dry stabilization composition in an amorphous glassy state for a bioactive material, a method to pro

专利摘要:
DRY STABILIZATION COMPOSITION IN A AMORPHUS VITREOUS STATE FOR A MATERIALBIOACTIVE, METHOD TO PRODUCE THE SUCH COMPOSITION AND PRODUCTS PREPARED WITH THE SAME. The present invention relates to dry stabilization compositions for bioactive materials that include sugars and hydrolyzed proteins. The present invention also relates to a method for producing said composition, as well as products which comprise such as a reconstituted liquid, a ground powder, a tablet, a pellet or a capsule, among others. The invention also refers to a tablet, a pill or a pellet that are produced by consolidating a bioactive material soaked in a glassy and amorphous dry composition to a method for producing the tablet, pill or pellet and the products that comprise them, such as a drink, human food, animal feed, nutraceutical agent, pharmaceutical agent, agricultural product or vaccine.
公开号:BR112014023234B1
申请号:R112014023234-2
申请日:2013-03-22
公开日:2020-12-08
发明作者:Moti Harel；Qiong Tang；Trisha Rice；Kimberly Jennings；Brian Carpenter；Roger Drewes；Elizabeth Raditsis
申请人:Advanced Bionutrition Corporation；
IPC主号:

专利说明:

CROSS REFERENCE TO RELATED REQUESTS
[001] This patent application is partly a continuation of US Patent Application 13 / 378,106, filed on March 29, 2012, which is the entry of the National Stage of International Application No. PCT / US11 / 22821, filed on 28 January 2011, which claims priority for US Provisional Application 61 / 299,315, filed on January 28, 2010. This patent application is also partly a continuation of US Patent Application 13 / 208,459, filed on August 12, 2011 , which claims priority for Provisional Order US 61 / 373,711, filed on August 13, 2010. This patent application additionally claims priority for Provisional Order US 61 / 614,994, filed on March 23, 2012, Provisional Order US 61 / 642,094, deposited on May 3, 2012, and Provisional Order US 61 / 646,337, deposited on May 13, 2012. The content of all of the above orders is hereby incorporated by reference for all purposes. BACKGROUND OF THE INVENTION
[002] The preservation of the structure and function of biological materials during long-term storage at high temperature and humidity is of fundamental importance for the food, nutraceutical and pharmaceutical industries. Sensitive biological materials such as proteins, enzymes, cells, bacteria and viruses must often be preserved for long-term storage for later use. Although many methods have been tried to stabilize biological materials under storage, many are not suitable for sensitive bioactives, such as live or attenuated bacteria and viruses. For example, traditional freeze-drying with leaf 1a / 74 cold step of this process can have undesirable effects, such as denaturation of proteins and enzymes, and can break cells.
[003] There is a need for a stabilization composition that is useful for a wide range of biological materials and that provides superior stabilization and preservation of biological materials over extended periods of time at elevated temperatures and varying degrees of moisture, as can be found. during the transport and storage of materials, while maintaining a significant amount of activity under rehydration. There is also a need to stabilize compositions that can be used in tablet forming applications without excessive loss of activity of biological materials, many of which are sensitive to the high pressures and temperatures encountered during tablet forming. SUMMARY OF THE INVENTION
[004] In one aspect, the invention provides a dry stabilizing composition for a bioactive material, including a carbohydrate component including between about 10% and 80% oligosaccharide, between about 5% and 30% disaccharide and between about 1 % and 10% polysaccharide; and a protein component including between about 0.5% and 40% hydrolyzed animal or plant proteins; based on the total weight of the composition. The composition can be combined with a bioactive material.
[005] In another aspect, the invention provides a method of producing the above composition combined with the bioactive material, including: (a) combining the bioactive material with at least the carbohydrate component and the protein component in an aqueous solvent to form a viscous paste; (b) instantaneous freezing of the paste in liquid nitrogen to form solid frozen particles, beads, droplets or chains; (c) primary drying by removing water under vacuum from the product of step (b) keeping it at a temperature above its cold temperature; and (d) secondary drying of the product from step (c) at maximum vacuum and a temperature of 20 ° C or higher for a time sufficient to reduce the water activity to below 0.3 Aw.
[006] In another aspect, the invention provides a tablet, pill or pellet made by consolidating a sensitive bioactive material embedded in a dry amorphous glassy composition including one or more sugars and one or more hydrolyzed proteins, in which the sugars include about 10% and 60% and hydrolyzed proteins include between about 1% and 40% based on the total dry weight of the composition.
[007] In yet another aspect, the invention provides a method for producing the aforementioned tablet, pill or pellet, including compacting the sensitive bioactive material embedded in the dry amorphous glassy composition, wherein the dry amorphous glassy composition is made by a process including: (a) combining a bioactive material with at least one or more sugars and one or more proteins hydrolyzed in an aqueous solvent to form a viscous paste; (b) instantaneous generation of the paste in liquid nitrogen to form solid particles, beads, droplets or frozen chains; (c) primary drying by removing water under vacuum from the product of step (b) keeping it at a temperature above its cold temperature; and (d) secondary drying of the product from step (c) at maximum vacuum and a temperature of 20 ° C or higher for a time sufficient to reduce the water activity to below 0.3 Aw. BRIEF DESCRIPTION OF THE DRAWINGS
[008] Figure 1 shows the acceleration stability of commercially available probiotic bacteria and probiotic bacteria in dry composition of the present invention.
[009] Figure 2 shows the effect of various molar ratios between the glass intensifiers and the mixture of carbohydrates on the composition under probiotic stability (L. paracasei) under accelerated storage conditions (37 ° C and 33% RH).
[0010] Figure 3 shows the effect of the composition of the current invention under stability under storage of the probiotic bacteria L. acidophilus. The stability of dry probiotic bacteria was tested under accelerated storage conditions of 24 ° C and 33% RH for 537 days.
[0011] Figure 4 shows the effect of several compounds of glass enhancers under stability under storage of the probiotic bacteria L. acidophilus. The stability of dry probiotic bacteria was tested under accelerated storage conditions of 24 ° C and 43% RH for 180 days.
[0012] Figure 5 shows the effect of various ratios of protein / sugar hydrolyzate under storage stability (35 ° C and 43% RH) of the probiotic bacteria Bifidobacterium lactis.
[0013] Figure 6 shows the pH optimization for maximum stability of L. rhamnosusprobiotic (acceleration storage conditions at 40 ° C and 33% RH for 8 weeks).
[0014] Figures 7 and 8. Visual and microscopic observations of the different dried compositions containing various matrices and glass-forming agents as a frozen solid bead according to the method of the present invention.
[0015] Figure 9. The effect of the L. rhamnosus culture form as fresh, frozen beads or dry powder cultures on their initial CFU counts in a dry composition.
[0016] Figure 10. The effect of the cold temperature of a composition containing L. rhamnosus as solid beads frozen in liquid nitrogen or intense freezer at -80 ° C and as viscous non-frozen paste at + 4 ° C in the initial bacterial CFU counts years in the dry composition. The results show only the effect of the cold temperature of the paste without an additional purging step before drying.
[0017] Figure 11. The effect of the cold temperature of a composition containing Bifidobacterium animalis as solid beads frozen in liquid nitrogen and as viscous non-frozen paste at + 4 ° C on the initial bacterial CFU counts in the dry composition. The results show only the effect of the cold temperature of the paste without the additional purging step before drying.
[0018] Figure 12. The effect of the vacuum purging duration of frozen solid beads on initial L. rhamnosus CFU counts in a dry composition.
[0019] Figure 13. The drying profile in a freeze dryer of the composition according to the method of the invention.
[0020] Figure 14. Drying process and losses of L. rhamnosus in the drying compositions and methods of the invention.
[0021] Figure 15. The stability trends of dry probiotic bacteria, composition of L. rhamnosus in storage at 40 ° C and 33% relative humidity.
[0022] Figure 16. Stability under shelf storage at 40 ° C and 43% RH for L. acidophilus sp. commonly freeze-dried or after formulating in the composition and methods of the present invention.
[0023] Figure 17. Stability under shelf storage at 40 ° C and 43% RH and 30 ° C and 60% RH of L. rhamnosus sp. commonly freeze-dried or after formulating in the composition and methods of the present invention.
[0024] Figure 18 demonstrates the effect of compression in tablet press in viability and stability under storage at 40 ° C and 43% RH of the stabilized and protected L. rhamnosusprobiotic in the composition of the present invention.
[0025] Figure 19 shows the effect of tablet formation with a mixture of multivitamins and minerals and exposure under storage at 40 ° C and 43% RH on the viability of stabilized and protected L. rhamnosusprobiotic in the composition of the present invention.
[0026] Figure 20 illustrates the effect of compression in a tablet press on the activity of the enzymes protease and lipase in a free or protected form in the composition of the present invention. The enzymes were either individually formed into tablets or mixed in equal amounts and then formed into tablets. DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS
[0027] It is to be understood that the terminology used here 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", "o" and "a" include plural referents unless clearly stated otherwise. Thus, for example, reference to "a protein" includes a single protein or a combination of two or more proteins; reference to "enzyme", "bacterium", etc., includes singular or mixtures of various types, and others.
[0028] When describing and claiming the present invention, the following terminology will be used according to the definitions set out below.
[0029] "Bioactive ingredient", "bioactive material" and "biological material" all refer to microorganisms or ingredients that allow biological activity. Bioactive materials suitable for use with the present invention include, but are not limited to, peptides, proteins, enzymes, hormones, nucleic acids, antibodies, drugs, vaccines, yeast, fungus, bacteria (probiotic or not), dirt microbes, viruses and / or cell suspensions.
[0030] "Biological composition" refers to preparations that are in such a form to allow the biological activity of the bioactive ingredients or agents to be unequivocally effective.
[0031] "Glass intensifier", "glass intensifier compound", and "glass forming agent" are used interchangeably here to denote a chemical compound with the ability to form amorphous or glassy structure below a critical temperature, the temperature of glass transition (Tg). During the formation of the glass structure, biological substances can be soaked within the glass structure. Glass intensifiers suitable for use with the present invention include, but are not limited to, organic acid salts such as lactic acid, ascorbic acid, maleic acid, oxalic acid, malonic acid, malic acid, succinic acid, citric acid , gluconic acid, glutamic acid, and others. Salts can include cations such as sodium, potassium, calcium, magnesium, phosphate and others. Other useful glass enhancers include proteins, protein hydrolysates, polypeptides and amino acids. A combination of glass-forming agents is also contemplated within a simple composition. The process used to obtain a glassy structure for the purpose of this invention is in general a solvent sublimation and / or evaporation technique. Ideally, compounds that are composed of GRAS are preferred over those that are not GRAS.
[0032] "Sugars" refer to saccharides predominantly composed of carbon, hydrogen, and oxygen. Useful saccharides include reduced and unreduced sugars and sugar alcohols and disaccharides. Two monosaccharides linked together form a disaccharide. The two monosaccharides used to form a disaccharide can be the same or different. Examples of disaccharides that can be used in the composition of the present invention include sucrose, treabil, lactose, maltose, isomaltose. Sulphated disaccharides can also be used.
[0033] "Carbohydrates" or "polyhydroxy compound" refer to saccharides predominantly composed of carbon, hydrogen, and oxygen. A saccharide typically composed of a sugar main chain of repeated structural units linked in a linear or non-linear manner, some of which contain positively or negatively charged chemical groups. Repeated units can range from two to several million. Useful saccharides include reduced or non-reduced sugars and sugar alcohols, disaccharides, oligosaccharides, water-soluble polysaccharides and derivatives thereof. Two linked monosaccharides form a disaccharide. The two monosaccharides used to form a disaccharide can be the same or different. Examples of disaccharides that can be used in the carbohydrate mixture of the present invention include sucrose, treatyl, lactose, maltose, isomaltose. Sulphated disaccharides can also be used. Small number of bound monosaccharides (typically three to twenty) forms an oligosaccharide. The monosaccharides used to form an oligosaccharide can be sugars of the same or different components. Examples of oligosaccharides suitable for use include inulin, maltodextrins, dextrans, fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), mannanoligosaccharides (MOS) and their combinations. A large number of linked monosaccharides (typically more than twenty) form a polysaccharide. The monosaccharides used to form a polysaccharide can be sugars of the same or different components. Examples of polysaccharides suitable for use include, but are not limited to, methylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, and hypellellosis; soluble starches or fractions of starch, xanthan gum, guar gum, pectins, carrageenan, galactomannan, gellan gum, including any derivatives thereof, cellulose acetate phthalate (TAMPA), carboxymethylcellulose, sodium alginate, alginic acid salts , hydroxyl propyl methyl cellulose (HPMC), acacia gum, locust bean gum, chitosan and chitosan derivatives, collagen, poly (glycolic acid), modified starches and cyclodextrins.
[0034] "Hydrolyzed protein" refers to the protein that has been subjected to partial or total acid or enzymatic hydrolysis to yield a hydrolyzed protein having a molecular weight of about 1 kDa to about 50 kDa. In some embodiments, referred to here as "extensively hydrolyzed protein", at least 20% of the protein substrate is converted into peptides having molecular masses of 200 to 2000 daltons. Hydrolyzed protein has approximately the same amino acid composition as total protein and can be obtained from any number of commercial sources. Being hypoallergenic, hydrolyzed protein can be advantageously used in a certain food for hypersensitive consumers such as infants and elders.
[0035] A "stable" formulation or composition is one in which the biologically active material essentially retains its physical stability, chemical stability, and / or biological activity under storage. Stability can be measured at selected temperature and humidity conditions for a selected period of time. Trend analysis can be used to estimate an expected shelf life before a material is actually in storage for that period of time. For live bacteria, for example, stability is defined as the time it takes to lose 1 CFU / g log of the dry formulation under predefined conditions of temperature, humidity and time period.
[0036] "Viability" with respect to bacteria, refers to the ability to form a colony (CFU 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 host cell lawn.
[0037] Temperatures or "ambient" conditions are those at any given time in a certain environment. Typically, the ambient temperature is 22-25 ° C, ambient atmospheric pressure, and ambient humidity are easily measured and will vary, depending on the season, climate and climatic conditions, altitude, etc.
[0038] "Water activity" or "Aw" in the context of dried formulation compositions refers to the availability of water and represents the energy state of water in a system. It is defined as the vapor pressure of water above a sample divided by that of pure water at the same temperature. Distilled pure water has a water activity of exactly one, that is, Aw = 1.0.
[0039] "Relative humidity" or "RH" in the context of stability under storage refer to the amount of water vapor in the air at a given temperature. Relative humidity is usually less than that required to saturate the air and expressed in percent saturation humidity.
[0040] "Drying" and its variations refer to a physical state that is dehydrated or anhydrous, that is, substantially devoid of liquid. Drying includes, for example, spray drying, fluid bed drying, lyophilization, and vacuum drying.
[0041] "Freeze drying" or freeze drying refers to the preparation of a composition in dry form by instant freezing and dehydration in the frozen state (sometimes referred to as sublimation). Freeze drying takes place at a temperature that results in the crystallization of the sugars. This process can take place under sufficient vacuum to keep the product frozen, in some embodiments, less than about <2 mm Hg (2000 mTORR).
[0042] "Primary water removal" or "primary drying" or "liquid drying" step, with respect to the processes described here, refer to the drying by dehydration that takes place from the thawing time of the frozen particles to the point where the secondary drying. Typically, the volume of the primary drying occurs by extensive evaporation, while the product temperature has remained significantly lower than the temperatures of the heat source. This process can take place under sufficient vacuum to keep the product defrosted, in some modalities, greater than about> 2 mm Hg (2000 mTORR).
[0043] "Secondary drying", with respect to the processes described here, refers to a drying step that takes place at a temperature of the formulation close to the temperature of the heat source. This process can take place under sufficient vacuum to reduce the water activity of a formulation, in some embodiments less than about <1 mm Hg (1000 mTORR). In a typical formation drying process, a secondary drying step reduces the water activity of the formulation to an Aw of 0.3 or less.
[0044] The present invention includes compositions and drying methods to preserve sensitive bioactive materials, such as peptides, proteins, hormones, nucleic acids, antibodies, drug vaccines, yeast, bacteria (probiotic or not), viruses and / or cell suspensions, under storage.
[0045] The compositions and drying methods of the present invention solve the problem of providing a cost-effective, industrial-scale dry formation containing sensitive bioactive materials, such as peptides, proteins, hormones, nucleic acids, antibodies, drugs, vaccines, yeast , bacteria, viruses and / or cell suspensions, with a significantly extended life in the dry state. The invention provides a preservation composition and a drying method comprising a biological material surrounded by an amorphous structure of highly soluble compounds. The drying process comprises: mixing the biological material and the composition in a liquid paste, said composition paste instantly frozen in liquid nitrogen to form droplets, chains or beads, followed by drying the bioactive material in a glassy formation of sugar by evaporating the moisture under a reduced pressure regime while providing heat to the composition.
[0046] The present invention is based on the remarkable discovery that biological materials can be protected in the glassy structure while retaining substantial activity. When the biological material is combined with the composition mixture and dried in accordance with the present invention, superior stability has been achieved during extended time exposure to severe temperature and humidity conditions. The present invention includes compositions containing a biological material, a mixture of soluble carbohydrates and glass-enhancing carboxylic acid salts. The compositions of the invention are inherently different in their physical structure and function from non-viscous or concentrated sugary compositions, which were simply dried under a typical freeze-drying process. For example, Pat. US 6,919,172 discloses an aerosolized powder composition for pulmonary administration containing a mixture of various carbohydrates and sodium citrate. However, the composition described in the patent lacks the additional protein compound which is essential for added stability and for the formation of a desirable physical structure during the drying of solutions having a high sugar concentration. The composition described in this patent also lacks viscosity or hydrogel structure, which allows efficient drying of the thawed or unfrozen solution for the intensified glass formation. In contrast, the composition and drying process of the present invention addressed all of these problems by achieving superior stability of the biological material. The prior art also lacks the additional carboxylic component that acts in synergism with hydrolyzed proteins to protect and stabilize biological material.
[0047] Enhanced glass structure was usually achieved in the prior art by foaming or boiling the solution under vacuum to facilitate effective drying. The foaming step in general resulted in extensive boiling and eruption of the solution which is an inevitable consequence of drying the unfrozen solution, and as a result, only very low solution loading capacity in a flask or a vessel can be achieved ( see, for example, US Pat. 6,534,087, where the thickness of the final foam product is less than 2 mm). The drying compositions and methods of the present invention prevent boiling and foaming of the formulation thus allowing much higher loading of the material per drying area and, as a result, can be easily increased to produce large quantities of material without the use of vases and specifically designed trays or equipment.
[0048] A wide range of biological materials can be used with the inventive composition to form an aqueous preservation medium according to the invention. This preservation medium can then be subjected to the drying processes of the present invention to produce a stable dry powder of biological material. These biological materials include, without limitation: enzymes, such as pancreatic enzymes, lipases, amylases, protease, phytase, lactate dehydrogenase; proteins, such as insulin; vaccines; viruses, such as adenoviruses; cells, including prokaryotic cells (including bacteria and fungi) and eukaryotic cells, other biological materials, including drugs, nucleic acids, peptides, hormones, vitamins, carotenoids, minerals, antibiotics, microbiocides, fungicides, herbicides, insecticides, spermicides , antibodies and lipid vesicles.
[0049] Probiotic bacteria have been shown to benefit particularly from the compositions and drying methods of the present invention. The stable dry probiotic powder is prepared according to the compositions and methods of the invention including mixing fresh, frozen or dried cultures of probiotic bacteria with a mixture of carbohydrates and glass intensifying compounds, instantly freezing the viscous formulation in liquid nitrogen to form frozen droplets, chains or solid beads, and dry by applying sufficient vacuum initially to raise the formulation's temperature above the cold temperature and provide a 20 ° C and higher heat source to facilitate primary water removal. Maintaining the formulation temperature above the freezing point can be accomplished by adjusting the vacuum and conducting or radiating heat to the formulation. To complete the drying process and still reduce the water activity of the formulation to below Aw 0.3 or less, a secondary drying step is applied at maximum vacuum and the temperature raised to up to 70 ° C. Such a composition can remain stable under storage conditions of 40 ° C and 33% RH for 30 days or more, as shown in Figure 15.
[0050] Living microorganisms such as probiotic bacteria in compressed tablets have been shown to benefit particularly from the compositions and drying methods of the present invention. The dry, stable biological powder is prepared according to the compositions and methods of the invention including mixing fresh, frozen or dried cultures of single-celled organisms with a mixture of sugars, hydrolyzed proteins and an antioxidant and potentially including additional amounts of polysaccharides and oligosaccharides. glass intensifying compounds and compounds, instantly freeze the viscous formulation in liquid nitrogen to form droplets, chains or frozen solid beads, which evaporate the water by initially applying sufficient vacuum to raise the formulation's temperature above its cold temperature and provide a heat source of 20 ° C and higher to facilitate the removal of primary water. Maintaining the formulation temperature above the freezing point can be accomplished by adjusting the vacuum and conducting or radiating heat to the formulation. To complete the drying process and still reduce the water activity of the formulation to below Aw 0.3 or less, a secondary drying step is applied at maximum vacuum and the temperature raised to up to 70 ° C. COMPOSITIONS OF THE INVENTION
[0051] In some embodiments, the formulation comprises a mixture of di, oligo and polysaccharide carbohydrates, in which the bioactive material is embedded. Examples of a suitable polysaccharide 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, gellan gum, guar gum, acacia gum, xanthan gum, locust bean gum, chitosan and chitosan derivatives, collagen, poly (glycolic acid), modified starches. Examples of a suitable oligosaccharide include, but are not limited to, cyclodextrins, fructans, inulin, FOS, maltodextrins, dextrans, etc .; and combinations thereof. Examples of a suitable disaccharide include, but are not limited to, lactose, trehalose, sucrose, etc. In a particular embodiment, a suitable exemplary polysaccharide is sodium alginate or ice gum. In other embodiments, the formulation comprises, in weight percent of total dry substance, 0.1 - 20% sodium alginate.
[0052] In some embodiments, the carbohydrate mixture comprises, in weight percent of total dry substance, 0, 1-10% of polysaccharides, 1-10% of oligosaccharides and 10-90% of disaccharides. In an additional embodiment, the carbohydrate mixture comprises di, oligo and polysaccharides in a 10: 0.14: 0.1-2 weight ratio, or where the disaccharide / oligosaccharide / polysaccharide weight ratio is about 10: 0.2: 0.1 to about 10: 2: 1.
[0053] In some embodiments, the disaccharide fraction in the carbohydrate mixture includes various sugars and sugar alcohols. Suitable disaccharides are those that do not crystallize and / or damage or destabilize the biologically active material in the formulation at cold temperatures (for example, less than -20 ° C) and during the removal of water. For example, the bioactive material can be dried in the presence of glass-forming sugars such as sucrose, lactose or trellis to promote retention of the molecular structure throughout the drying process and give structural rigidity to the amorphous matrix in the dry state. A suitable disaccharide would effectively replace the hydration 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 disaccharide in the composition may include protecting the bioactive material from exposure to harmful light, oxygen, oxidizing agents and moisture. A suitable disaccharide must be easily dissolved in a solution. Trealose is a particularly attractive protector because it is a non-reducing disaccharide found in living plants and organisms (eg, bacteria, fungi and invertebrates such as insects and nematodes) that remain in an inactive state during periods of drought. In some cases, it may be beneficial to include two or more different disaccharides such as a mixture of trehalose and sucrose to inhibit crystal formation, to enhance the stability of the dry bioactive material formulation under storage conditions for extended periods of time and to reduce costs.
[0054] In some embodiments, the oligosaccharide fraction in the carbohydrate mixture includes inulin, maltodextrins, dextrans, fructo-oligosaccharide (FOS), galacto-oligosaccharide (GOS), mannan-oligosaccharide (MOS) and combinations thereof. Oligosaccharides mitigate several problems associated with using trehalose alone as a protector for a variety of preserved biological materials. Although very effective in protecting biological material during dehydration and rehydration, trehalose alone does not provide stability under desirable storage as a stabilizer for extended periods of time, especially in high temperatures and / or humid environments. This problem was solved in the present invention with the addition of oligosaccharides, for example inulin, to the carbohydrate mixture.
[0055] An adequate exemplary mass ratio of the saccharides in the carbohydrate mixture is 10: 0.1 -10: 0.1-2 disaccharides / oligosaccharides / polysaccharides and, in some embodiments, where the disaccharide weight ratio / oligosaccharides / polysaccharides is from about 10: 0.2: 0.1 to about 5: 10: 1. In some embodiments, the carbohydrate mixture comprises, in weight percent of total dry substance, 10-90% disaccharides, 1-10% oligosaccharides and 0.1-10% polysaccharides. In other modalities, the mixture of carbohydrates comprises in percent by weight of total dry substance, 10-50% of disaccharides, 10-80% of oligosaccharides and 0.1-10% of polysaccharides.
[0056] In a particular embodiment, the formulation comprises a mixture of oligosaccharides. The oligosaccharide mixture mitigates several problems associated with the use of a simple oligosaccharide alone as a glass intensifying material in the composition. Although very effective in raising the glass transition temperature, oligosaccharides tend to crystallize quickly and precipitate and thus fragment the glassy amorphous structure, especially at high temperatures and / or humid environments. This problem was solved in the present invention with the addition of a mixture of oligosaccharides instead of a single type of oligosaccharide, in some embodiments a mixture of low DE fructans and dextrins. In some embodiments, the carbohydrate mixture comprises, in weight percent of total dry substance, 5-40% fructans and 5-40% low DExtrins.
[0057] A suitable composition comprises from about 0.5% to about 90% of a carbohydrate component including at least one di, oligo and polysaccharide and a protein component comprising from about 0.5% to about 40% of a hydrolyzed protein. In some embodiments, the composition comprises about 30% to about 70% of the carbohydrate component and about 1% to about 40% of a glass-enhancing component such as a protein hydrolyzed with protein and carboxylic acid, wherein the carbohydrate component comprises about 10% to 90% or about 40% to 80% of a disaccharide; about 1% to about 10% or about 5% to 10% of an oligosaccharide; and about 0.1 to about 10% or about 5% to about 10% of a polysaccharide. The composition further comprises a salt of an organic acid which is considered to be another glass-enhancing component and comprises between about 0.5% and 20% of carboxylic acid, based on the total weight of the composition.
[0058] In an additional embodiment, the composition comprises a mixture of sodium alginate and oligosaccharide in a weight ratio of 1: 1-10, or 1: 1-5, of sodium alginate / oligosaccharides.
[0059] In yet another embodiment of the present invention, the composition is cross-linked with divalent metal ions to form a firm hydrogel. In some embodiments, the crosslinked hydrogel formulation is formed by atomizing or extruding the paste in a bath containing bivalent metal ion solution or by adding bivalent metal ions directly into the paste and allowing the formulation to harden and form a hydrogel. The hydrogel formulation is then instantly frozen and dried according to the drying methods of the invention.
[0060] In other embodiments, the composition comprises significant amounts of glass intensifying compounds including salts of organic acids such as lactic acid, ascorbic acid, maleic acid, oxalic acid, malonic acid, malic acid, succinic acid, citric acid, gluconic acid, glutamic acid, and others. Salts can include cations such as sodium, potassium, calcium, magnesium, and others. Examples include sodium citrate, sodium lactate, sodium maleate, magnesium gluconate, sodium ascorbate, and others. Salts having a high glass transition temperature (Tg) and high solubility are preferred. Exemplary organic acids include citric acid and its salts (eg, sodium or potassium citrate, trisodium citrate dehydrate) and ascorbic acid and its salts ( eg sodium ascorbate, potassium ascorbate, magnesium ascorbate). For example, in some embodiments, the composition of the invention includes a mixture of di, oligo and polysaccharide carbohydrates and organic acid ions such as citric acid and / or ascorbic acid.
[0061] The amount of glass intensifiers used in the composition will vary, depending on the general composition and its intended drying storage conditions. In general, the amount of the glass-intensifying compound in the composition is higher than two (2) weight percent of total dry substance while the pH of the solution or dispersion is kept slightly alkaline (pH 7-7.5). Without being bound by theory, it is believed that the function of the glass-intensifying compound in relatively high content as described here is not only to contribute to the desirable amorphous and rigid glass structure of the resulting dry composition, but also to protect the material bi-active from exposure to harmful light, oxygen, oxidizing agents and moisture. A suitable exemplary composition comprises, in weight percent of total dry substance, 1-20% or about 2-10% of glass intensifying compound by weight of total dry substance.
[0062] Other suitable glass intensifiers that are included in the composition to also increase its stability include proteins, protein hydrolysates, polypeptides and amino acids. These include gelatin, albumin, serum protein, soy protein, casein, caseinate, immunoglobulins, soy protein, pea protein, cottonseed protein or other food and dairy or vegetable proteins and / or their hydrolysates, or any other protein hydrolyzed. Examples of poly (amino acids) include polyalanine, polyarginine, polyglycine, poly (glutamic acid) and others. Useful amino acids include lysine, glycine, alanine, arginine or histidine, as well as hydrophobic amino acids (tryptophan, tyrosine, leucine, phenylalanine, etc.) and a methylamine such as betaine.
[0063] In some embodiments, casein or pea protein or hydrolyzed casein or hydrolyzed pea proteins are used. In some embodiments, the hydrolyzed protein fraction in the composition mixture includes partially hydrolyzed or extensively hydrolyzed proteins, polypeptides and amino acids. As used herein, extensively hydrolyzed proteins are those obtained through extensive enzymatic hydrolysis using proteases for the modification (breakdown) of proteins. In some embodiments, hydrolyzed animal or vegetable proteins such as casein, whey, soy, or pea proteins, or extensively hydrolyzed casein or pea proteins. Some modalities employ extensively hydrolyzed proteins having more than 80% of short chain peptides with a molecular weight of about 1 kDa to about 50 kDa and at least 20% of the protein substrate are converted into peptides having molecular masses of 200 to 2000 daltons. Not wishing to be bound by theory, it is believed that a mixture is the result of an extensively hydrolyzed sugar and protein as described here allows for faster drying and contributes to the desirable amorphous and rigid glass structure of the resulting dry composition. A hydrolyzed enzyme protein can be prepared by methods known to those skilled in the art or can be obtained from a commercial source. A suitable exemplary composition comprises, in weight percent of total dry substance, 540% extensively hydrolyzed proteins.
[0064] An adequate total exemplary amount of protein, hydrolyzed protein or extensively hydrolyzed protein and amino acids in the dry composition is about 1% to about 40%, or about 5% to about 40%, or about 10% to about 30% of the total dry mix mass.
[0065] It should be noted that the appropriate amount of glass intensifiers in the composition may depend on the desired characteristics of the dry composition. For example, a composition containing a mixture of carbohydrate and protein or protein hydrolysates can be used to enhance the chemical stability of a biological material while being stored under moderate temperature and relative humidity, such as 25 ° C and 25% RH. The determination of the appropriate amount of glass intensifiers, and particularly the relative ratio of disaccharides to oligosaccharides, should be done according to the desired storage conditions. For example, a composition containing a high disaccharide / oligosaccharide ratio can be used to enhance the chemical stability of a biological material being stored under moderate temperature and relative humidity, such as 25 ° C and 25% RH. A composition containing a low disaccharide / oligosaccharide ratio can be used to enhance the chemical stability of a biological material being stored under high temperature and relative humidity, such as 30 ° C and 40% RH or above.
[0066] Ascorbic acid ions may be preferred in some modalities such as glass intensifiers to obtain the added benefit of stabilization at a higher temperature and exposure to moisture. Alternatively, in some embodiments, a combination of citrate and / or ascorbate ions with another glass enhancer, such as protein or protein hydrolyzate, is more preferred.
[0067] In some modalities, the formulation comprises a mixture of sugars and hydrolyzed proteins, in which the bioactive material is soaked. Examples of suitable sugars include, but are not limited to, disaccharides such as lactose, trehalose, sucrose and a mixture thereof. Examples of suitable hydrolyzed proteins include, but are not limited to, extensively hydrolyzed gelatin, albumin, serum protein, soy protein, casein, caseinate, immunoglobulins, soy protein, pea protein, cottonseed protein or any other protein extensively hydrolyzed from dairy, of animal or vegetable origin and a mixture thereof. A suitable exemplary total amount of sugars in the dry composition is about 10% to about 80% of the total dry mass, or about 10% to about 60% of the dry mass.
[0068] In an exemplary embodiment, the glass-forming agent comprises a mixture of a disaccharide and a hydrolyzed protein. In a particular embodiment, a suitable exemplary glass-forming agent is a mixture of trehalose and hydrolyzed protein. In some embodiments, the formulation comprises, in weight percent of total dry substance, 10-90% trehalose and 0.1-30% hydrolyzed protein, or 20-80% trehalose and 0.1-20% hydrolyzed protein, or 40-80% trehalose and 0.1-20% hydrolyzed protein.
[0069] Ideally, compounds that are compounds that are generally recognized as safe (GRAS) are preferred over those that are not GRAS. Others include an excipient salt such as magnesium sulfate; a polyol such as higher trihydric or sugar alcohols, (for example glycerin, erythritol, glycerol, arabitol, xylitol, sorbitol, and mannitol); propylene glycol; polyethylene glycol; pluronic; surfactants; and combinations thereof.
[0070] In some embodiments, the biological 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 Bifidobacterium, Clostridium genera , Fusobacterium, Melissococcus, Propionibacterium, Streptococcus, Enterococcus, Lactococcus, Kocuriaw, Staphylococcus, Peptostrepococcus, Bacillus, Pediococcus, Micrococcus, Leuconostoc, Weisella, Aerococcus, Oenococcus and Oenococcus. 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. licheniformis, B. mesentericus, B. pumilus, B subtilis, B. natto, Bacteroides amylophilus, Bac. capillosus, Bac. ruminocola, Bac. suis, Bifidobacterium adolescentis, B. animalis, B. breve, B. bifidum, B. infantil, B. lactis, B. longum, B. pseudolongum, B. thermophilum, Candida pintolepesii, Clostridium butyricum, Enterococcus cremoris, E. di- acetylactis, E faecium, E. intermedidas, E. lactis, E. muntdi, E. thermophilus, Escherichia coli, Kluyveromyces fragilis, Lactobacillus acidophilus, L. alimentarius, L. amylovorus, L. crispatus, L. brevis, L. Case, L. curvatus, L. cellobiosus, L. delbrueckii ss. bulgaricus, L farciminis, L. fermentum, L. gasseri, L. helveticus, L. lactis, L. plantarum, L. johnsonii, L. reuteri, L. rhamnosus, L. sakei, L. salivarius, Leuconostoc mesenleroides, P. cereviseae (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. METHODS OF MAKING COMPOSITIONS
[0071] A suitable process for mixing biological material and composition is to add the total dry composition mixture to a concentrated culture or media solution containing the biological material. The weight by weight of biological material in the culture media is typically between about 5% and 30% w / v, or between about 10% and 20% w / v. The weight by weight added of the composition mixture in the culture media is typically between about 10% and about 60%, or between about 20% and 40%. The final solid content in the mixed paste is about 20% to about 60% and more specifically about 30% to about 50%. In some embodiments, the solution is mixed at room temperature or slightly heated to help solubilize the materials in the viscous solution (for example, from 20 ° C to 40 ° C). In a variation of the present invention, the total amount of the carbohydrate mixture in the formulation is adjusted to achieve a desired formulation viscosity and density that allowed for efficient drying avoiding excessive rubber or foam formation that may occur during the drying step. A suitable exemplary paste viscosity is about 1,000 cP to about 500,000 cP, or about 5,000 cP to about 300,000 cP. A desired viscosity and density of the final paste can be achieved by any means known in the art, for example, by slightly adjusting the amount of polysaccharides in the carbohydrate mixture or by degassing or injecting gas such as air, nitrogen, carbon dioxide, argon etc.
[0072] The biological material slurry of the present invention is typically instantly frozen to between -30 ° C to -180 ° C, or the formulation is instantly frozen in liquid nitrogen by atomization, dripping or injection in a liquid nitrogen bath. Collect the particles, beads, chains or droplets from the liquid nitrogen bath and dry in a freeze dryer or vacuum dryer, or alternatively store them in an intense freezer (between -30 ° C and -80 ° C) for use afterwards in a frozen form or for later drying, for example, by spray drying.
[0073] In general, drying process techniques that are useful include spray drying; or evaporative drying of an unfrozen solution in a vacuum oven or centrifugal evaporator at a temperature above the cold temperature of the slurry (-20 to 50 ° C), followed by grinding to the desired particle size. The resulting powder particles are vitreous with most of the vitreous materials coating the biological material. The advantage of coating the biological material with glassy materials is to increase the physical stability of the product and to reduce harmful intermolecular reactions within the particle. In a suitable exemplary embodiment, the frozen particles are loaded into trays and immediately transferred to a vacuum drying chamber where the drying process proceeds in three main steps including: (1) An optional short purging and water stabilization step frozen particle structure under a vacuum pressure of less than <2 mm Hg (2000 mTORR), (2) Primary drying step under vacuum of more than> 2 mm Hg (2000 mTORR) and at a temperature above the point freezing of the paste, and (3) Secondary and final drying step of the amorphous vitreous material under full vacuum pressure and high temperature for a time sufficient to reduce the water activity of the dried formulation to 0.3 Aw or less.
[0074] In a particular embodiment of the present invention, the dried formulation is granulated with a mixture of melted fats to obtain enhanced preservation in short periods of exposure to extreme conditions of temperature and humidity.
[0075] The dry and stable biological composition can be used directly as a flake, or ground into a powder and sieved for an average particle size of about 10 μm to about 1000 μm. The formulation can be administered directly to an animal, including man, as a concentrated powder, as a reconstituted liquid, (for example, a drink), or it can be incorporated into a flake or powder form in an existing animal feed product or human or agricultural.
[0076] In some embodiments, compositions for the preparation of frozen or dry stable powder of biological materials according to the invention include a mixture of carbohydrate and glass intensifier. Such materials, when mixed with the bioactive material, form beads, chains and droplets in liquid nitrogen and can be dried efficiently in an amorphous glassy structure according to the methods of the invention and to provide large quantities of stable dry compositions for storage and administration of the said bioactive material. (See Figures 7 and 8 for visual and microscopic observations and the water activity (Aw) of the different formulations after drying). The carbohydrate mixture provides structural stability to the formulation at high temperature and humidity such as above 30 ° C and 40% RH and / or physical and chemical protective benefits for bioactive materials and prevents or reduces adverse effects upon reconstitution or rehydration.
[0077] The polysaccharide fraction in the carbohydrate mixture can provide thick viscosity to the formulation and better control on the density properties of the vacuum formulation and increased structural resistance to the dried formulation compositions of the invention. (See Figure 8 - Images 4, 4b, 4c for the glassy and dry structure of that particular formulation). Suitable polysaccharides, particularly for living organisms, are water-soluble gums, because of their distinctive characteristic for forming viscous gel at moderate temperatures. Gums at a certain concentration have also been found to stabilize the formulation's structure effectively under vacuum, providing the formulation with appropriate viscosity and density and allowing the formulation to dry effectively during the primary water removal step at a particular viscosity. Certain gums can also form hydrogels by crosslinking with bivalent or multivalent cations (eg alginates, pectins, chitosan) or through changes in temperature or pH (eg gelatins, CMC, CAP, gellan gum). Hydrogel-treated solutions would prevent the problems associated with vacuum drying non-frozen solutions. Gums in a certain concentration have also been found to stabilize the formulation effectively and facilitate the formation of an amorphous glassy structure and to intensify the vacuum drying profile (see Figure 7 - images 3a, 3b, 3c, 4, and Figure 8 - 4c and Figure 13 ).
[0078] Notably seeing the images in Figure 7 in combination with the results set out below in Table 1, it is evident that samples 3b, 3c, 4, 5, and 6 were all sufficiently dried to provide some porosity in the amorphous glass structures. Table 1

[0079] For example, a dry form of bioactive material can be formulated into a solution or suspension containing the powder mixture of the composition. The composition mixture can be dissolved in a warm aqueous solution with low shear stirring before cooling and mixing with the bioactive material. Bioactive material, such as cultured viruses or bacteria, can be concentrated and separated from the culture media by centrifugation or filtration before resuspension in the formulation. Alternatively, all or a portion of the water in the formulation is supplied in the liquid of the concentrated biological material. The suspension is maintained at a temperature slightly above room temperature and the powder mixture of the dry composition is slowly added to the warm suspension (25 ° C to 40 ° C) containing the biological material. The suspension is gently agitated in a planetary mixer until all components are completely dispersed or dissolved and a uniform paste is obtained.
[0080] The viscous paste can then be cross-linked to form a hydrogel (depending on the properties of the polysaccharide) by adding metal ions or changing the temperature or pH of the paste and then dried according to the drying methods of the invention. Alternatively, the paste can be instantly frozen by atomizing through a spout, dripping or injecting in dry ice or liquid nitrogen bath to form small particles or chains of droplets or solid beads. The frozen solid particles can be stored in an intense freezer between -30 ° C and -80 ° C for later use as a stable frozen product or until dry. A suitable exemplary drying method is vacuum drying where the temperature of the product is kept slightly above its cold temperature. The droplets or frozen beads are placed in trays at a loading capacity of about 0.1 kg / 0.92 m2 (square foot) to about 1.5 kg / 0.92 m2 (square foot) and dried according to with the method of the invention. In some embodiments, the drying process is initiated by a short purging step that allows the product to acclimatize to the initial temperature and the structure of the frozen particles to relax and stabilize and degassed excess air. Typically, the purging step takes between 1 and 60 minutes depending on the product's viscosity and tray loading. The beads or particles should remain in a solid frozen form during the entire purging step. The temperature of the product is then brought above its cold temperature and the primary water removal step is followed until all free water is evaporated from the product. Once the formulation temperature has reached the desired temperature, heat is adjusted to maintain that temperature and the primary liquid drying step by progressed evaporation. In this stage, the formulation is already defrosted and the accelerated evaporation of the water occurs without any boiling or foaming. The drying process is completed with an additional secondary drying phase at maximum vacuum and high temperature.
[0081] Typical methods in the prior art involve extensive foaming and / or spray and violent boiling which can be harmful to sensitive biological agents and cause difficulties for industrial escalation at high loading capacity (see for example Pat. US 6,534,087, where the applied vacuum pressure results in violent boiling and defoaming). However, current compositions and methods prevent the formulation from bubbling or foaming at the same time achieving a significantly faster drying rate and allowing high formulation loading capacity. In addition, complete and efficient degassing of viscous slurries is difficult and may require an extended period of time. These obstacles have all been resolved in the present invention using a suitable composition that allows for effective primary water removal while a glassy structure is formed without boiling and excessive foaming. Loading of solid frozen particles in a different tray of paste or viscous syrup allows for much higher loading capacity per drying area in trays than achieved according to the prior art.
[0082] In a suitable example of the present invention, the biological material is a culture of live concentrated probiotic bacteria. A powder composition mixture, in some embodiments, contains 1-4% sodium alginate or gellan gum, 50-75% trehalose, 110% inulin or FOS, 10-20% protein hydrolysates, such as hydrolysates casein, whey, pea, soy or cottonseed and 1-10% sodium citrate or sodium ascorbate. The probiotic culture can be fresh, frozen or already dried in a dry powder form.
[0083] In another suitable example of the present invention, the biological material is a concentrated culture of living microorganisms. A powder composition mixture is prepared by mixing 1-4% sodium alginate or gellan gum, 5-30% trehalose, 5-40% inulin, 5-40% low DE maltodextrin, 10-30% protein extensively hydrolyzed, such as casein protein, whey, peas, soybeans or cottonseed. 0.1-10% of additional glass enhancers such as sodium citrate, sodium glutamate or sodium ascorbate can also be included in the composition, as an option. The micro-organism or spore culture can be fresh, frozen or already dried in a dry powder form.
[0084] The composition mixture is added to the concentrated culture media to bring the solid content of the solution mixture to 40-60% (w / w) and the pH adjusted to 6.5-7.5 with ions of phosphate or citrate. The solution is mixed at a temperature slightly above room temperature (typically between 25 ° C - 37 ° C) until all components are completely dissolved. The viscous paste is dripped in liquid nitrogen to form droplets or small beads which are then removed from the liquid nitrogen, accumulated in bags and stored in an intense freezer at -80 ° C until dry.
[0085] A typical drying method for live probiotic bacteria includes spreading the frozen solid beads in trays in a uniform layer to a loading capacity between 1001500 g / 0.92 m2 (square foot), and the trays are immediately placed in a freeze dryer. Vacuum is then applied to about 1 mm Hg (1000 mTORR) or lower and depending on the size of the freeze dryer and the type of heat source, the shelf temperature is adjusted to keep the particles at about -20 to about -30 ° C. The frozen solid beads are allowed to purge for about 1 to about 60 minutes and the vacuum adjusted to between 2 and 10 mm Hg (2000 and 10,000 mTORR) and increased heat transfer to raise the formulation temperature to between -20 ° C and 0 ° C, or between -10 ° C and 0 ° C, typically about -10 ° C. These temperature and vacuum pressure conditions are maintained during the primary water removal step, which can last from a few hours to up to 24 hours depending on tray loading. At some point during the primary drying process, the evaporation rate of the solvent decreases and the temperature of the formulation begins to increase due to the excess heat supply in the drying chamber. This point indicates the end of the primary drying step in this invention. As the solvent is removed from the formulation, the glass-forming compounds in the solution become concentrated and thicker until they stop flowing like a liquid and form an amorphous and / or stable glassy structure.
[0086] A secondary drying step is then followed at maximum vacuum and the temperature of the formulation 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 under storage for an extended period of time at room 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.
[0087] The drying methods of the invention result in a biologically active material that is embedded within an amorphous glassy structure, thus preventing the unfolding or denaturation of proteins and significantly reducing molecular interactions or cross-reactivity, due to the greatly reduced mobility of the molecules composed and others within the amorphous glassy composition. As long as the amorphous solid structure is maintained at a temperature below its glass transition temperature and the residual humidity remains relatively low, probiotic bacteria can remain relatively stable. See Figure 15. It should be noted that achieving a glassy structure is not a prerequisite for long-term stability since some biological materials can do better in a more crystalline state.
[0088] The dried glass structure can be used en bloc, cut into desired shapes and sizes, or can be crushed and ground into a free flowing powder, which provides easy downstream processing such as wet or dry agglomeration, granulation, tabletting, consolidation , pelletizing or any other type of release process. Crushing, grinding, crushing 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. A suitable exemplary particle size is less than about 1000 μm and in some embodiments less than 500 μm.
[0089] In another example of the present invention, the dry stable powder containing bioactive material is agglomerated with melted fats. The dry powder is placed in a planetary mixer at 40 ° C and the melted fats such as cocoa butter, natural waxes or hydrogenated oil or a mixture of them are slowly added to the warm powder under mixing and the mixture is cooled down of the melting temperature of the fats while continuous mixing until a visually uniform size of agglomerated powder is achieved. The weight mass of the melted fat mixture in the composition is between about 20% and about 70%, in some embodiments about 30-50%. The final product can be consumed in agglomerated form or compressed in a tablet compression machine and consumed in a tablet form.
[0090] In a particular example, the dry powder is compressed in a tablet compression machine to form a tablet in a desirable shape and size. The dry, stable biological composition is optionally mixed with a filler to adjust the potency of the tablet to a desirable dosage. The filler may include, but is not limited to, maltodextrin, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, colloidal silica dioxide, and combinations thereof. Optionally, a disintegration promoting agent is also included in the tabletting mixture. Examples of a disintegrating promoting agent may include, but are not limited to, croscarmellose sodium, crospovidone (insoluble polyvinylpyrrolidone), sodium starch glycolate, sodium starch glycate, pregelatinized starch and the like. As used herein, the tabletting mixture can optionally include flow agents. Flow agents can include, but are not limited to, magnesium stearate, calcium stearate, zinc state, stearic acid and smoked silica such as hydrophilic or hydrophobic smoked silica.
[0091] Suitable methods for producing tablets from stable biological composition and other tablet ingredients include standard tablet tabletting methods, including those conventionally used to produce multilayer tablets. Compressive compression pressure of up to 50 kN / cm2, corresponding to a tensile strength below 100N (Erweka equipment) is preferable, however exposure to temperature should be limited to below 60 ° C in those cases where the biological material is a living microorganism.
[0092] The tablets can be designed to be swallowed whole, chewed or consumed as tablets of effervescent drink. When the tablets disintegrate under consumption, whether in the mouth, drink or stomach, the biological material is exposed to other active materials from which they have been kept separate by the tablet structure. This can potentially harm biological material if the local concentration of harmful materials is too high. Therefore, it is preferred, in some modalities, that the disintegration of the biological active material is delayed to allow the contents of other active components in the tablet to be diluted and dispersed. This problem was solved in the present invention by forming a hardened or crosslinked structured composition as described herein. In some embodiments, biological materials remain intact within the composition matrix when mixed with water. In some embodiments, the biological material is released unscathed from the composition matrix at a desired location of action throughout the animal's digestive tract.
[0093] Tablets according to the invention can be packaged in such a way as to keep their initial state of dryness within acceptable limits. This may involve packing the tablets in a moisture-impervious compartment such as a tube or blister pack or a container containing a desiccant such as silica gel to absorb water to reduce water activity within the container. For oxygen protection, such a package may also contain an oxygen decontaminating material such as FreshPax®, Ageless ™, ascorbyl palmitate or other ascorbates, propyl gallates or other gallates, alpha-tocopherol, magnesium or sodium sulfite, hydroxyanisole butylated or butylated hydroxytoluene and others.
[0094] The compositions and methods described here stabilize the biological material and preserve its activity during a period of extended storage at above room temperature and relative humidity. For example, the compositions are tested for stability by subjecting them to high temperature (for example, 40 ° C) and high humidity (for example, 33% RH, or 43% RH) and measuring the biological activity of the formulations. As an example for live probiotic bacteria, results from these studies demonstrate that the bacteria formulated in these compositions are stable for at least 60 days. Stability is defined as time for a CFU / g loss of power loss. Such formulations are even stable when high concentrations of the biologically active material are used. Thus, these formulations are advantageous in that they can be transported and stored at room temperature, or above, for long periods of time. EXAMPLES EXAMPLE 1 Preparation of dry and stable composition Basic carbohydrate mix
[0095] About 70 g of trehalose (Cargill Mineápolis, MN), about 5 g of instant inulin (Cargill Mineápolis, MN) and about 3 g of sodium alginate (ISP Corp., Wayne, NJ) were uniformly mixed in dry form.
[0096] Basic mixture of glass intensifiers
[0097] About 17 g of casein hydrolyzate or pea hydrolyzate (ultrafiltered hydrolysates, Marcor, Carlstadt, NJ) and 5 g of sodium citrate or sodium ascorbate (Sigma, St. Louis, MO) were uniformly mixed in dry.
[0098] Stabilization of probiotic bacteria
[0099] Fresh Lactobacillus rhamnosus concentrate (100 ml at 10% solids, straight from the fermentation harvest) was added in a mixer and kept at 35 ° C. About 78 g of basic carbohydrate mixture and about 22 g of the basic mixture of glass enhancers were slowly added to the probiotic culture and the mixing was carried out at 35 ° C for 10 minutes. The viscous paste was then transferred to a vessel having a perforated bottom and allowed to drip into a bath containing liquid nitrogen. The beads were then removed from the liquid nitrogen and immediately transferred to dry.
[00100] Drying of frozen beads containing probiotic bacteria
[00101] The frozen beads were spread out on a tray at a loading capacity of 200 g / 0.92 m2 (square foot) and immediately placed on a shelf in a freeze dryer (Model 25 SRC, Virtis, Gardiner, NY) .
[00102] Vacuum was then adjusted to between 2-2.7 mm Hg (2000-2700 mTORR) and the shelf temperature raised to + 30 ° C. These vacuum temperature and pressure adjustments were maintained for 5 hours. Optionally, the temperature of the frozen beads was acclimated to about -20 ° C before starting the primary liquid drying by applying a vacuum pressure to about 1 mm Hg (1000 mTORR) and allowing the solid frozen beads to purge for about 10 minutes. minutes. The primary drying step was then followed by adjusting the vacuum pressure to between 2-2.7 mm Hg (2000-2700 mTORR) and the shelf temperature raised to + 30 ° C. These vacuum temperature and pressure settings were maintained for 5 hours. A secondary drying step was then followed at a total vacuum of 0.15-0.2 mm Hg (150-200 mTORR) and the shelf temperature maintained between 30 ° C and 50 ° C for an additional 3 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 Storage stability of dry probiotic bacteria
[00103] Figure 1 shows the stability under storage under two accelerated storage conditions other than 40 ° C and 33% RH and 30 ° C and 43% RH of the dry, stable probiotic bacteria from Example 1 and commercially available dry probiotic bacteria (Cul - turelle, Amerifit, Inc., Cromwell, CT). Commercial probiotic bacteria lost their viability completely within the first few weeks under accelerated storage conditions, while the dry composition of the probiotic bacteria of the present invention lost only 1.18 log after 60 days at 30 ° C and 43% RH and only 1, 09 log at 40 ° C and 33% RH. EXAMPLE 3 Scale production of the stable dry composition containing probiotic bacteria Lactobacillus rhamnosus
[00104] Lactobacillus rhamnosus (400 g of frozen concentrate from a commercial source) was thawed at 37 ° C in a jacketed dual planetary mixer (DPM, 1qt, Ross Engineering, Inc. Savana, GA,) and the solid content adjusted to 10 % w solids with distilled water). About 212 g of trehalose (Cargill Mineápolis, MN), about 20 g of instant inulin (Cargill Mineápolis, MN), about 12 g of sodium alginate (ISP Corp., Wayne, NJ), about 136 g of casein hydrolyzate (ultrafiltered hydrolysates, Marcor, Carlstadt, NJ) and about 20 g of sodium ascorbate (Sigma, St. Louis, MO) were uniformly mixed in dry form. The powder mixture was slowly added to the probiotic culture and the mixture was carried out at 40 RPM and 37 ° C for 10 minutes. The paste was then transferred to a vessel having a perforated bottom and allowed to drip in a bath containing liquid nitrogen. The beads were then removed from the liquid nitrogen, placed in a bag lined with sealed aluminum foil and stored in an intense freezer at -80 ° C for several weeks.
[00105] To dry, the frozen beads were spread uniformly on trays at a loading capacity ranging from 500 to 1500 g / 0.92 m2 (square foot) and the trays placed on shelves in a freeze dryer (Model 25 SRC, Virtis, Gardiner, NY). A primary liquid drying step was started by adjusting the vacuum pressure to between 2-2.7 mm Hg (20002700 mTORR) and the elevated and stabilized product temperature between -10 and -5 ° C. Over time (about 10-16 h), the temperature of the product increased to about 20 to 25 ° C, at which point a secondary drying step started at maximum vacuum (0.150.2 mm Hg ( 150-200 mTORR)) and the product temperature maintained between 30 to 40 ° C for an additional 14 hours. The formulation was completely dried and its water activity measured at 0.23 Aw. EXAMPLE 4 Scale production of the stable dry composition containing probiotic bacteria Bifidobacterium lactis
[00106] Bifidobacterium lactis (400 g frozen concentrate from a commercial source) was thawed at 37 ° C in a jacketed dual planetary mixer (DPM, lqt, Ross Engineering, Inc. Savana, GA.). About 212 g of trehalose (Cargill Mineápolis, MN), about 20 g of instant inulin (Cargill Mineápolis, MN), about 12 g of sodium albinate (ISP Corp., Wayne, NJ) and about 20 g of sodium ascorbate (Sigma, St. Louis, MO) were uniformly mixed in dry form. The powder mixture was slowly added to the probiotic culture. About 136 g of pea hydrolyzate (ultra-filtered hydrolysates, Marcor, Carlstadt, NJ) were dissolved in 80 g of distilled water and the mixture briefly microwaved or heated in a 60 ° C water bath until dissolved complete and then cooled to about 35 ° C. Dry mix powder and solution containing pea protein hydrolyzate were added to the probiotic concentrate and mixing was performed at 40 RPM and 37 ° C for 20 minutes. The paste was then transferred to a vessel having a perforated bottom and allowed to drip into a bath containing liquid nitrogen. The beads were then removed from the liquid nitrogen, placed in a bag lined with sealed aluminum foil and stored in an intense freezer at -80 ° C for several weeks.
[00107] For drying, the frozen beads were spread evenly on trays with a loading capacity of 800 g / 0.92 m2 (square foot) and the trays placed on shelves in a freeze dryer (Model 25 SRC, Virtis, Gardiner , NY). A primary liquid drying step was initiated by adjusting the vacuum pressure to between 2-2.7 mm Hg (2000-2700 mTORR) and the temperature of the product elevated and stabilized between -10 and -5 ° C. Over time (about 10-16 h), the temperature of the product increased to about 20 to 25 ° C at which point a secondary drying step started at maximum vacuum (0.15-0.2 mm Hg (150-200 mTORR)) and the product temperature maintained between 30 to 40 ° C for an additional 14 hours. The formulation was completely dried and its water activity measured at 0.23 Aw. EXAMPLE 5 Preparation of a hydrogel formulation containing probiotic bacteria Bifidobacterium lactis
[00108] Bifidobacterium lactis concentrated probiotic paste is prepared according to Example 1. To the basic formulation, 0.5 g of dibasic calcium phosphate is added, followed by 0.5 g of glucono-lactone. The slurry is allowed to harden at room temperature for the next 2 hours to form a solid hydrogel. The firm gel is sliced into thin, long filaments using a commercially available slicer / defibrillator. The thin filaments are either directly loaded onto wet trays or instantly frozen in liquid nitrogen and loaded into a tray for a loading capacity of 500 g / 0.92 m2 (square foot) and placed in a freeze dryer for drying as described in Example 3. The water activity (Aw) of the formulation is 0.05 (Measured by Hygro-Palm Aw1, Rotonic Huntington, NY). The dry formulation is further ground to fine powder using standard hammer milling equipment and sieved through 50-250 micron screens. EXAMPLE 6 Optimization of the molar ratio between the mixture of glass and carbohydrate intensifiers
[00109] Various compositions containing various molar ratios of the mixture of glass and carbohydrate intensifiers were prepared according to Example 1. A concentrated culture of the probiotic L. paracasei bacteria was obtained from a commercial source and prepared in a dry composition as described in Example 1 except that the paste was immediately loaded onto trays in a wet form without instant freezing and purging steps. The pulp was dried in the primary and secondary stages as described in Examples 1 and 3, except that the shelf temperature was raised to 40 ° C during the primary and secondary drying stages. The stable powder was subjected to accelerated storage conditions at 37 ° C and 33% RH for 84 days. Figure 2 shows the effect of various molar ratios on the stability of dried bacteria. Results suggested that the optimal molar ratio between the mixture of glass and carbohydrate intensifiers is about 0.12-0.15. EXAMPLE 7 Effect of the composition of the current invention on the storage stability of probiotic bacteria L. acidophilus
[00110] A composition containing mixture of carbohydrates and mixture of glass intensifiers as described in Example 1 was prepared. A concentrated culture of the probiotic L. acidophilus bacteria was obtained from a commercial source and prepared in a dry composition as described in Examples 1 and 3 and the stable powder was subjected to accelerated storage conditions at 24 ° C and 33% RH for 537 days. Figure 3 demonstrates the superior stability of the probiotic formulated with the composition of the current invention. Results show that the probiotic viability reduced by only 0.18 log for 537 days of shelf storage under the specified conditions. EXAMPLE 8 Effect of various glass intensifier compounds on the storage stability of probiotic bacteria L. acidophilus
[00111] Various compositions containing mixture of carbohydrates as described in Example 1 and mixture of glass intensifiers containing casein hydrolyzate and sodium citrate or sodium ascorbate or a combination of both were prepared. A concentrated culture of the probiotic L. acidophilus bacteria was obtained from a commercial source and prepared in a dry composition as described in Example 1 except that the paste was immediately loaded into trays in a wet form without the instant freezing and purging steps. The slurry was dried in primary and secondary stages as described in Examples 1 and 3 and the stable powder was subjected to accelerated storage conditions at 24 ° C and 43% RH for 180 days. Figure 4 shows the effect of several glass-enhancing compounds on the stability of dried bacteria. Results suggested that a better significant stability was obtained by including an additional glass intensifier on top of the protein hydrolyzate. In particular, the inclusion of equal amounts of sodium acetate and sodium ascorbate provided the most stable composition. Results from both Examples 5 and 6 also suggested that several glass enhancers may be more effective or may even act as a destabilizing agent depending on the bacterial strain. EXAMPLE 9 Effect of various protein / sugar hydrolyzate ratios on storage stability of probiotic bacteria Bifidobacterium lactis
[00112] Various compositions containing mixture of carbohydrates and glass intensifiers as described in Example 1 and compositions containing equal amounts, but in various ratios of pea / trehalose with or without sodium ascorbate were prepared. The concentrated culture of the probiotic Bifidobacterium lactis bacteria was obtained from a commercial source and prepared in a dry composition as described in Examples 1 and 3 and the stable powder was subjected to accelerated storage conditions at 35 ° C and 43% RH for 7 weeks . Figure 5 shows the effect of 1: 4, 1: 2.5 and 1: 1.5 ratios of pea hydrolyzate / trehalose with or without sodium ascorbate on the stability of dried bacteria. Results suggested that better significant stability was obtained in increasing ratios of pea hydrolysate / trehalose. In particular, a 1: 1.5 ratio of pea hydrolyzate / trehalose provided a more stable composition. Inclusion of sodium ascorbate due to higher pea / trehalose hydrolyzate resulted in superior stability compared to compositions without sodium ascorbate. EXAMPLE 10 Optimizing the pH for maximum stability of L. rhamnosus probiotic
[00113] Various compositions containing mixture of carbohydrates and glass intensifiers as described in Example 1 at different pHs were prepared. A concentrated culture of the probiotic bacteria L. rhamnosus was obtained from a commercial source and prepared in a dry composition as described in Examples 1 and 3. The stable powder was subjected to accelerated storage conditions at 40 ° C and 33% RH for 8 weeks. Figure 6 shows the effect of the paste's pH on the stability of dried bacteria. Results suggested that optimal stability was achieved at neutral pH (~ 7). EXAMPLE 11 Stable dry powder containing an enzyme
[00114] A hydrogel formula containing 40 weight percent phytase (BASF, GmBH) is prepared by mixing 400 g of the carbohydrate mixture and 200 g of the glass intensifier mixture as described in Examples 1 and 4 and 400 g of phytase in 1000 ml of water. The hydrogel formulation cut into strips is instantly frozen in liquid nitrogen and dried in a vacuum oven at a primary and secondary drying temperature of 50 ° C. To determine loading and storage stability of the dried formula: a dry sample is accurately weighed (<100 mg) in a microcentrifuge tube. A 200 μl portion 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 NaOH, 0.5% SDS and 0.075 M citric acid (tri-sodium salt) are added. The tubes are sonicated for 10 min at 45 ° C, followed by a brief centrifugation at 5,000 rpm for 10 min. The aliquots of the clear DMSO / NaOH / SDS / Citrate solution are washed in wells of a microplate and analyzed for protein content using the Bradford assay method. The stability of the dry stable enzyme composition after exposure to 95 ° C for 20 min is significantly higher than a dry enzyme without the composition of the present invention. EXAMPLE 12 Stable dry powder containing an infectious salmon anemia virus (ISAV) vaccine
[00115] ISAV vaccine concentrate paste (Novozyme, Denmark) is prepared according to Example 4 except that 20 ml of 4% chitosan solution in 0.5% acetic acid were added to the paste containing the ISAV vaccine concentrate, the mixture of carbohydrates and the glass intensifiers. 0.5 g of dibasic calcium phosphate is added, followed by 0.5 g of gluconolactone. The slurry is allowed to harden at room temperature for the next 2 hours to form a solid hydrogel. The firm gel is cut into long, thin filaments using a commercially available slicer / defibrillator. Thin filaments are either directly loaded onto trays in a wet form or instantly frozen in liquid nitrogen and loaded into a tray at a loading capacity of 1500g / 0.92 m2 (square foot) and placed in a freeze dryer for drying as described in Example 3. The water activity (Aw) of the formulation is 0.25. The dry formulation is additionally ground to fine powder using standard hammer milling equipment and sieved through 50-150 micron screens. The stable dry ISAV composition is used for oral vaccination by covering a commercial animal feed with the dry composition and feeding Atlantic salmon. EXAMPLE 13 Preparation of invasive bait
[00116] Pelletized bait for invasive species specifically targeted in accordance with the present invention is prepared containing a pesticide. 200 g of a formulation as described in Example 9 are prepared and added to 200 gm of water. To this solution, 90 gm of Rotenone and 0.5 gm of dibasic calcium phosphate are added, followed by 0.5 gm of gluconolactone. The paste is immediately spray dried in a standard industrial spray dryer, and the dry formulation is used to target specific invasive species with no deleterious effect of the toxin on the environment or close to the ecosystem. EXAMPLE 14 Preparation of a formulation of protected plant probiotics
[00117] A biological control agent such as Rhizobacteria is prepared in a dry composition according to Example 4. The effectiveness of the dry Rhizobacteria composition is assessed on lettuce growth under gnotobiotic conditions. Doses of 100 mg of dry Rhizobacteria composition per plant are inoculated in jars with sand and planted with pre-germinated lettuce seedlings (24 h). A 5 ml nutrient dose of sterile Hoagland's solution is applied to the plants in the jar. The jars are randomly placed in a growth chamber maintained at 28 ° C with 12 h of photoperiod. During each 7-day interval after inoculation, adherent plants and 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. EXAMPLE 15 Preparation of dry and stable probiotic substance Basic Formulation
[00118] A 75 g portion of trehalose (Cargill Minneapolis, MN) and 22 g of extensively hydrolyzed casein (Marcor, Carlstadt, NJ) was uniformly mixed with 3 g of sodium alginate (ISP Corp., Wayne, NJ) in dry form. Fresh concentrate of Lactobacillus acididophylus (100 ml containing at least 10% solids, straight from the 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 vessel having 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 dry. Drying of frozen beads of the basic formulation
[00119] The frozen beads were spread evenly on a tray at a loading capacity of 100 g / 0.92 m2 (square foot) and immediately placed on a shelf in a freeze dryer (Model 25 SRC, Virtis, Gardiner, NY ). Vacuum pressure was then applied to 1 mm Hg (1000 mTORR) and the frozen solid beads were allowed to purge for 10 minutes. Vacuum was then adjusted to 2.7 mm Hg (2700 mTORR) and the 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 (0.15-0.2 mm Hg (150200 mTORR)) and the shelf temperature raised to 30 ° C for an additional 2 hours. The formulation was completely dried and its water activity measured by a Hygropalm Aw1 instrument (Rotational Instrument Corp., Huntington, NY.) At Aw = 0.23. EXAMPLE 16 Stable dry composition containing probiotic bacteria Lactobacillus rhamnosus LGG
[00120] Lactobacillus rhamnosus LGG (500 g of frozen concentrate from a commercial source) was thawed at 37 ° C in a jacketed dual planetary mixer (DPM, lqt, Ross, Engineering, Inc. Savana, GA). Two glass-forming agents; trehalose (387 g, Cargill Minneapolis, MN) and extensively hydrolyzed casein (83 g, Mar- cor, 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 vessel having a perforated bottom and allowed to drip into a vessel containing liquid nitrogen. The beads were then removed from the liquid nitrogen, placed in a bag lined with sealed aluminum foil and stored in an intense freezer at -80 ° C for several weeks.
[00121] For drying, the frozen beads were spread evenly on trays at a loading capacity ranging from 100 to 500 g / 0.92 m2 (square foot) and the trays placed on shelves in a freeze dryer (Model 25 SRC , Virtis, Gardiner, NY). Vacuum pressure was applied at 1000 mTorr and the shelf temperature adjusted to + 20 ° C. The frozen solid beads were allowed to purge for a period of time ranging from 1 to 30 minutes. The purging step was followed by a primary drying step after adjusting the vacuum pressure to 2.7 mm Hg (2700 mTORR)) and the shelf temperature raised to + 30 ° C. This vacuum temperature and pressure was maintained for 12 hours. A secondary drying step was then followed at full vacuum (0.15-0.2 mm Hg (150-200 mTORR)) and the shelf temperature maintained at 30 ° C for an additional 4 hours. The formulation was completely dried and its water activity measured at 0.23 Aw. Figure 13 shows the drying profile of the probiotic formulation.
[00122] Losses of viability 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 Figures 10, 11 and 14. Viability losses for the entire process were generally less than <1 log depending on the type of bacterial culture (frozen or dry cultures) and the cold temperature of the viscous paste. Results show that instantaneous freezing of probiotic bacteria in liquid nitrogen (-180 ° C) was a less harmful process than freezing at -80 ° C.
[00123] Figures 12 & 15 show the effect of various purging time periods ranging from 0 min (no purging) to 30 min on the initial counts of probiotic bacteria in the dry composition and stability under storage under 40 ° accelerated storage conditions C and 33% RH. Results suggest that a longer purging time generally improves the initial bacterial count in the dry formulation, but had no effect on storage stability of the probiotic formulation. EXAMPLE 17
[00124] Trealose (752 g, Cargill Minneapolis, MN), extensively hydrolyzed Pea protein (167g, Marcor, Carlstadt, NJ), sodium alginate (30g, ISP Corp., Wayne, NJ) and 50g instant Inulin, Cargill Mineápolis, MN) were homogeneously mixed in dry form. The dry mixture was slowly added to 1000 ml of hot deionized water at 80 ° C in a dual jacketed planetary mixer (DPM, lqt, Ross Engineering, Inc. Savana, GA,) and the mixture was performed at 40 RPM for 10 minutes. The temperature of the mixture was reduced to 37 ° C and 100 g of dry powder of Lactobacillus rhamnosus LGG obtained from a commercial source were slowly added and mixing continued for 20 minutes. The paste was then extruded through a 2 mm orifice needle in a bath containing liquid nitrogen. The filaments / beads were then removed from liquid nitrogen, placed in a bag lined with sealed aluminum foil and stored in an intense freezer at -80 ° C for several weeks. For drying, the frozen filaments / beads were spread evenly on trays at a loading capacity ranging from 100 to 500 g / 0.92 m2 (square foot) and the trays placed on shelves in a freeze dryer (Model 25 SRC, Virtis, Gardiner, NY) and dried as described in Example 16. All formulations were retained within the tray and no splash or foam was observed at all loading levels. The formulation was even completely dried at the highest loading capacity and the water activity measured at 0.26 Aw and lower for all samples. EXAMPLE 18 Preparation of a hydrogel formulation containing probiotic bacteria Bifidobacterium sp.
[00125] Concentrated probiotic paste of Bifidobacterium sp. it is prepared according to Example 15. For the basic formulation, 0.5 g of dibasic calcium phosphate is added, followed by 0.5 g of glucanolactone. The slurry was allowed to harden at room temperature for the next 2 hours to form a solid hydrogel. The firm gel was sliced into thin, long filaments using a commercially available slicer / defibrillator. The thin filaments are instantly frozen in liquid nitrogen and loaded into a tray at a loading capacity of 700 g / 0.92 m2 (square foot) and placed in a freeze dryer for drying as described in Example 16. The water activity (Aw) of the formulation was 0.05 (Measured by HygroPalm Aw1, 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 19 Allergen-free composition containing probiotic bacteria Lactobacillus acidophylus
[00126] Trealose (752 g, Cargill Minneapolis, MN), extensively hydrolyzed Pea protein (167 g, Marcor, Carlstadt, NJ), sodium alginate (30 g, ISP Corp., Wayne, NJ) and Inulin 50 g instant, Cargill Mineápolis, MN) were homogeneously mixed in dry form. The dry mixture was slowly sterilized by adding 1000 ml of hot deionized water at 80 ° C in a jacketed dual planetary mixer (DPM, lqt, Ross Engineering, Inc. Savana, GA,) and the mixture was performed at 40 RPM for 10 minutes until smooth and clear paste is formed. The temperature of the mixture was reduced to 37 ° C and 1000 g of frozen beads containing Lactobacillus acidophylus obtained from a commercial source were slowly added and mixing continued for 10 minutes. The paste was then extruded through a 2 mm orifice needle in a bath containing liquid nitrogen. The filaments / beads were then removed from the liquid nitrogen placed in a bag lined with sealed aluminum foil and stored in an intense freezer at -80 ° C for several weeks. For drying, the frozen filaments / beads were spread evenly on trays at a loading capacity of 1000 g / 0.92 m2 (square foot) and the trays placed on shelves in a freeze dryer (Model 25 SRC, Virtis, Gardiner, NY) and dried as described in Example 16. The initial CFU counts of probiotic bacteria in the dry composition were 10.53 logs / g, and the loss of viability after 42 days storage under accelerated storage conditions of 40 ° C and 33 % RH was 0.69 log CFU / g. EXAMPLE 20 Infant formula containing the dry formulation of the present invention
[00127] A dry stable formulation containing Lactobacillus rhamnosus was prepared according to Example 16 followed by sieving in two groups of particle size (above 50 μm and below 150 μm). 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 μm and 150 μm). The final product contains about 108 cfu of Lactobacillus rhamnosus per 100 g of infant formula. EXAMPLE 21 Probiotic supplement containing the stable dry formulation of the invention
[00128] A stable dry composition containing Lactobacillus acididophylus is formulated in oral dosage forms, such as tablets, microcapsules, or capsules. Orange-flavored tablets containing 99.9 g of a compression agent (dextrose) and 0.1 g of dry formulation particles in the size range between 50 μm and 150 μm are prepared by direct compression on a rotating machine using a stamping standard 1.27 cm (1/2 ") round concavity. The final product contains about 108 cfu / unit dose. Hardness of the tablets is in the range of 8-10 kp and disintegration times are about 20 seconds. compressed tablets are packaged in 180 cc HDPE bottles of 100 tablets each and exposed to a controlled temperature / humidity of 40 ° C / 33% RH.The product is subjected to the monthly microbiological stability test over a period of 12 months or until a reduction in the assay count below 1 x 106 / unit dose is observed. EXAMPLE 22 A functional beverage beverage containing the stable dry formulation of the present invention
[00129] A dry mixture containing (% by weight) 71% sucrose, 14% maltodextrin, 10% inulin, 2% dextrose, 1% citric acid 0.3% anhydrous acacia gum, 0.3% of flavors, 0.3% of tricalcium phosphate and 0.1% of dry probiotic formulation particles (L. acidophilus) in the size range between 50 μm and 250 μm is prepared. The final product contains about 109 cfu / unit dose (30 g dry mix). The product is packaged in bags coated with small aluminum foil (30 g unit dose / bag) to drink by stirring in 340 thousand water. The stability of the probiotic bacteria in the dry drink mix is subjected to the monthly microbiological stability test over a period of 12 months or until a reduction in the assay count below 1 x 107 / unit dose is observed. EXAMPLE 23 Probiotic pet food preparation
[00130] A commercially available lump pet food for dogs is dried in a convection oven to a water activity of 0.1, and then coated with the dry stable probiotic formulation prepared as described in Example 17. The pellets dried are sprayed with about 5% fat-based moisture barrier (a mixture of 40% chicken fat, 40% cocoa butter and 20% beeswax), mixed in a rotating drum with the formulation dry powder (usually 0.10.5% of total pet food providing a dosage of 10.sup.8 CFU / g), and finally sprayed with additional fat-based moisture barrier coating. The total amount of the coating is about 15% (of pet food). The coating time is about 30 min. EXAMPLE 24 Preparation of fish feed with various probiotic microorganisms
[00131] Pelleted fish food according to the present invention is prepared with a mixture of various probiotics. A stable dry probiotic formulation containing a mixture of L. rhamnosus, L. acidophylus and Bifidobacterium lactis is prepared as described in Example 15. A commercially available starter feed for salmon (Zeigler Bros., Gardners, PA) is first dried in a convection oven at a water activity of 0.1, and then coated with the formulation of probiotics in a rotating drum. The pellets (1000 g) are sprayed first with about 5% by weight of a moisture-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 107 cfu / g of food), and finally sprayed with additional fat-based moisture barrier coating. The total amount of coating is about 10% (of the fish food). EXAMPLE 25 Stable dry powder containing an enzyme
[00132] A hydrogel formula containing 40 weight percent Savinase (Novozymes, Denmark) is prepared by mixing 600 g of the formulation described in Example 18 and 400 g of savinase in 1000 g of water solution. The hydrogel formulation cut into strips is instantly frozen in liquid nitrogen and dried in a vacuum oven at a drying temperature of the formulation of 50 ° C. To determine loading and storage stability of the dried formula: a dry sample is accurately weighed (<100 mg) in a microcentrifuge tube. 200 μl 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 NaOH, 0.5% SDS and 0.075 M citric acid (tri-sodium salt) are added. The tubes are sonicated for 10 min at 45 ° C, followed by a brief centrifugation at 5,000 rpm for 10 min. The aliquots of the clear DMSO / NaOH / SDS / Citrate solution are placed 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 higher than a dry enzyme without the formulation of the present invention. EXAMPLE 26 Stable dry powder containing vitamin A
[00133] A formulation containing 30 weight percent Vitamin A is prepared by mixing 320 g of instant Inulin, 320 g of maltodextrin DE-1 (Tate & Lyle, London, GB), 50 g of sodium carboxymethylcellulose (Ashland Aqualon 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 frozen instantly in liquid nitrogen, then spread out on trays at a loading capacity of 1000 g / 0.92 m2 (square foot) and dried as described in Example 16. The vitamin A composition is stable (> 80%) at 40 ° C and 75% RH for 3 months. EXAMPLE 27 Carotene preparation in a protected formulation with enhanced bioavailability
[00134] A formulation that protects and enhances the bioavailability of carotene that would otherwise be subject to oxidation through other ingredients in a food 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 are 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 tripolyphosphate. The hydrogel-treated microparticles or filaments are allowed to harden at room temperature for 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-laden particles are instantly frozen and immediately placed in 500 g / 0.92 m2 (square foot) trays and freeze-dried in a freeze dryer until the water activity has reduced to less than 0, 3. The dry formulation is further milled to desired size distribution and packaged. EXAMPLE 28 Preparation of invasive bait
[00135] Pelleted bait for specifically targeted invasive species are 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 90 gm of Rotenone and 0.5 gm of dibasic calcium phosphate are added, followed by 0.5 gm of gluconolactone. The paste is allowed to harden at room temperature for 2 hours. The firm gel is sliced into thin, long filaments through a slicer / defibrillator. The thin filaments are loaded onto a tray and placed in a freeze dryer. Shelf temperature is set at -30 ° C and the formulation allowed to freeze before full vacuum is applied and the shelf temperature has risen to + 60 ° C for overnight drying. The dry formulation is ground to the appropriate size distribution for specifying the bait size for the specific target species. EXAMPLE 29 Preparation of a protected pesticide in a water-insoluble formulation
[00136] A granular formulation protected from a pesticide that would otherwise be subject to decomposition by other ingredients in a formulation during storage or after application in 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 are 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 allowed to harden at room temperature for 4 hours, and then sliced into thin, long filaments through a slicer / defibrillator. The thin filaments are loaded onto trays and dried in a freeze dryer to achieve 0.1 water activity. The dry formulation is further ground to the desired size distribution and packaged. EXAMPLE 30 Preparation of a formulation of protected plant probiotics
[00137] A biological control agent such as Rhizobacteria is prepared in a dry composition according to Example 18. The effectiveness of the dry Rhizobacteria composition is evaluated on the growth of lettuce under gnotobiotic conditions. Doses of 100 mg of dry Rhizobacteria composition per plant are inoculated in jars with sand and planted with pre-germinated lettuce seedlings (24 h). A 5 ml nutrient dose of sterile Hoagland's solution is applied to the plants in the jar. The jars are randomly arranged in the growth chamber maintained at 28 ° C with a 12 h photoperiod. During each 7-day interval after inoculation, the plants and adherent 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. EXAMPLE 31 Production of dry stable composition containing probiotic bacteria Lactobacillus acidophylus (DSM-20356)
[00138] Cold bacterial concentrate (10 g obtained from a local fermentation process) was thawed at 37 ° C in a water bath and the solid content adjusted to 10% solids moistened with distilled water). About 5 g of hydrolyzed pea protein (ultrafiltered hydrolysates, Marcor, Carlstadt, NJ) are completely dissolved in 50 g of warm water and added to the thawed bacterial culture. About 2.5 g of trehalose (Cargill Mineápolis, MN), about 5 g of instant Inulin, about 5 g DE-l maltodextrin (Cargill Mineápolis, MN) and about 1.5 g of sodium alginate (ISP Corp., Wayne, NJ) were uniformly mixed in dry form. The powder mixture was slowly added to the bacterial culture and mixing was carried out using a small spatula at 37 ° C for 20 minutes. The paste was then allowed to drip into a bath containing liquid nitrogen. The beads were then removed from the liquid nitrogen, placed in a bag lined with sealed aluminum foil and stored in an intense freezer at -80 ° C until dry.
[00139] For drying, the frozen beads were spread evenly on trays at a loading capacity ranging from 500 to 1500 g / 0.92 m2 (square foot) and the trays placed on shelves in a freeze dryer (Model 25 SRC , Virtis, Gardiner, NY). A primary liquid drying step was initiated by adjusting the vacuum pressure to between 20-2.7 mm Hg (2000-2700 mTORR)) and the product temperature being raised and stabilized between -10 -5 ° C. Over time (about 10-16 h) the temperature of the product increased to about 20-25 ° C at which point a secondary drying step started at maximum vacuum (0.15-0.2 mm Hg (150-200 mTORR)) and the product temperature maintained between 30-40 ° C for an additional 14 hours. The formulation was completely dried and its water activity measured below 0.3 Aw. The formulation was ground using a commercially available hammer mill and the particles sieved below 100 microns.
[00140] The viability of the stable probiotic bacteria along with a powder commonly dried by freezing the bacteria was monitored following standard dilution procedures and plating weekly on LMRS agar plates. Figure 16 shows that after 14 days at 40 ° RH, the stability of the probiotic bacteria that were formulated in the composition of the present invention was two (2) logs higher than the stability of bacteria commonly freeze-dried. These results demonstrate that the stability of probiotic bacteria is dramatically improved under conditions of high humidity and non-refrigerated storage when using the compositions and methods of the present invention. EXAMPLE 32
[00141] Production of agglomerated composition of stable dry melted fats containing probiotic bacteria Lactobacillus acidophylus (DSM-20356). Ten (10) g of dry powder composition were produced as described in Example 31. The dry powder was placed in a beaker in a 40 ° C water bath. 10 g of melted fat mixture containing eight (8) portions of cocoa butter and two (2) stearate portions (27-Stearine, Loders Croklaan, Channahon, IL) were slowly added to the warm powder under mixing. The mixture was cooled to 10 ° C while the mixture was continued until a visually uniform size of agglomerated powder was achieved. EXAMPLE 33 Stability under shelf storage at 40 ° C and 43% RH or 30 ° C and 60% RH of a dry composition containing probiotic bacteria Lactobacillus rhamnosus sp.
[00142] Ten (10) g of dry powder composition containing the probiotic bacteria- Lactobacillus rhamnosus sp. (obtained from a local fermentation source) were produced as described in Example 31. The dry stable composition was placed in a desiccator and exposed to 40 ° C and 43% RH or 30 ° C and 60% RH. The viability of the stable probiotic together with a powder commonly dried by freezing the bacteria was monitored following standard dilution procedures and plated weekly on LMRS agar plates. Figure 17 shows that after 14 days at 40 ° C and 43% RH, the stability of the probiotic bacteria that were formulated in the composition of the present invention was three (3) logs higher than the stability of bacteria commonly freeze-dried. After 7 days at 30 ° C and 60% RH, the stability of the probiotic bacteria that were formulated in the composition of the present invention was also three (3) logs higher than the stability of bacteria commonly freeze-dried. These results demonstrate that the stability of probiotic bacteria is dramatically improved under conditions of uncooled storage and high humidity when using the compositions and methods of the present invention. EXAMPLE 34 Production of animal feed containing stable dry composition containing probiotic bacteria against pathogenic microorganisms
[00143] About 10 kg of commercially available animal feed for oxen or chickens are covered in a rotating drum with 3% oil mixture containing a portion of the ground biological material as described in Example 31 or 32 and two (2) portions of vegetable oil such as corn oil. The CFU count of probiotic bacteria is IE9 / g of feed. The coated feed is placed in a chamber at 43% relative humidity at 40 ° C and after 14 days of storage in these extreme conditions; the loss of viability of probiotic bacteria is less than one (1) log of the initial CFU. Another coated feed is placed in a chamber at 33% relative humidity at 30 ° C and after six (6) months of storage under these conditions; the loss of viability of probiotic bacteria is less than one (1) log of the initial CFU. This example demonstrates that microorganisms, such as Lactobacillus sp., Used to treat various animals including pets, can be preserved in the composition and drying methods of the present invention and then coated in feeds for long-term shelf storage or at least at least two (2) weeks in a feed hopper under typical humidity and temperature conditions that uncoated feed is stored. EXAMPLE 35 Production of dry stable composition containing unicellular fungi S. cerevisiae
[00144] Fresh baker's yeast paste (100 g obtained from a local distributor) is placed in a 10 ° C water bath, about 50 g of hydrolyzed pea protein (ultrafiltered hydrolysates, Brown, Carlstadt, NJ ) are completely dissolved in 500 g of warm water. The solution is cooled to 10 ° C and added to the yeast paste while mixing. About 25 g of sucrose (obtained from a local supermarket), about 50 g of instant inulin, about 50 g of maltodextrin DE-1 (Cargill Mineápolis, MN), about 12 g of sodium ascorbate (Sigma) and about 15 g of sodium alginate (ISP Corp., Wayne, NJ) are uniformly mixed in a dry form. The powder mixture is slowly added to the yeast culture and mixing is carried out at 40 RPM and 10 ° C for 20 minutes. The paste is then transferred to a vessel having a perforated bottom and allowed to drip into a bath containing liquid nitrogen. The beads are then removed from the liquid nitrogen, placed in a bag lined with sealed aluminum foil and stored in an intense freezer at -80 ° C for several weeks. Drying and grinding are carried out as described in Example 31. EXAMPLE 36 Spray drying of the stable dry composition containing unicellular fungi S. cerevisiae
[00145] Yeast paste is prepared as described in Example 34. The paste is further diluted with cooled distilled water (10 ° C) to obtain a viscosity of about 1000-2000 cP. The diluted slurry is spray dried (Mobile Minor spray dryer, GEA Niro Inc., Columbia, MD), using an inlet / outlet temperature setting of 80 ° C / 60 ° C. EXAMPLE 37 Corn seed coating with stable dry composition containing single-celled fungi
[00146] About 10 kg of commercially available maize seeds are coated on top at 40 ° C in a rotating drum with 3% melted oil mixture containing a portion of the ground biological material as described in Example 34 or Example 35 and two (2) portions of vegetable oil such as palm or coconut oil. The yeast CFU count is 1E8 / g of seed. The coated seeds are placed in a chamber at 60% relative humidity at 30 ° C and after three (3) months of storage in these extreme conditions, the loss of viability of the yeast is less than one (1) log of the initial CFU. This example demonstrates that microorganisms used as agricultural inoculants such as various strains of Penicillium sp. they can be preserved in the composition and drying methods of the present invention and then coated on grains for long term storage under typical humidity and temperature conditions that the uncoated seeds are stored. EXAMPLE 38 Preparation of a hydrogel composition containing probiotic bacteria Bifidobacterium sp.
[00147] Concentrated probiotic paste from Bifidobacterium sp. is prepared according to Example 31. To the powder mixture, 5 g of dibasic calcium phosphate are added. The powder mixture is added to the probiotic culture under mixing followed by 5 g of gluconolactone. The slurry is allowed to harden at room temperature for the next two (2) hours to form a solid hydrogel. The firm gel is sliced into thin, long filaments using a commercially available slicer / defibrillator. Thin filaments are either directly loaded onto trays in a wet form or frozen instantly in liquid nitrogen and loaded into a tray at a loading capacity of 500g / 0.92 m2 (square foot) and placed in a freeze dryer for drying as described in Example 31. The dry formulation is ground to fine powder using standard hammer milling equipment and sieved through a 50 micron screen. EXAMPLE 39 Production of stable fermented milk containing probiotic bacteria
[00148] One hundred (100) grams of plain culture pasteurized milk (Dannon, obtained from a local supermarket) are added with medium (0.5) gram of cross-linked powder containing stable probiotic as described in Example 37. The initial CFU count in fermented milk it is IE9 / g of fermented milk. Fermented milk is stored in a refrigerator at 4 ° C for six (6) weeks. The loss of viability of probiotic bacteria in refrigerated fermented milk is less than one (1) log of the initial CFU. This example demonstrates that probiotic bacteria such as various strains of Lactobacillus and Bifidobacterium can be preserved in the composition and drying methods of the present invention. Then the probiotic bacteria in the compositions can be completely hydrated and can remain active in the dairy products for the extended period of time under typical unpreserved conditions that the probiotic bacteria will not survive. EXAMPLE 40 Stable dry composition containing an enzyme
[00149] A hydrogel formula containing 40 weight percent phytase (Marcor, Carlstadt, NJ) is prepared by mixing 250 g of the powder mixture as described in Example 34 and 200 g of phytase in 500 ml of water solution containing about 50 g of pea hydrolyzed protein. The hydrogel formulation cut into strips is instantly frozen in liquid nitrogen and dried in a vacuum oven at a primary and secondary drying temperature of 50 ° C. To determine loading and storage stability of the dried composition: a dry sample is accurately weighed (<100 mg) in a microcentrifuge tube. 200 μl 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 NaOH, 0.5% SDS and 0.075 m citric acid (tri-sodium salt) are added. The tubes are sonicated for 10 min at 45 ° C, followed by a brief centrifugation at 5,000 rpm for 10 min. The aliquots of the clear DMSO / NaOH / SDS / Citrate solution are taken into wells of a microplate and analyzed for protein content using the Bradford assay method. The stability of the dry stable enzyme composition after exposure to 95 ° C for 20 min is significantly higher than a dry enzyme without the composition of the present invention. EXAMPLE 41 Stable dry composition containing a biological plant control agent
[00150] A biological control agent such as Rhizobacteria is prepared in a dry composition according to Example 34. The effectiveness of the dry Rhizobacteria composition is evaluated on the growth of lettuce under gnotobiotic conditions. Doses of 100 mg of dry Rhizobacteria 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 the growth chamber maintained at 28 ° C with 12 h of photoperiod. During an interval of 7 days after inoculation, the plants and adherent 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. Lettuce seedlings treated with Rhizobacteria composition show an increased growth than untreated seedlings. EXAMPLE 42 Production of tablets containing stable dry composition of probiotic bacteria Lactobacillus rhamnosus sp.
[00151] Cold bacterial concentrate (10 g obtained from a local fermentation process) was thawed at 37 ° C in a water bath and the solid content adjusted to 10% by weight of solids with distilled water. About 5 g of hydrolyzed pea protein (ultra-filtered hydrolysates, Marcor, Carlstadt, NJ) were completely dissolved in 50 g of warm water and added to the thawed bacterial culture. About 5 g of trehalose (Cargill Minneapolis, MN) and about 2.5 g of sodium ascorbate were uniformly mixed in dry form. Optionally, about 5 g of instant inulin, about 5 g of maltodextrin DE-1 (Cargill Minneapolis, MN) and about 1.5 g of sodium alginate (ISP Corp., Wayne, NJ) were also added to form a viscous paste at a desirable viscosity of about 50,000 cP and to further enhance the glassy structure of the dry material. The powder mixture was slowly added to the bacterial culture and the mixing was carried out at 37 ° C for 20 minutes. The viscous bacterial suspension was then slowly dripped into a bath of liquid nitrogen. The frozen beads were then removed from the liquid nitrogen, placed in a bag lined with sealed aluminum foil and stored in an intense freezer at -80 ° C until dry.
[00152] For drying, the frozen beads were spread evenly on trays at a loading capacity ranging from 500 to 1500 g / 0.92 m2 (square foot) and the trays placed on shelves in a freeze dryer (Model 25 SRC , Virtis, Gardiner, NY). A primary moisture removal step was started by adjusting the vacuum pressure to between 20-2.7 mm Hg (2000-2700 mTORR)) and allowing the product temperature to rise and stabilize between minus -10 and -5 ° C. Over time (about 10-16 h), the temperature of the product increased to about 2025 ° C at which point a secondary drying step started at maximum vacuum (0.05-0.2 mm Hg (50-200 mTORR)) and the product temperature maintained between 30-45 ° C for an additional 14 hours. The formulation was completely dried and its water activity measured below 0.3 Aw. The formulation was ground using a coffee grinder and the particles sieved below 250 microns.
[00153] For tabletting, the dry and stable probiotic composition (100 mg) was mixed with 400 mg of DE-1 maltodextrin containing 2% w / w of magnesium stearate and 2% w / w of smoked hydrophilic silica (AEROSIL® 200 , Evonik Industry) and compressed in manual pill press equipment (using a 1.27 cm (% ") tablet diameter housing.) Similar tablets containing powder commonly dried by freezing probiotic bacteria (free probiotic) were also prepared and used for comparison with tablets containing protected probiotic bacteria.
[00154] Viability before and after formation in tablets and during storage at 40 ° C and 43% RH of the stable p robiotic bacteria together with free probiotic was weekly monitored, following standard dilution procedures and placing on LMRS agar plates . Figure 18 shows that the free probiotic bacteria lost in a viability log in the tabletation process while the viability of the protected bacteria remained essentially the same after the tabletation process. After 14 days storage at 40 ° C and 43% RH, the viability of the probiotic bacteria that were formulated in the composition of the present invention was slightly reduced by about 0.3 log while the viability of the freeze-dried bacteria further reduced about 0.6 log. These results demonstrate that the composition and methods of the present invention provide significant protection against the compression pressure and associated heat during the tableting of probiotic bacteria and during storage in conditions of high humidity and non-refrigerated storage. EXAMPLE 43 Preparation of multivitamin / probiotic tablets containing stable dry composition of probiotic bacteria Lactobacillus rhamnosus sp.
[00155] The protection of the compositions and methods as disclosed here was further explored in tablets containing multivitamin ingredients. Ten (10) g of dry powder composition were produced as described in Example 42. For tabletting, the dry and stable probiotic composition (100 mg) was mixed with 400 mg of commercially available multivitamin powder (Centrum®, Pfizer) containing 2% w / w of magnesium stearate and 2% w / w smoked hydrophilic silica (AEROSIL® 200, Evonik Industries) and compressed in manual pill press equipment (using a 1.27 cm (^ ") tablet diameter housing Similar tablets containing freeze-dried powder of probiotic bacteria (free probiotic) were also prepared and used for comparison with tablets containing protected probiotic bacteria.The resulting tablets were then tested for total probiotic counting, the results are shown in Figure 19.
[00156] As shown in Figure 19, the free probiotic bacteria lost more than two (2) viability logs in the tabletation with the multivitamin ingredients process while the viability of the protected bacteria reduced by less than one log. After 14 days of storage at 40 ° C and 43% RH, the viability of the probiotic bacteria that were formulated in the composition of the present invention remained essentially the same while the viability of the freeze-dried bacteria dropped an additional three (3) logs . These results demonstrate that the composition and methods of the present invention also provide significant protection to sensitive biological materials from other harmful compounds in the tablet mixture, thus allowing to mix a variety of biological materials in a tablet without affecting its overall potency. EXAMPLE 44 Tableting a stable dry composition containing protected enzymes
[00157] Dry and stable compositions containing a protease or a lipase (both from Sigma) were prepared as described in Example 42. The final dry compositions contained 10% protease or lipase, 40% trehalose, 20% pea protein extensively hydrolyzed, 10% sodium ascorbate. In addition, 6% sodium alginate and 14% inulin were also included in the composition.
[00158] For tabletation, the dry enzyme compositions (50 mg each) were mixed with 450 mg of maltodextrin DE-1 containing 2% w / w of magnesium stearate and 2% w / w of smoked hydrophilic silica and compressed with a manual pill press equipment (using a 1.27 cm (% ") tablet diameter housing). Tablets containing equal amounts of both protected enzymes were also prepared by mixing and 25 mg of protease and 25 mg of lipase with 450 mg of DEI maltodextrin mixture Similar tablets containing dry enzyme powder in a free form (free enzyme or a mixture of both) were also prepared and used for comparison with tablets containing the protected enzymes.
[00159] The remaining protease and lipase activity after tabletation with respect to their activity in the powder mixture before tabletation was determined according to methods known in the art using Azocasein and pNP-palmitate as substrates, respectively.
[00160] As shown in Figure 20, tablet-free protease either alone or in combination with free lipase resulted in about 40% loss of activity while the protected protease lost no activity when turned into a tablet alone and only about 17 % when transformed into a tablet in a mixture with protected lipase. Tabletation-free or protected lipase did not result, however in any significant loss of activity, tabletation-free lipase in the presence of free protease resulted in 64% loss of activity, whereas tabletation-protected lipase in the presence of protected protease resulted in only 33% loss of activity. These results demonstrate that the composition and methods of the present invention provide significant protection against compression pressure and associated heat during enzyme tabletation. Results also show that the composition and methods of the present invention provide protection from other digestive enzymes in the tablet mixture, thus allowing to mix in a tablet a variety of desired enzymes without affecting its general activity. EXAMPLE 45 Animal feed tabletation containing stable dry composition containing probiotic bacteria against pathogenic microorganisms
[00161] The protection of the compositions and methods as disclosed here is further explored in tablets containing animal feed ingredients. About 100 g of dry and stable compositions containing the probiotic bacteria L. acidophilus sp. they are prepared and dried as described in Example 42. The final dry compositions contained 10% dry bacterial cell biomass, 54% treacle, 20% extensively hydrolyzed pea protein, 10% sodium asorbate. In addition, 6% sodium alginate is also included in the composition.
[00162] About 10 kg of finished feed pellets for commercially available dogs and chickens are air dried overnight at 40 ° C and then finely ground to free-flowing powder. The stable dry probiotic composition is mixed with the feed powder and compressed in manual pill press equipment (using 1/8 - 7/8 "(0.31-2.22 cm) pill diameter housings) to form about 200-2000 mg of sized pills containing about ten (10) billion live cells per gram of feed. For chicken treatment, probiotic feed pills are slowly poured into 100 kg of standard commercial feed while mixing. finished treated feed is ready to feed the birds and reinforces resistance to pathogens such as salmonella.For stability tests, probiotic pills are placed in a chamber at 43% relative humidity at 40 ° C and after 14 days of storage in these extreme conditions, the loss of viability of probiotic bacteria is less than one (1) log of the initial CFU. This example demonstrates that the microorganisms used for animal trawlers including pets such as as several Lactobacillus sp. they can be protected in the composition and drying methods of the present invention and then compressed in a tablet press and be supplied with standard feeds in a typical feed hopper under typical humidity and temperature conditions. EXAMPLE 46 Preparation of effervescent drink tablets containing stable dry composition of probiotic bacteria
[00163] About 10 g powder of dry and stable compositions containing the probiotic bacteria L. acidophilus sp. or Bifidobacterium sp. they are prepared and dried as described in Example 42 and 45.
[00164] Effervescent tablets such as Alka Seltzer®, Fizzies® or sports drinks are finely ground to free flowing powder. The dry stable probiotic composition is mixed with the effervescent powder and compressed in manual pill press equipment (using a 2.22 cm (7/8 ") tablet diameter housing) to form about 2000 mg of sized tablets containing about ten (10) billion live cells per tablet. For stability testing, the probiotic effervescent tablets are placed in a 43% relative humidity chamber at 33 ° C and after 90 days of storage in these extreme conditions, the loss viability of probiotic bacteria is less than one (1) log of the initial CFU This example demonstrates that sensitive biological materials such as live probiotic bacteria can be protected and stabilized in the composition and drying methods of the present invention and then compressed in a press tablets and stored under severe humidity and temperature consumption conditions. EXAMPLE 47 Preparation of tablets containing stable dry composition of probiotic bacteria to treat vaginal infections such as yeast or bacterial vaginosis
[00165] About 15 g of powder of dry and stable compositions containing the probiotic bacteria L. acidophilus sp. they are prepared and dried as described in Examples 1 and 4.
[00166] The dry probiotic composition is mixed with 74 g of lactose, 10 g of corn starch, 0.5 g of magnesium stearate, 0.01 g of sodium carboxymethylcellulose, 0.01 g of polyvinylpyrrolidine and 0.01 g of hydrophobic smoked silica and mixed for 15 minutes. The powder mixture is compressed in a manual tablet press equipment. The weight of the resulting tablet is about 1.5 g. Maximum tablet hardness is 6 to 8 kg. The tablet disintegrated in the water in about 30 seconds. EXAMPLE 48 Production of oil suspension containing stable dry composition of probiotic bacteria Lactobacillus acidophylus (DSM-20356)
[00167] Frozen, concentrated L. acidophylus (200 g obtained from a local fermentation process) was thawed at 37 ° C in a water bath and added with 200 g of 3% pea hydrolyzed protein solution (ultrafiltered hydrolysates, Marcor, Carlstadt, NJ). The bacterium suspension was centrifuged at 4000 g for 15 min (Sorvall RC-5B, Du-Pont Company, Wilmington, DE) and the supernatant decanted. The bacterial precipitate was brought to its original weight (200 g) with 3% hydrolyzed pea protein solution. Additional 50 g of hydrolyzed pea protein was completely dissolved in 80 g of warm water, pH adjusted to 9 with 20% NaOH solution and added to the bacterial culture. Eighty-five (85.6) g of sucrose (obtained from a local market), 30 g of Cyclodextrin-7 (Cargill Mineápolis, MN), 20 g of sodium ascorbate (Sigma) and 15 g of sodium alginate ( ISP Corp., Wayne, NJ) were uniformly mixed in the dry form. The powder mixture was slowly added to the bacterial culture and the mixture was loaded into a 1-room planetary mixer (Charles Ross & Son Company, Hauppauge, New York) at 37 ° C for 20 minutes. The paste was then slowly dripped into a bath containing liquid nitrogen. The frozen beads were then removed from the liquid nitrogen, placed in a bag lined with sealed aluminum foil and stored in an intense freezer at -80 ° C until dry.
[00168] For drying, the frozen beads were spread evenly on trays at a loading capacity ranging from 500 to 1500 g / 0.92 m2 (square foot) and the trays placed on shelves in a freeze dryer (Model 25 SRC , Virtis, Gardiner, NY). A primary drying step was initiated by adjusting the vacuum pressure to between 20-2.7 mm Hg (20002700 mTORR)) and the temperature of the product raised and stabilized between -12 ° C and -5 ° C. Over time (about 1 0-16 h) the product temperature increased to about 20-25 ° C at which point a secondary drying step started at maximum vacuum (0.1-0.15 mm) Hg (100-150 mTORR)) and the product temperature maintained between 30-40 ° C for an additional 14 hours. The formulation was completely dried and its water activity measured below 0.3 Aw. The formulation was ground using a commercially available hammer mill and the particles sieved below 250 microns.
[00169] The viability of the stable composition of probiotic bacteria was tested at 40 ° C and 43% RH for 14 days in a dry powder form or in suspension of corn oil (1g dry powder mixed in 100 g of oil ) or after coating 10 g of the oil suspension in 45 g of chicken feed pellets (the feed pellets were first acclimated in a 33% RH humidity chamber for two weeks). After 14-day incubation at 40 ° C and 43% RH, probiotic bacteria lost only 0.5 log CFU / g when kept in a dry form, 0.34 log when mixed in oil suspension and 0.65 log when coated in chicken feed. These results demonstrate that the viability of probiotic bacteria is preserved in various feeding applications after exposure for 14 days in conditions of high humidity and non-refrigerated storage when using the compositions and methods of the present invention. EXAMPLE 49 Production of stable dry composition containing live phage against Vibrio anguillarum
[00170] Culture of concentrated live phage (100 g obtained from a manufacturer) is placed in a jacketed planetary mixer at 10 ° C. About 50 g of hydrolyzed pea protein (ultrafiltered hydrolysates, Marcor, Carlstadt, NJ) are completely dissolved in 300 g of warm water. The solution is cooled to 10 ° C and added to the phage culture while mixing. One hundred and seventy-four (174) g of sucrose (obtained from a local market), 60 g of Cyclodextrin-7 (Cargill Mineápolis, MN), 40 g of sodium ascorbate (Sigma) and 30 g of sodium alginate (ISP Corp., Wayne, NJ) are uniformly mixed in a dry form. The powder mixture is slowly added to the phage culture and the mixture is loaded into a 1-quarter planetary mixer at 10 ° C for 20 minutes. The paste is then slowly dripped into a bath containing liquid nitrogen. The frozen beads are then removed from the liquid nitrogen, placed in a bag lined with sealed aluminum foil and stored in an intense freezer at -80 ° C until dry. Drying and grinding are carried out as described in Example 48. Ten (10) grams of dry composition powder are mixed with 100 g of fish oil and the suspension coated in 10 kg of Atlantic salmon feed pellets. The coated feed is then stored under typical warehouse storage conditions. The viability of phages in fish feed is preserved after 14 days of exposure in conditions of high humidity and non-refrigerated storage when using the compositions and methods of the present invention.
[00171] Although the invention is illustrated and described here with reference to the specific embodiments, the invention is not intended to be limited to the details shown. Otherwise, several modifications can be made to the details within the scope and range of equivalents of the claims without abandoning the invention.

权利要求:
Claims (21)
[0001]
1. Dry stabilizing composition in an amorphous glassy state, characterized by the fact that it comprises a bioactive material, one or more disaccharides between 10 and 50%, one or more oligosaccharides between 10 and 80%, one or more polysaccharides between 0 , 1 and 10%, one or more hydrolyzed proteins between 0.5 and 40%, and one or more carboxylic acid salts, each percentage based on the total weight of the composition, where the one or more oligosaccharides consists of one or more cyclodextrins, in which the bioactive material consists of a living microorganism, and in which the composition exhibits a reduction in less than one log of Colony Forming Units per gram (CFU / g) after 14 days at 40 ° C and 43% humidity relative (RH).
[0002]
2. Composition according to claim 1, characterized by the fact that the one or more hydrolyzed proteins are selected from the group consisting of hydrolyzed casein, hydrolyzed whey protein, hydrolyzed pea protein, hydrolyzed soy protein, and a mixture of them.
[0003]
3. Composition according to claim 1, characterized by the fact that the one or more polysaccharides are selected from the group consisting of cellulose acetate phthalate (CAP), carboxymethyl-cellulose, pectin, alginic acid salts, hydroxyl propyl methyl cellulose (HPMC), methyl cellulose, carrageenan, gellan gum, guar gum, acacia gum, xanthan gum, locust bean gum, chitosan and chitosan derivatives, collagen, poly (glycolic acid), starches, modified starches, and a mixture of themselves.
[0004]
4. Composition according to claim 1, characterized by the fact that the one or more disaccharides are selected from the group consisting of trehalose, sucrose, lactose, and a mixture of them.
[0005]
5. Composition according to claim 1, characterized in that the carboxylic acid is selected from the group consisting of lactic acid, ascorbic acid, maleic acid, oxalic acid, malonic acid, malic acid, succinic acid, citric acid, gluconic acid , and glutamic acid.
[0006]
6. Composition according to claim 1, characterized by the fact that the living microorganism is a probiotic bacterium.
[0007]
7. Reconstituted liquid, ground powder, tablet, pellet, capsule, human food, animal feed, or coated seed product, characterized by the fact that it is prepared with the composition, as defined in claim 1.
[0008]
8. Nutraceutical product, agricultural pharmaceutical or vaccine product, characterized by the fact that it comprises the composition, as defined in claim 1.
[0009]
9. Human feed, human feed additive, animal feed, animal feed additive, nutraceutical, pharmaceutical, agricultural product or vaccine product, in the form of a bar, liquid formula, colloidal suspension, powder, tablet, capsule and seed coated, characterized by the fact that it is prepared with the composition, as defined in claim 1.
[0010]
10. Composition according to claim 1, characterized by the fact that hydrolyzed proteins are plant proteins.
[0011]
11. Dry stabilizing composition in an amorphous glassy state, characterized by the fact that it comprises a bioactive material, one or more disaccharides between 10 and 50%, one or more oligosaccharides between 10 and 80%, one or more polysaccharides between 0.1 and 10%, one or more hydrolyzed proteins between 0.5 and 40%, and one or more salts of carboxylic acid, each percentage based on the total weight of the composition, in which the bioactive material consists of a living microorganism , and where the composition exhibits a reduction in less than one log of Colony Forming Units per gram (CFU / g) after 14 days at 40 ° C and 43% relative humidity (RH), and in which the composition is prepared by a method comprising: (a) combining the bioactive material with the one or more disaccharides, the one or more oligosaccharides, the one or more polysaccharides, with one or more hydrolyzed proteins, and the one or more salts of carboxylic acid in an aqueous solvent to form a viscous paste; (b) instant freezing of the paste in liquid nitrogen to form solid frozen particles in the form of beads, droplets or chains; (c) primary drying of the frozen particles by vacuum evaporation at a temperature above freezing temperature of the particles to form a primary dry formulation; and (d) secondary drying of the primary dry formulation at maximum vacuum and a temperature of 20 ° C or higher for a time sufficient to reduce the water activity of the primary dry formulation to below 0.3 Aw, whereby the composition is ready.
[0012]
12. Composition, according to claim 13, characterized by the fact that microorganism is a probiotic bacterium. 13. Composition according to claim 12, characterized by the fact that the one or more disaccharides are selected from the group consisting of trehalose, sucrose, lactose, and a mixture of them.
[0013]
14. Composition according to claim 12, characterized by the fact that the one or more oligosaccharides are selected from the group consisting of cyclodextrins, inulin, maltodextrins, dextrans, fructo-oligosaccharide (FOS), galacto-oligosaccharide ( GOS), mannana-oligosaccharide (MOS), and a mixture of them.
[0014]
15. Composition according to claim 12, characterized by the fact that the one or more oligosaccharides consists of cyclodextrins.
[0015]
16. Composition, according to claim 12, characterized by the fact that the one or more polysaccharides are selected from the group consisting of cellulose acetate phthalate (CAP), carboxymethyl-cellulose, pectin, sodium alginate, salts of alginic acid, hydroxyl propyl methyl cellulose (HPMC), methyl cellulose, carrageenan, gum gum, guar gum, acacia gum, xanthan gum, locust bean gum, chitosan and chitosan derivatives, collagen, poly (glycolic acid), starches, modified starches, and a mixture thereof.
[0016]
17. Composition, according to claim 12, characterized by the fact that the one or more hydrolyzed proteins are selected from the group consisting of hydrolyzed casein, hydrolyzed whey protein, hydrolyzed pea protein, hydrolyzed soy protein, and a mixture of them.
[0017]
18. Composition according to claim 12, characterized by the fact that carboxylic acid is selected from the group consisting of lactic acid, ascorbic acid, maleic acid, oxalic acid, malonic acid, malic acid, succinic acid, citric acid , gluconic acid, and glutamic acid.
[0018]
19. Composition according to claim 12, characterized by the fact that hydrolyzed proteins are plant proteins.
[0019]
20. Method for producing a tablet, pill or pellet, characterized by the fact that it comprises compressing a dry stabilizing composition into an amorphous glassy state to form the tablet, pill or pellets, wherein the composition comprises a bioactive material, one or more disaccharides between 10 and 50%, one or more oligosaccharides between 10 and 80%, one or more polysaccharides between 0.1 and 10%, one or more hydrolyzed proteins between 0.5 and 40%, and one or more salts of carboxylic acid , each percentage based on the total weight of the composition, where the one or more oligosaccharides consists of one or more cyclodextrins, where the bioactive material consists of a living microorganism, and where the composition exhibits a reduction in less than a log of Colony Forming Units per gram (CFU / g) after 14 days at 40 ° C and 43% relative humidity (RH).
[0020]
21. Method according to claim 1, characterized by the fact that the one or more living microorganisms comprise a virus, a bacterium, a yeast or a mixture thereof.
[0021]
22. Method for producing a tablet, pill or pellet, characterized by the fact that it comprises compressing a dry stabilizing composition into an amorphous glassy state to form the tablet, pill or pellets, wherein the composition comprises a bioactive material, one or more disaccharides between 10 and 90%, one or more oligosaccharides between 1 and 10%, one or more polysaccharides between 0.1 and 10%, one or more hydrolyzed proteins between 0.5 and 40%, and one or more salts of carboxylic acid , each percentage based on the total weight of the composition, where the one or more oligosaccharides consists of one or more cyclodextrins, where the bioactive material consists of a living microorganism, and where the composition exhibits a reduction in less than a log of Colony Forming Units per gram (CFU / g) after 14 days at 40 ° C and 43% relative humidity (RH).

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法律状态:
2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-03-27| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2018-03-27| B15K| Others concerning applications: alteration of classification|Ipc: A61K 47/36 (2006.01), A61K 47/30 (2006.01), A61K 9 |

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2019-05-21| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2020-05-19| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-09-01| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2020-12-08| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 22/03/2013, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201261614994P| true| 2012-03-23|2012-03-23|

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US61/614,994|2012-03-23|

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US201261642094P| true| 2012-05-03|2012-05-03|

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US61/642,094|2012-05-03|

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US201261646337P| true| 2012-05-13|2012-05-13|

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US61/646,337|2012-05-13|

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PCT/US2013/033505|WO2013142792A1|2012-03-23|2013-03-22|Stabilizing composition for biological materials|

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