formulation under specific temperature, humidity and time conditions.

[0043] "Viability" with respect to bacteria, refers to colony forming ability (JTK or Colony Forming Unit) on a nutrient medium suitable for bacterial growth. Viral viability refers to the ability to infect and multiply in suitable host cells, resulting in platelet formation on host cells grown in the form of turf.

[0044] The compositions and methods of the present invention solve the problem of providing low cost and industrially scalable drying processes for formulations containing sensitive bioactive materials, such as peptides, proteins, enzymes, hormones, vitamins, carotenoids, minerals, drugs, antibiotics, microbicides, fungicides, herbicides, insecticides, spermicides, nucleic acids, antibodies, vaccines, bacterial, viral and / or cell suspensions, with a significantly extended shelf life in the dry state.

[0045] The invention provides a composition comprising a bioactive material and matrix- and glass forming agents in a solution mixture and a drying method comprising rapidly freezing said composition in liquid nitrogen to form an amorphous solid structure in the form of droplets, fibers, spheres and

The microfiltration and blowing of the frozen particles under high vacuum followed by fixing the bioactive material in the sugar glass formed by lyophilizing or evaporating moisture under a reduced pressure regime during the heat delivery to the composition.

[0046] Most loss of viability of microorganisms during drying processes can be attributed to a combination of ice crystals, high osmotic and oxidizing pressures, shear forces and energy release during cavitation of the bubbles associated with "boiling" and foaming of the composition at low drying and high temperature pressures. The present invention avoids such adverse effects and provides compositions and drying methods with minimal losses resulting in the formation of a protected bioactive material in the sugar glass matrix for difficult further storage and use conditions.

COMPOSITIONS OF THE INVENTION [0047] The present invention encompasses compositions of a bioactive material, a matrix forming agent and a sclerosing agent in a viscous solution. It has been found that the formulations of the invention are properly different in their physical structure and functions from non-sticky or concentrated formulations that have been dried with or without rapid freezing and purging. For example, the formulations known in the prior art were pre-foamed by boiling to facilitate effective drying. The foaming step generally results in a large boiling and ejection of the solution, which is an inevitable consequence of the liquid-state drying and as a result only a very low loading capacity of the material in the vial or tank can be achieved. (see, e.g., US Pat. No. 6,534,087, in which the thickness of the final foamed product is less than 2 mm).

[0048] The compositions and methods of drying according to the present invention avoid boiling and extensive foaming of the formulation therefore they allow much more material to be loaded onto the drying surface and, as a result, can be easily scaled to produce large quantities of material without the use of specially designed tanks, trays or equipment.

[0049] Probiotic bacteria have been shown to benefit particularly from the formulations and drying methods of the present invention. The formulation is prepared in accordance with the compositions and methods of the invention including mixing fresh, frozen or dry probiotic bacterial cultures with at least one matrix forming agent and at least two glazing agents, rapidly freezing a viscous liquid nitrogen formulation to form an amorphous structure of frozen solid drops, fibers or balls. During basic drying, sufficient vacuum pressure is used to blow and fix the structure of the frozen particles and then the frozen particles are lyophilized or evaporated under reduced vacuum and elevated temperatures above the freezing point of the formulation. Maintaining the formulation temperature above the freezing point can be achieved by removing heat by conduction from the formulation, and / or loss of latent heat due to evaporation of water. In order to complete the drying process and to further reduce the water activity of the formulation, a secondary drying step may be applied at a higher vacuum pressure of 1000 mTORR and below and at an increased temperature of 70 ° C to provide a final composition with Aw water activity of 0.3 or less. . Such a composition may remain stable under storage conditions of 40 ° C and 33% RH for 30 days or more, as shown in Figure 8. In order to complete the drying process and to further reduce the water activity of the formulation, a secondary drying step may be applied at a higher vacuum pressure of 1000 mTORR and below and at an increased temperature of 70 ° C to provide a final composition with Aw water activity of 0.3 or less. . Such a composition may remain stable under storage conditions of 40 ° C and 33% RH for 30 days or more, as shown in Figure 8. In order to complete the drying process and to further reduce the water activity of the formulation, a secondary drying step may be applied at a higher vacuum pressure of 1000 mTORR and below and at an increased temperature of 70 ° C to provide a final composition with Aw water activity of 0.3 or less. . Such a composition may remain stable under storage conditions of 40 ° C and 33% RH for 30 days or more, as shown in Figure 8.

Preparation of the Composition [0050] The materials mixed in a solution with favorable bioactivity for the preparation of dry powder compositions according to the invention comprise at least one matrix forming agent and at least two sclerosing agents. Such materials, when mixed with a beneficial bioactive material, form beads, microspheres, fibers or drops in liquid nitrogen and can be effectively lyophilized or dehydrated in an amorphous glass form according to the method of the invention and provide a large amount of stable dry compositions for storing and administering said bioactive material (see Figures 1A &amp; B with visual and microscopic ones)

EP 2 529 004 B1 for observations and water activity (Aw) of different formulas after drying). The matrix-forming agent provides structural stability of the formulation, improves the drying profile and / or physical and chemical benefits by protecting bioactive materials. The matrix-forming agent also provides an increase in the viscosity of the formulation and thus a better control of the formulation properties under vacuum pressure and enhances the structural strength of the dry compositions of the invention (see Figure 1B Figures 4, 4b, 4c with vitreous structures and dryness of these particular formulations). The matrix-forming agent comprises a mixture of polysaccharides and oligosaccharides. Preferred polysaccharides, especially for living organisms, are water-soluble gums, because their specific feature is the formation of viscous gel at mild temperatures.

[0051] In particular, when looking at Figures 1A in combination with the results of the fourth set shown in Table 1 below, it is evident that samples 3b, 3c, 4, 5 and 6 all have dried enough to provide some porous and amorphous glassy structures.

Table 1

<td></td><td></td><td></td><td colspan="6">Visual Evaluation of Different Dry Compositions</td><td></td>

<td></td><td></td><td>1</td><td>2</td><td>3a</td><td>3b</td><td>3c</td><td>4</td><td>5</td><td>6</td>

<td></td><td>Dryness</td><td>Niesucha</td><td>Niesucha</td><td>Niesucha</td><td>Dry</td><td>Dry</td><td>Dry</td><td>Dry</td><td>Dry</td>

<td></td><td>Porosity</td><td>Lack</td><td>Lack</td><td>Lack</td><td>Present</td><td>Present</td><td>Present</td><td>Lack</td><td>Partial</td>

<td></td><td>and</td><td>0847</td><td>0.923</td><td>0916</td><td>0216</td><td>0.183</td><td>0376</td><td>0.171</td><td>0.112</td>

<td></td><td>glassy structure</td><td>Lack</td><td>Lack</td><td>Lack</td><td>Present</td><td>Present</td><td>Present</td><td>Partial</td><td>Partial</td>

[0052] Schemi of the invention may include various sugars, non-reducing sugars, sugar alcohols, amino acids, proteins, hydrolysed proteins and peptides. The glass forming compound is preferably one of those non-crystallizing and / or destabilizing the biological activity of the formulation material at freezing temperatures (e.g., less than -20 ° C). For example, a bioactive material can be physically embedded in amorphous

A sugar glassy structure such as sucrose or trehalose to stimulate the retention of the molecular structure during the drying process and to reinforce the structural stiffness of the amorphous matrix in a dry state. The scleractic agent replaces the hydration water lost during drying to prevent damage to cell membranes and enzyme denaturation (see review by Crowe et al., 1998). Other functions of glass-forming agents may include the protection of bioactive materials from damage caused by exposure to light, oxygen, oxidizing agents and moisture. Most glass-forming agents must have been easily soluble in solution in an amount ranging from about 0.1 weight percent to about 80 weight percent. It is advantageous to cover two or more different glass-forming agents,

[0053] Pre-dried formulations include a significant amount of total solids (components without a solvent such as water). The main part of total solids consists of bioactive material, matrix forming agent and glass forming agents. For example, the bioactive material is present in the formulation at concentrations ranging from about 5-60 weight percent, the matrix forming agent from about 1-20 weight percent, and the glass forming agent from about 5-80 weight percent. In another example, the matrix-forming agent may be present in the formulation at concentrations ranging from about 0.5-10 percent by weight, and an oil-forming agent from about 10-50 percent by weight. Preferably, the wet formulation should have a solids content between about 5% and 80%, more preferably between 30% and 60%. The viscosity of the formulations according to the invention is usually higher than 1000 centipoise (cP), more preferably more than 5000 cP and most preferably more than 10,000 cP. The density of the formulations according to the invention is preferably between 0.9 and 1.2 g / ml.

METHODS FOR PREPARING PERMANENT DRY FORMULATION [0054] Methods for preparing stable dry formulations containing

EP 2 529 004 B1 1 bioactive materials include; (1) preparing the formulation by mixing the bioactive material with matrix forming agents and glass in solution, (2) quick freezing of the formulation to form solid frozen particles, (3) subjecting the frozen particles to high vacuum pressure for a short time to blow out particles and consolidate the structure , (4) removing water by lyophilizing and / or evaporating moisture under reduced pressure during which heat is delivered to the formulation to a temperature above the freezing point of the formulation, preferably above -10 ° C; (5) then reducing the water activity of the formulation to less than 0.3 Aw under full vacuum and at elevated temperature.

[0055] In one embodiment, for example, the formulations of the invention include bioactive materials contained in a solution or suspension comprising matrix and glass forming agents. The matrix forming agent and / or glass forming agents in high concentration are dissolved and disinfected in a hot solution under stirring before cooling and mixing with the bioactive material. The bioactive material, such as cultured viruses or bacteria, is concentrated and isolated from the culture medium by centrifugation and filtration before re-suspension in the formulation.

[0056] In one embodiment of the present invention, the total amount of water in the formulation is provided from the liquid of the concentrated living organisms and the suspension of living organisms is maintained at a temperature just above room temperature. The dry ingredients are mixed together and then slowly added to a warm (25 ° C to 40 ° C) suspension of living organisms. The formulation slurry is vigorously stirred in a planetary mixer until all ingredients are dispersed and a homogeneous suspension is obtained.

[0057] The viscous solution is then rapidly frozen by atomization, dropping or extrusion into a liquid nitrogen bath to form small solid drops, fibers or beads. Frozen particles can be stored in the freezer between -30 ° C and -80 ° C for drying or directly placed on trays and freeze-dried or dried according to the methods of the invention. Solid particles are blown for a short time, usually

Between 2 and 20 minutes, under a sufficient vacuum (e.g., &lt;2,000 mTORR). Generally, the particles remain in a solid frozen form at a temperature below -20 ° C during the blowing step. After the initial blowing step, the vacuum pressure is raised to between 2000 and 10000 mTORR and the heat can be delivered allowing the formulation temperature to rapidly rise to between -5 ° C and + 5 ° C and the particles begin to thaw. When the formulation temperature reaches the desired temperature, the heat is adjusted to maintain this temperature and the basic drying step proceeds. At this stage, the formulation is already thawed and the accelerated evaporation of water proceeds without any boiling or foaming.

[0058] Typical methods in the prior art include extensive foaming and / or spattering and rapid boiling, which can damage sensitive biological drugs and cause difficulties on an industrial scale at high load capacities (see, e.g., US Patent No. 6,534,087, where vacuum pressure is applied resulting in vigorous boiling and foaming), while the current compositions and methods prevent any boiling and excessive foaming of the formulation while achieving significantly faster drying rates and allow high loading capacities of the formulation. In addition, complete and effective degassing of the viscous liquid suspension is difficult and may require an extended period of time. All these difficulties have been solved in the present invention by using a suitable composition, which allows effective basic drying of the liquid that creates amorphous glassy creations without any boiling or excessive foaming. Unexpected and important was mainly that this was achieved by quick freezing of the respective composition and before introducing a short purging step before starting the basic drying step of the liquid. Loading solid frozen particles into a tray, in contrast to a slurry or viscous syrup, allows a considerably higher payload per unit area than was achieved in accordance with the prior art. After completing the basic drying step, the stable amorphous glassy formulation is maintained at elevated secondary drying temperatures (between 20 ° C and 70 ° C) and a vacuum pressure of less than 1000 mTORR

EP 2 529 004 B1 to a reduction of water activity in a short period of time.

[0059] Another embodiment of the invention provides methods for preparing a hydrogel formulation of a composition for protecting bioactive materials. For example, a formulation containing a bioactive material and matrix and glass forming agents are mixed in solution and crosslinked to form dense hydrogel particles by atomization or extrusion into a divalent metal bath or the addition of divalent metal ions directly to the slurry and slicing the cured hydrogel sheet into small fibers or pieces . The hydrogel particles are then dried according to the drying methods of the invention as described below.

[0060] In one particular embodiment of the invention, for example, the formulation contains live probiotic bacteria in a solution of 1-4% sodium alginate and 10-40% trehalose. Proteins and especially hydrolysed proteins such as casein, whey, pea, soy or cottonseed are added to the formulation to 5-10% to improve the drying process and formation of an amorphous vitreous solid structure of the formulation (see Figure 1A, Figures 4,5 and 6 ). Probiotic cultures can be fresh, frozen or already dried as a dry powder (see Figure 2 with the amount of JTK of different probiotic bacterial culture forms in a dry formulation) The solution is stirred at just above room temperature (usually between 25 ° C-37 ° C) until all the ingredients are completely dissolved. The slurry formulation is atomized,

[0061] A typical method of drying live probiotic bacteria includes; spreading frozen solids on a tray into a homogeneous layer at a load between 100-1000 g / square foot, then the trays are placed directly in the freeze dryer. The vacuum pressure is then set to approximately 1000 mTORR and depending on the size of the freeze dryer and the type of heat source, the shelf temperature is set to + 20 ° C or to a temperature sufficient to keep the particles at -20 ° C. Solid frozen

The beads are left to blow on the wheel for 5-30 minutes and the vacuum is set at between 2000 and 10000 mTORR and the heat transfer increases to raise the formulation temperature to between -5 ° C and + 5 ° C. These vacuum temperature and pressure conditions are maintained during the basic drying step of the liquid, which can last from several up to 24 hours depending on the load on the tray, preferably from about 3 to 10 hours. At some point during the basic drying process, the solvent evaporation rate drops and the formulation temperature begins to rise due to excessive heat supply in the drying chamber. This point indicates the end of the basic drying step. Due to the solvent being removed from the formulation,

[0062] Next, a secondary drying step is carried out at full vacuum and 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 to provide a composition that is stable in storage for an extended period of time at ambient temperatures. The secondary drying step may take several hours and its end point is when the formulation is completely dry and its water activity is less than 0.3 Aw.

[0063] The drying processes according to the invention give a biologically active material that is encapsulated in an amorphous vitreous matrix, therefore, protects the unfolded proteins and significantly reduces molecular interactions or cross-reactions, resulting in reduced mobility of the compounds and other molecules in the amorphous glassy composition. As long as the amorphous solid is at a temperature below its glass transition temperature, the residual moisture remains sufficiently low (e.g., below Aw equal to 0.3), the bioactive material remains sufficiently stable (see Figure 8). It should be noted that achieving a glassy state is not a prerequisite for long-term durability, because some bioactive components may feel better in a more crystalline state.

EP 2 529 004B1

Preparation of Dry Powder [0064] The dry formulation can be used as a whole, cut into desired shapes and sizes or crushed and ground into a free flowing powder that provides easy further processing such as wet or dry agglomeration, granulation, tableting, packing, pellet formation or mixing with food or feed products or any other type of delivery process. Crushing, grinding, pulping or powdering processes are well known in the art. For example, a hammer mill, air mill, impact mill, jet mill, circular mill, Wiley mill, or similar grinding equipment may be used. Preferably, the size of the milled particles is less than 1000 Pm and more preferably less than 500 Pm.

[0065] The compositions and methods described herein protect the biological activity of closed biologically active materials. For example, the stability of the composition is examined by subjecting them to elevated temperature (e.g., 40 ° C) and high humidity (e.g., 33% RH) and measuring the biological activity of the formulation. As an example for live probiotic bacteria, the results of these studies show that the bacteria contained in such formulations are stable for at least 20 days (see Figure 8). Durability is defined as the time in which one log JTK / g loss of activity is lost. Such formulations are stable when high concentrations of biologically active materials are used. Therefore, such formulations are advantageous because they can be transported and stored at room temperatures or above for extended periods of time.

EXAMPLES [0066] EXAMPLE 1 [0067] Preparation of dry and stable probiotic substances

Basic formulation [0068] 75 g of trehalose (Cargill Minneapolis, MN) and 22 g of exactly hydrolyzed casein (Marcor, Carlstadt, NJ) were mixed to obtain homogeneity from 3 g of sodium alginate (ISP Corp., Wayne, NJ) in a dry form. Fresh Lactobacillus acidophilus concentrate (100 ml containing at least 10% solids, harvested directly from fermentation) was added to

The mixer was stirred and kept at 35 ° C. A dry blend of gum, sugar and hydrolyzed protein was slowly added to the probiotic cultures and mixing was carried out at 35 ° C for 10 minutes. The viscous slurry was then transferred to a mixer containing perforated and allowing to drip into a nitrogen-containing bath. The beads were then removed from liquid nitrogen and directly transferred for drying.

Drying the frozen beads of the basic formulation [0069] Frozen balls were evenly distributed on a tray with a load capacity

100 g / square foot and directly placed on the shelf in a lyophilizer (Model 25 SRC, Virtis, Gardiner, NY). The vacuum pressure was then set to 1000 mTORR and the solid frozen beads were allowed to blow for 10 minutes. The vacuum was then set to 2,700 mTORR and the shelf temperature was raised to + 30 ° C. This temperature and vacuum pressure were maintained for 3 hours. The secondary drying step was then carried out at maximum 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 as measured by Hygropalm Aw1 (Rotonic Instrument Corp., Huntington, NY.) Was Aw = 0.23. EXAMPLE 2

A stable dry composition containing probiotic bacteria Lactobacillus rhamnosus LGG [0070] Lactobacillus rhamnosus LGG (500 g frozen concentrate from a commercial source) was thawed at 37 ° C in a double jacketed planetary mixer (DPM, 1qt, Ross Engineering, Inc. Savannah, GA) ). Two glass forming agents; trehalose (387 g Cargill Minneapolis, MN) and a thoroughly hydrolyzed casein (83 g, Marcor, Carlstadt, NJ) were mixed to obtain homogeneity in a dry form with two matrix forming agents; Sodium alginate (15 g, ISP Corp., Wayne, NJ) and inulin "instant" (25 g, Cargill Minneapolis, MN). The dry blend was slowly added to thawed probiotic bacteria and mixing was carried out at 40 rpm and 37 ° C for 10 minutes. The viscosity of the suspension was adjusted to 12,000 cP by adding 50-200 ml of water.

EP 2 529 004B1 of a reservoir containing perforated and allowing for dropping into a tank containing a nitrogen bottom. The beads were then removed from liquid nitrogen, placed in a sealed aluminum bag and stored in a freezer at -80 ° C for several weeks.

[0071] For drying, the frozen balls were evenly distributed in trays with capacities ranging from 100 to 500 g / square foot and the trays were placed on shelves in a freeze drier (Model 25 SRC, Virtis, Gardiner, NY). The applied vacuum pressure was 1000 mTORR and the shelf temperature was set to + 20 ° C. Solid frozen beads were allowed to blow for a short period of time ranging from 1 to 30 minutes. Then, after the blowing step, the basic drying step followed by setting the vacuum to 2,700 mTORR and raising the shelf temperature to + 30 ° C. This temperature and vacuum pressure were maintained for 12 hours. The secondary drying step was then carried out at full vacuum (150-200 mTORR) and shelf temperature maintained at 30 ° C for an additional 4 hours. The formulation was completely dry and its water activity measured was 0.23. Figure 6 shows the drying profile of the probiotic formulation.

[0072] The loss of viability after freezing the slurry at different temperatures (+ 4 ° C, - 80 ° C and -180 ° C) and after the drying process including the preparation of frozen beads, and drying in the freeze dryer is shown in Figure 3, 4 and 7. Losses the viability of the whole process was generally lower than &lt;1 log depending on the type of bacterial culture (frozen or dry cultures) and the freezing point of the viscous slurry. The results show that the rapid freezing of probiotic bacteria in liquid nitrogen (-180 ° C) was a less damaging process than freezing at -80 ° C.

[0073] Figures 5 &amp; 8 show the effect of different blowing times ranging from 0 min (no blow) to 30 minutes on the initial amount of probiotic bacteria in the dry composition and on storage stability under accelerated storage conditions of 40 ° C and 33% RH. The results indicate that the longer blowing time generally improves the initial amount of bacteria in the dry formulation, but does not affect the storage stability of the probiotic formulation.

EP 2 529 004B1

EXAMPLE 3 Trehalose (752 g, Cargill Minneapolis, MN), finely hydrolyzed pea protein (167 g, Marcor, Carlstadt, NJ), sodium alginate (30 g, ISP Corp., Wayne, NJ) and inulin "instant" 50 g, Cargill Minneapolis, MN) were homogeneously mixed in a dry form. The dry blend was slowly added to 1000 mL of hot deionized water at 80 ° C in a double jacketed planetary mixer (DPM, 1qt, Ross Engineering, Inc. Savannah, GA) and mixing was carried out at 40 rpm for 10 minutes. The temperature of the mixture was reduced to 37 ° C and 100 g of dry Lactobacillus rhamnosus LGG powder obtained from a commercial source was slowly added and stirring was continued for 20 minutes. The suspension was then extruded through a 2 mm hole of the liquid nitrogen containing bath needle. The fibers / beads were removed from liquid nitrogen placed in a sealed aluminum foil bag and stored in the freezer at -80 ° C for several weeks. For drying, frozen fibers / beads were evenly distributed in trays with a loading capacity of 100 to 500 g / square foot and trays were placed on the shelves in a freeze dryer (Model 25 SRC, Virtis, Gardiner, NY) and dried as described in Example 2. All the formulations were satisfactorily maintained on trays and no spattering or foaming was observed at all load levels. The formulation was completely dried even at higher loadings and the measured water activity was 0.26 Aw and less for all tests. the frozen fibers / beads were evenly distributed in trays with a loading capacity of 100 to 500 g / square foot and trays were placed on the shelves in a freeze dryer (Model 25 SRC, Virtis, Gardiner, NY) and dried as described in Example 2. All formulations were satisfactorily maintained on trays and no spattering or foaming was observed at all load levels. The formulation was completely dried even at higher loadings and the measured water activity was 0.26 Aw and less for all tests. the frozen fibers / beads were evenly distributed in trays with a loading capacity of 100 to 500 g / square foot and trays were placed on the shelves in a freeze dryer (Model 25 SRC, Virtis, Gardiner, NY) and dried as described in Example 2. All formulations were satisfactorily maintained on trays and no spattering or foaming was observed at all load levels. The formulation was completely dried even at higher loadings and the measured water activity was 0.26 Aw and less for all tests. All formulations were satisfactorily maintained on trays and no spattering or foaming was observed at all load levels. The formulation was completely dried even at higher loadings and the measured water activity was 0.26 Aw and less for all tests. All formulations were satisfactorily maintained on trays and no spattering or foaming was observed at all load levels. The formulation was completely dried even at higher loadings and the measured water activity was 0.26 Aw and less for all tests.

EXAMPLE 4

Preparation of a hydrogel formulation containing probiotic bacteria Bifidobacterium lactis (Bb12):

[0075] A concentrated probiotic suspension of Bifidobacterium lactis (Bb12) was prepared according to Example 1. 0.5 g of dibasic calcium phosphate was added to the basic formulation, followed by 0.5 g of gluconolactone. The suspension was allowed to cure at room temperature for another 2 hours to form a solid hydrogel. The compact gel has been cut into thin and long fibers using a commercially available one

EP 2 529 004B1 shredder / shredder. The thin fibers were quickly frozen in liquid nitrogen and loaded into trays with a capacity of 700 g / square foot and placed in a freeze dryer for drying as described in Example 2. The water activity (Aw) of the formulation was 0.05 (measured HygroPalm Aw1, Rotonic Huntington, NY ). The dry formulation was then ground to a fine powder using standard hammer mill equipment and sieved through a 50-250 micron grid.

EXAMPLE 5

An allergen-free composition containing Lactobacillus acidophilus bacteria.

Trehalose (752 g, Cargill Minneapolis, MN), finely hydrolyzed pea protein (167 g, Marcor, Carlstadt, NJ), sodium alginate (30 g, ISP Corp., Wayne, NJ) and inulin "instant" 50 g, Cargill Minneapolis, MN) were homogeneously mixed in a dry form. The dry mix was sterilized by slowly adding to 1000 ml of hot deionized water at 80 ° C in a double jacketed planetary mixer (DPM, 1qt, Ross Engineering, Inc. Savannah, GA) and mixing at 40 rpm for 10 minutes to the moment of creating a smooth and transparent suspension. The temperature of the mixture was reduced to 37 ° C and 1000 g of frozen Lactobacillus acidophilus containing beads obtained from a commercial source was slowly added and stirring was continued for 10 minutes. The suspension was then extruded through a 2 mm hole into a bath containing liquid nitrogen. The fibers / beads were then removed from liquid nitrogen placed in a sealed aluminum foil bag and stored in the freezer at -80 ° C for several weeks. For drying, frozen fibers / beads were evenly distributed in a tray with a capacity of 1000 g / square foot and trays were placed on shelves in a lyophilizer (Model 25 SRC, Virtis, Gardiner, NY) and dried as described in Example 2. Initial amount of JTK probiotic bacteria in the dry composition it was 10.53 log / g and the loss of viability after 42 days of storage under accelerated storage conditions of 40 ° C and 33% of the WW was 0.69 log JTK / g The fibers / beads were then removed from liquid nitrogen placed in a sealed aluminum foil bag and stored in the freezer at -80 ° C for several weeks. For drying, frozen fibers / beads were evenly distributed in a tray with a capacity of 1000 g / square foot and trays were placed on shelves in a lyophilizer (Model 25 SRC, Virtis, Gardiner, NY) and dried as described in Example 2. Initial amount of JTK probiotic bacteria in the dry composition it was 10.53 log / g and the loss of viability after 42 days of storage under accelerated storage conditions of 40 ° C and 33% of the WW was 0.69 log JTK / g The fibers / beads were then removed from liquid nitrogen placed in a sealed aluminum foil bag and stored in the freezer at -80 ° C for several weeks. For drying, frozen fibers / beads were evenly distributed in a tray with a capacity of 1000 g / square foot and trays were placed on shelves in a lyophilizer (Model 25 SRC, Virtis, Gardiner, NY) and dried as described in Example 2. Initial amount of JTK probiotic bacteria in the dry composition it was 10.53 log / g and the loss of viability after 42 days of storage under accelerated storage conditions of 40 ° C and 33% of the WW was 0.69 log JTK / g

EXAMPLE 6

EP 2 529 004B1

An infant formula containing a dry formulation according to the present invention:

[0077] A stable dry formulation containing Lactobacillus GG (Valio Corp.,

Finland) was prepared according to Example 2 then sieved into two fractions with a particle size (above 50 μm and 150 μm). The infant formula was prepared by mixing 99.9 g of Nutramigen (Mead Johnson, Evansville, IL) with 0.1 g of dry formulation particles in a size range between 50 μm and 150 μm). The finished product contained about 10⁸ jtk Lactobacillus GG per 100 g preparation for infants.

EXAMPLE 7

A probiotic supplement containing a stable dry formulation according to the present invention.

[0078] A stable dry composition comprising Lactobacillus acidophilus has been formulated in oral form, such as tablets, capsule-shaped or capsule-coated tablets. Orange flavored tablets containing 99.9 g of filler (dextrose) and 0.1 g of dry formulation particles in a size range between 50 μm and 150 μm were prepared by direct compression on a rotary machine using a 1/2 "round standard set of concave punch and die The final product contains about 10⁸ cfu / unit dose. The tablet hardness is in the range of 8-10 kp, and the disintegration time is approximately 20 seconds. Compressed tablets are packed into HDPE bottles with a volume of 180 cm³ 100 tablets per each and exposed to a controlled temperature / humidity of 40 ° C / 33% RH. The product is subjected to monthly microbiological stability tests for a period of 12 months or until a reduction in the amount of less than 1 × 10 is observed⁶ dose units.

EXAMPLE 8

A functional beverage comprising a stable dry formulation according to the present invention:

[0079] A dry mix was prepared containing (wt%) 71% sucrose, 14% maltodextrin, 10% inulin, 2% dextrose, 1% anhydrous citric acid, 0.3% gum arabic, 0.3% flavor, 0.3 %

And the dry probiotic particle size (L. acidophilus) in the size range between 50 Pm and 250 Pm. The final product contained about 10⁹ jtk / unit dose (30 g dry mix) The product is packed in small bags of aluminum foil (30 g unit dose / bag) for drinking it is mixed with 340 ml of water. The persistence of probiotic bacteria in a dry beverage blend has been subjected to monthly microbiological stability tests for a period of 12 months or until a reduction in the amount of less than 1 x 10 is observed⁷ unit dose.

EXAMPLE 9

Preparation of probiotic animal feed [0080] Commercially available animal feed for dogs in the form of pellets is dried in convection ovens up to a water activity of 0.1 and then coated with a stable dry formulation prepared as described in Example 3. Dry pellets are sprayed with a 5% fatty layer of a moisture barrier (a mixture of 40% chicken fat, 40% cocoa butter and 20% beeswax), mixed in a blender for driers with a dry powder formulation (usually 0.1-0.5% of the total feed for animals, which provides a dose of 10 sup.8 JTK / g), and finally sprayed with an additional layer of a barrier fat layer protecting against moisture. The total amount of coating is about 15% (pet food). The coating time is about 30 minutes.

EXAMPLE 10

Preparation of fish food with several probiotic microorganisms [0081] Pellet fish feed in accordance with the present invention is prepared with a mixture of several probiotics. A persistent dry probiotic formulation containing a mixture of L. rhamnosus, L. acidophilus and Bifidobacterium lactis is prepared as described in Example 1. A commercially available starting food for salmon (Zeigler Bros., Gardners, PA) is first dried in a convection oven to a water activity of 0 , 1, and then coated with a probiotic formulation in a mixer

EP 2 529 004B1 for barrels. The pellets (1000 g) were first sprayed with 5% by weight of the moisture barrier film (a mixture of 40% fish oil, 40% cocoa butter and 20% beeswax), then mixed with 1 g of a stable dry probiotic formulation (to achieve a dose of 10⁷ cfu / g of food), and finally sprayed with an additional coating of a barrier fat layer protecting against moisture. The total amount of coating is about 10% (food for fish).

EXAMPLE 11

A stable dry powder containing the enzyme:

[0082] A hydrogel formulation containing 40 weight percent Savinase (Novozymes, Denmark) was prepared by mixing 600g of the formulation described in Example 4 and 400g of savinase in 1000g of aqueous solution. The shredded hydrogel is quickly frozen in liquid nitrogen and dried in a vacuum oven at a drying temperature of the formulation of 50 ° C. To determine the loading capacity and durability during storage of the dry preparation: the dry sample is accurately weighed (&lt;100 mg) in a microfuge tube. 200μl of dimethyl sulfoxide was added to the test tube. The formulation was dissolved in DMSO buffer by vortexing. To this sample was added 0.8 ml of a solution containing 0.05 N NaOH, 0.5 SDS and 0.075 M citric acid (trisodium salt). The tubes were sonicated for 10 min at 45 ° C, then briefly centrifuged at 5,000 rpm for 10 min. Portions of pure DMSO / NaOH / SDS / citrate solution were added to the wells of the microplate and the protein content was analyzed using the Bradford assay. The storage stability of a stable enzyme formulation is significantly higher than that of the dry enzyme without the formulations of the present invention.

EXAMPLE 12

Persistent Dry Powder Containing Vitamin A:

[0083] A formulation containing 30 weight percent of vitamin A is prepared by mixing 320 g of "instant" inulin, 320 g of maltodextrin DE-1 (Tate &amp; Lyle, London, UK), 50 g of sodium carboxymethylcellulose (Ashland Aqualon Functional Ingredients, Wilmington, DE), 10 g

Sodium ascorbate and 300 g of crystalline vitamin A (BASF Corp., Florham Park, NJ) in 1000 g water. The wet formulation is spray dried in a Mobile-Minor spray dryer (GEA Process Engineering Inc., Columbia MD) at an inlet and outlet temperature of 180 ° C and 80 ° C, or quickly frozen in liquid nitrogen, then spread over trays of capacity 1000g / square foot and dried as described in Example 2. The composition containing Vitamin A is stable (&gt; 80%) at 40 ° C and 75% RH for 3 months. EXAMPLE 13

Preparation of carotenes in a protective formulation with improved bioavailability:

[0084] A formulation that protects and improves the bioavailability of carotenes that would otherwise be subjected to oxidation by other components in the diet during storage or after feeding the organism is prepared in accordance with the formulations and the method of the present invention. The formulation containing 6 g of water-soluble chitosan (LSK BioPartners, Inc. Salt Lake City, Utah) is dissolved in 200 g of water. 90 g of natural astaxanthin (Naturose ™, Cyanotech Corp., Kailua-Kona, HI) was added to this solution and the suspension was atomized or extruded into a bath containing 5% sodium tripolyphosphate. Hydrogel microparticles or fibers were allowed to cure at room temperature for 4 hours. The particles were removed from the crosslinking bath, washed with water and mixed with a dry mix of 90 g sucrose and 10 g of exactly hydrolysed casein. The sugar / protein loaded particles are quickly frozen and directly placed on trays with a capacity of 500 g / square foot and freeze-dried in a freeze dryer until the water activity is below 0.3. The dry formulation is then milled to the desired size distribution and packaged.

EXAMPLE 14

Preparation of Baits for Invasive Species [0085] A pellet bait specifically targeting invasive species is prepared in accordance with the present invention. 200 g of the formulation containing the pesticide as described in Example 1 was

EP 2 529 004 B1 was prepared and 200 gm of water was added to it. To this solution was added 90 gm of Rotenone and 0.5 gm of dibasic calcium phosphate, followed by 0.5 gm of gluconolactone. The suspension was allowed to cure at room temperature for 2 hours. The compact gel is cut into thin and long fibers by a slicer / shredder. Thin fibers are loaded into trays and placed in a lyophilizer. Shelves temperature is set at -30 ° C and the formulation is allowed to freeze before applying a full vacuum and raising the shelf temperature to + 60 ° C during the overnight drying. The dry formulation is ground to an appropriate size distribution for the bait size specification targeted at specific species.

EXAMPLE 15

Preparation of a pesticide protected in a water-insoluble formulation:

[0086] A protected granular formulation of a pesticide that is otherwise capable of being degraded by other ingredients in the formulation during storage or after being used in an environment is prepared with the formulation and the method of the present invention. The formulation containing 6 g of pectin and 102 g of sucrose is added to 200 g of water. 90 g of a dry formulation of a sensitive pesticide and a mixture containing 1.5 g of calcium phosphate dibasic and 0.5 g of calcium chloride are added to this solution, followed by 0.85 g of gluconolactone. The suspension was allowed to cure at room temperature for 4 hours and then cut into thin, long fibers through a shredder / shredder. Thin fibers were loaded into trays and dried in a lyophilizer to achieve a water activity of 0.1.

EXAMPLE 16

Preparation of a protected plant probiotic formulation:

[0087] A biological control agent such as Rhizobacteria is prepared in a dry composition according to Example 4. The effectiveness of the dry Rhizobacteria of the composition has been evaluated with the growth of lettuce under gnotobiotic conditions. Doses of 100 mg Rhizobacteria dry composition per plant

EP 2 529 004B1 were inoculated in a jar of sand and planted with previously germinated (24-hour) seedlings of lettuce. A dose of 5 ml of the nutrient solution of the Hoagland sterile solution was given to the plant in a jar. During each 7-day period after inoculation, the plants and attached sand are gently removed from the jar. The roots are washed with sterile phosphate buffer (pH 7.0) and root length measurements are recorded. REFERENCES [0088] The following references and all references cited herein are related by reference to all purposes herein.

References to patents:

[0089]

6.190.701 Composition and method for stable injectable liquids, March 1999, Roser et al.

6,964,771 Method for stably incorporating substances in 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 induction bubble nucleation, April 2005, Bronshtein.

7,153,472 Preserving 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 environmental temperature preservation, October 2001, Bronshtein et al. .

7381425 Preservation of bioactive materials by freeze dried foam, September 2006, Truong-le, Vu.

Other References:

[0090]

Morgan, CA, Herman, N., White, PA, Vesey, G. 2006. Preservation of

EP 2 529 004B1 micro-organisms by drying; a review. J. Microbiol. Methods. 66 (2): 18393.

Capela, P., Hay, TKC, &amp; Shah, NP 2006. Effect of cryoprotectants, prebiotics and microencapsulation on survival of probiotic organisms in yoghurt and freeze-dried yoghurt. Food Research International, 39 (3) 203-211).

Annear, 1962. The Preservation of Leptospires by Drying From the Liquid State, J. Gen. Microbiol., 27: 341-343.

Crowe, JF, Carpenter, JF and Crowe, LM 1998. THE ROLE OF VITRIFICATION IN ANHYDROBIOSIS. Annu. Rev. Physiol. 60: 73-103.

EP 2 529 004B1