Probiotic oral dosage forms and method of enhancing the stability, thereof

Abstract

Provided are probiotic compositions that have enhanced stability under various storage conditions. The stability of the probiotic compositions is enhanced through the addition of various agents and excipients. Examples of agents and excipients that may be used to enhance the stability of probiotic compositions include prebiotics, such as the sugar alcohols mannitol, sorbitol, and lactitol, and/or phytonutrients such as oligomeric proanthocyanidins (OPCs). The probiotic compositions may be formulated into oral dosage forms such as tablets, caplets, and capsules, or manufactured as a chewing gum or as a powder formulation that may be dissolved in a liquid such as water, milk, juice, or yogurt.

Claims

1. A method of enhancing stability of a uniformly blended probiotic composition comprising combining at least one probiotic species, mannitol, sorbitol, at least one prebiotic wherein the prebiotic is a sugar alcohol selected from the group consisting of erythritol, lactitol, maltitol, and xylitol, at least one disintegrant, and at least one glidant; wherein the amount of mannitol in the composition is greater than the amount of the sorbitol, greater than the amount of the at least one disintegrant and greater than the amount of the at least one glidant, wherein the amount of sorbitol in the composition is greater than the amount of the at least one disintegrant and greater than the amount of the at least one glidant, wherein the amount of the at least one disintegrant in the composition is greater than the amount of the at least one glidant.

2. The method of claim 1, wherein the disintegrant is selected from the group consisting of sodium croscarmellose, crospovidone, gellan gum, hydroxypropyl cellulose, starch, and sodium starch glycolate.

3. The method of claim 1, wherein the glidant is selected from the group consisting of silicon dioxide, colloidal silicon dioxide, and talc.

4. The method of claim 1, further comprising combining a lubricant in the composition.

5. The method of claim 4, wherein the lubricant is selected from the group consisting of calcium stearate, magnesium stearate, stearic acid, sodium stearyl fumerate, and vegetable based fatty acids.

6. The method of claim 1, wherein the mannitol or sorbitol is present in the composition in a range of approximately 30% w/w to approximately 98% w/w.

7. The method of claim 1, wherein the at least one probiotic species is selected from the group consisting of Lactobacillus acidophilus, L. bulgaricus, L. casei, L. paracasei, L. fermentum, L. plantarum, L. rhamnosus, L. salivarius, Bifidobacterium bifidum, B. infantis, B. animalis subsp. lactis, B. longum, Streptococcus thermophilis, Enterococcus faecalis, and E. faecium.

8. The method of claim 7, wherein the at least one probiotic species is selected from L. acidophilus, L. salivarius, B. animalis subsp. lactis, and S. thermophilis.

9. The method of claim 1, wherein the composition comprises at least two probiotic species selected from Lactobacillus acidophilus, Bifidobacterium animalis subsp. lactis, and Streptococcus thermophilis.

10. The method of claim 1, wherein the at least one probiotic species is present in the composition in a range of approximately 10.sup.6 to approximately 10.sup.10 colonies per dosage unit.

11. The method of claim 1, further comprising combining a phytonutrient in the composition.

12. The method of claim 11, wherein the phytonutrient is an oligomeric proanthocyanidin (OPC).

13. The method of claim 12, wherein the OPC is extracted from grape seed or pine bark.

14. The method of claim 12, wherein the OPC is present in the composition in a range of approximately 0.5% w/w to approximately 10% w/w.

15. The method of claim 1, further comprising combining a vitamin in the composition, wherein the vitamin is selected from the group consisting of vitamin A (retinol), vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine), vitamin B, (biotin), vitamin B, (folic acid), vitamin B12 (cyanocobalamin), vitamin C (ascorbic acid), vitamin D1 (lamisterol), vitamin D2 (ergocalciferol), vitamin D3 (dihyrotachysterol), vitamin D4 (7-dehydrositosterol), vitamin E (tocopherol), and vitamin K (naphthoquinone).

16. The method of claim 1, further comprising combining a dietary mineral in the composition, wherein the dietary mineral is selected from the group consisting of calcium, chloride, magnesium, phosphorous, potassium, sodium, and sulfur.

17. The method of claim 1, further comprising combining a trace mineral in the composition, wherein the trace mineral is selected from the group consisting of chromium, cobalt, copper, fluorine, iodine, iron, manganese, molybdenum, selenium, and zinc.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a bar graph showing the results of the L. acidophilus stability tests described in Examples 11 and 12 (Tables 32 and 34).

DETAILED DESCRIPTION OF THE INVENTION

(2) Following is a description of exemplary embodiments of the present invention. The definitions set forth below are provided solely for the purpose of describing the exemplary embodiments and are not intended to limit the scope of the present invention. As the embodiments described herein are exemplary, it is to be understood that the invention as described contemplates modifications in the function, purpose, and/or structure of the exemplary embodiments.

(3) As used in this specification and the appended claims, the singular forms a, an, and the include plural referents unless the context clearly dictates otherwise.

(4) As used herein, the term animal refers to a non-human species and the term organism refers to both animal and human species. While the term animal as used herein will typically be used to refer mammals, the term is not exclusive to mammals and where appropriate may include both mammals and non-mammals.

(5) The term excipient refers to an inert substance that is typically used as a diluent or vehicle for a drug or dietary supplement.

(6) The term probiotic refers to bacterial genera that have a beneficial effect in animal organs, such as the human GI and vaginal tracts. The bacterial genera used most often as probiotics are lactobacilli and bifidobacteria; however, other beneficial bacterial species, such as S. thermophilis are also probiotics. After passage through the stomach and small intestine, some probiotics survive and become established transiently in the large bowel, where the colon's fermentation capacity is positively modified. See, e.g., Roberfroid, AM J CLIN NUTR 71(SUPPL):1682S-1687S (2000).

(7) The probiotic species that may be used in the compositions of the present invention may be any known probiotic, such as, for example, L. acidophilus, L. bulgaricus, L. casei, L. paracasei, L. fermentum, L. plantarum, L. rhamnosus, L. salivarius, B. bifidum, B. infantis, B. animalis subsp. lactis, B. longum, S. thermophilis, E. faecalis, and E. faecium. It is to be understood that the foregoing list is intended only to be illustrative and not a limiting representation of the probiotics that may be included in the probiotic compositions of the present invention. In this respect, any additional probiotic species may also be used in the compositions of the present invention, such as, for example, any additional known and/or available lactobacillus or bifidobacterium species.

(8) The term prebiotic refers a nondigestible food ingredient that beneficially affects the host by selectively stimulating growth and/or activity of one or more probiotic species in the colon, such as for example, lactobacilli and/or bifidobacteria. Because prebiotics have a chemical structure that resists digestion through the alimentary tract, they reach the colon as intact molecules where they are able to elicit systemic physiological functions and act as fermentable substrates for colonic microflora. Examples of prebiotics include fructooligosaccharides, such as inulin, which is extracted from chicory, artichokes, asparagus, dandelions, dahlias, endive, garlic, leeks, lettuce, and onions; trans-galactosyloligosaccharides; glactosyllactose; isomaltooligosaccharides; raffinose; lactulose; lactitol (a sugar alcohol); and partially hydrolyzed guar gum. The most commonly used prebiotic is inulin. While inulin has been found to be a good substrate for bacteroides and bifidobacterium species (B. bifidum and B. longum excluded) it is not an excellent substrate for other probiotics. Roberfroid et al., NUTR. REV. 51:137-146 (1993). Where a prebiotic is combined with a probiotic, the resulting supplement is sometimes referred to as a synbiotic.

(9) An example of a prebiotic that may be used with the probiotic compositions of the present invention is the sugar alcohol lactitol, which is currently commercially used as a replacement sweetener for low calorie foods. Lactitol has two calories (9 kilojoules) per gram and approximately 40% of the sweetness of sugar. The United States Food and Drug Administration (FDA) classifies lactitol, along with other sugar alcohols, which may or may not be prebiotics, i.e., erythritol, maltitol, mannitol, sorbitol, and xylitol, as generally recognized as safe (GRAS).

(10) As noted above, the choice of the prebiotic to be combined with a particular probiotic will be determined according to which prebiotic provides the most suitable substrate for a particular probiotic. When considering the use of a particular prebiotic, synergy between one or more prebiotics and/or synergy between one or more prebiotics and other ingredients should always be considered. Synergy between the prebiotics lactitol and OPC with the sugar alcohols mannitol and sorbitol is illustrated in the experiment set forth in Example 10.

(11) In Example 10, the mannitol and sorbitol sugar alcohols described in the carbohydrate carrier system disclosed in U.S. Patent Publication No. 2003/0118642 A1 to Norman et al., which is incorporated by reference herein, were combined with L. acidophilus to form two probiotic batches (Table 27), which were tested for stability (Table 28). As a control, the PHARMABURST product (SPI Polyols, New Castle, Del.), which is a commercially available product covered by the Norman et al. Patent Publication, was used as a carrier to prepare an L. acidophilus probiotic; PHARMABURST includes the sugar alcohols mannitol and sorbitol. As shown in Table 28, mannitol and sorbitol alone were incapable of keeping L. acidophilus viable under the harsh storage conditions of 40 C./75% RH and as shown in Table 30, while PHARMABURST was capable of keeping L. acidophilus viable for 2 weeks under the mild storage conditions of 25 C./60% RH, the viability was limited. By contrast, as shown in Tables 31 and 32 (Example 11), when lactitol and/or OPC were added to the L. acidophilus probiotic formulation of Table 29, L. acidophilus maintained a high level of stability for two weeks under the harsh storage conditions of 40 C./75% RH without a desiccant (see, Table 32, Batch E), indicating a synergistic effect between the sugar alcohols mannitol and sorbitol and the prebiotics lactitol and/or OPC. The foregoing indicates that L. acidophilus probiotics that are formulated to include at least the sugar alcohols mannitol and sorbitol in combination with lactitol and/or OPC results in probiotic compositions that have a long shelf-live.

(12) To determine if lactitol has a significant effect on the stability of L. acidophilus, three batches of L. acidophilus probiotic tablets were prepared as set forth in Table 33 (Example 12), one batch with L. acidophilus and lactitol (Batch I), a second with L. acidophilus and PHARMABURST (Batch J), and a third with L. acidophilus, lactitol, and PHARMABURST (Batch K). The results of Table 34 (Example 12) show that under the harsh conditions of 40 C./75% RH, lactitol alone was not effective in maintaining the stability of L. acidophilus whereas PHARMABURST alone and the combination of a relatively low weight percent of PHARMABURST in combination with lactitol showed some viable colonies of L. acidophilus after two weeks. A log graph comparing the stability test results of Batch E (Table 32) with Batches I, J, and K (Table 34) is shown in FIG. 1. As is shown in the graph, under the harsh storage conditions of 40 C./75% RH, after two weeks, 100% lactitol failed to stabilize L. acidophilus; 100% PHARMABURST showed limited viability of L. acidophilus; a combination of 10% PHARMABURST and 90% lactitol showed limited viability of L. acidophilus; and a combination of 87% PHARMABURST and 13% lactitol showed significant viability of L. acidophilus. The results of the stability tests as shown in FIG. 1 indicate that at an optimal ratio, a synergistic effect occurs between the PHARMABURST ingredients and lactitol.

(13) The term phytonutrient refers to a nutrient derived from a plant source that has a beneficial effect on the health of the organism, i.e., the animal or human, taking the phytonutrient. Phytonutrients differ from nutrients in that they are not required for normal metabolism of the organism. Many phytonutrients are antioxidants that impart bright colors to fruits and vegetables. For example, lutein makes corn yellow, lycopene makes tomatoes red, carotene makes carrots orange, and anthocyanin makes blueberries blue. Both the bright colors and the antioxidant properties of phytonutrients are due to alternating single-bonded and double-bonded carbons. Phytonutrients that may be included in the probiotic formulations of the present invention include without limitation catechins, which are found in tea; polyphenols, which are found in fruit skins such as grape skin, apple skin, and orange skin; and oligomeric proanthocyanidins (OPCs) which are found in fruits, vegetables, nuts, seeds, flowers, and bark, but which are generally extracted from grape seeds and/or pine bark. The isolation of OPC is described in U.S. Pat. Nos. 3,436,407 and 4,698,360, both to Masquelier et al. Tables 31 and 32 (Batch H) of Example 11 shows that the addition of OPC (without lactitol) to the L. acidophilus probiotic composition described therein maintained the stability of L. acidophilus at a very high level for two weeks under the harsh storage conditions of 40 C./75% RH without a desiccant. Within the context of the present invention, OPCs may be added to the probiotic compositions in a wide range, such as for example, from approximately 0.05% w/w to approximately 25% w/w; generally; however, a range of approximately 0.5% w/w to approximately 10% w/w should be sufficient for the OPC to show effect.

(14) In addition to the foregoing, the present invention also includes the addition of at least one probiotic species in combination with at least one prebiotic and at least one OPC. As shown in Table 18 of Example 7, the addition of the prebiotic lactitol and the phytonutrient OPC to the L. acidophilus and B. animalis subsp. lactis probiotic composition described therein resulted in the highest stability count for the probiotic composition at 6 months under the storage conditions of 25 C./60% RH with a desiccant. Further, Batch I of Tables 31 and 32 of Example 11 demonstrate that the addition of the prebiotic lactitol and the phytonutrient OPC to the L. acidophilus probiotic composition described therein maintained the stability count of the L. acidophilus probiotic composition at a very high level for two weeks under the harsh storage conditions of 40 C./75% RH without a desiccant.

(15) Additional ingredients that may be included with the probiotic combinations described above include vitamins, dietary minerals, trace minerals, and other phytonutrients. See Example 8, Tables 20-22.

(16) Vitamins that may be included in the probiotic composition of the present invention include without limitation, vitamins such as vitamin A (retinol), vitamin B.sub.1 (thiamine), vitamin B.sub.2 (riboflavin), vitamin B.sub.3 (niacin), vitamin B.sub.5 (pantothenic acid), vitamin B.sub.6 (pyridoxine), vitamin B.sub.7 (biotin), vitamin B.sub.9 (folic acid), vitamin B.sub.12 (cyanocobalamin), vitamin C (ascorbic acid), vitamin D.sub.1 (lamisterol), vitamin D.sub.2 (ergocalciferol), vitamin D.sub.3 (dihyrotachysterol), vitamin D.sub.4 (7-dehydrositosterol), vitamin E (tocopherol), and vitamin K (naphthoquinone). The foregoing listing includes vitamin salts, such as for example, retinol acetate, retinol palmitate, and thiamine mononitrate.

(17) Dietary minerals that may be included in the probiotic compositions of the present invention include without limitation, calcium, chloride, magnesium, phosphorous, potassium, sodium, and sulfur.

(18) Trace minerals that may be included in the probiotic compositions of the present invention include without limitation, chromium, cobalt, copper, fluorine, iodine, iron, manganese, molybdenum, selenium, and zinc.

(19) The probiotic compositions of the present invention may be manufactured into various formulations, such as for example, oral dosage forms, such as tablets, caplets, or capsules; chewing gum; or powders to be dissolved in a liquid.

(20) As noted above, the probiotic compositions of the present invention may be formulated into oral dosage forms, such as tablets, caplets, or capsules. The probiotic tablets and caplets of the present invention may be formulated for swallowing or for chewing. In the latter case, the tablets and caplets should be prepared with flavoring. Where necessary, the flavored chewable tablets and caplets may include a sweetener, which may be an artificial or natural sweetener or both. The probiotic capsules of the present invention will be manufactured primarily for swallowing.

(21) Procedures for preparing tablets, caplets, and capsules are known to those of ordinary skill in the art and include without limitation wet granulation, dry granulation, and direct compression (for tablets and caplets).

(22) Wet and dry granulation is used to manufacture tablets, caplets, or capsules. With granulation techniques, a chilsonation is used to manufacture the powder for the dosage forms. A chilsonator houses grooved, rotating rollers that are pressed tightly against one another by hydraulic pressure. Raw materials are placed into the hopper of the chilsonator and are fed by a system of horizontal and vertical screws into the rollers. As materials pass through the grooves in the rollers, it is compacted under very high pressure and emerges from the chilsonator as dense sheets. The sheets are milled into a fine granular powder using a Fitz mill and then passed through a screen to produce a uniform free flowing granule. The chilsonation process results in a finished powder that is two to four times denser than the starting material, a feature that permits the ingredients to be fashioned into the desired dosage form.

(23) With dry granulation, the powder may be incorporated into a gelatin capsule or it may be mixed with gelatin to form a tablet or caplet. With wet granulation, the powder is moistened thus creating large chunks of material that are subsequently dried and milled to convert the chunks to particles of a desired size for the manufacturing process. Once the particles of a desired size are obtained, the particles are incorporated into a gelatin capsule or mixed with gelatin to form a tablet or caplet.

(24) Most direct compression formulations consist of three types of ingredients: an inert carrier that provides volume to the final dosage form; a lubricant that aids in the compression process; and the active ingredients. Carriers may be present in dosage forms in many ranges, from 0.5% w/w to 95% w/w. Within the context of the present invention, carriers used to formulate the probiotic compositions of the present invention will generally be in the range of approximately 30% w/w to approximately 98% w/w. The carrier used in the Examples is a PHARMABURST carrier (SPI Polyols, New Castle, Del.).

(25) With direct compression, the ingredients are mixed in a batch blender, such as a twin-shell or V-blender, and discharged into a bin (usually portable), which then feeds a chute to the tableting process. As an alternative to batch blending, in-bin blending is also used. With in-bin blending, the unmixed material is placed in a portable bin, which may contain internal baffles, and is tumbled; in-bin blending avoids the need to transfer the material from the blender to the portable container. In direct compression formulations, there is a wide particle-size distribution with the active agent usually being at the fine end of the range. To avoid segregation of the particles, reliable flow must be maintained in the bins at all time. To ensure that all tablets are of the highest quality, tablets must be sampled at regular intervals during production for active ingredient concentration, tablet hardness, and dissolution rates.

(26) Direct compression may be used to make tablets and caplets for swallowing and also chewable tablets and caplets. With the latter, the hardness of the tablets and caplets will need to be reduced and the ingredients of the tablets and caplets will need to be adjusted to ensure that the tablets and caplets have a pleasant taste and pleasant mouth-feel. The taste of the chewable tablets and caplets may be adjusted through the use of various flavorings and sweeteners and the mouth-feel of the chewable tablets and caplets may be adjusted though the use of varying prebiotics, such as inulin, lactitol, mannitol, etc. The mouth feel of the chewable tablets must be adjusted to ensure that the when the tablet or caplet body is crushed, it folds into the flavored matrix; if the body is too brittle, it may fracture causing an uncomfortable sensation in the user's mouth. For chewable tablets, where taste is significant to user acceptability, the product should also have a pleasant odor when the bulk package is opened. Each of the features of the chewable tablet may be attained by adjusting the excipients until the desired properties are achieved.

(27) Depending on the final size and characteristics of the tablets and caplets, i.e., size, hardness, brittleness, mouth feel for chewing, etc., two different direct compression machines may be used to manufacture the dosage forms. Single punch presses typically exhibit low compression speeds while rotary presses exhibit high compression speeds. The Examples describe the use of both a single punch press (Examples 1 and 2) and rotary presses (also called rotary machines; Examples 2-4 and 7-10).

(28) The probiotic compositions of the present invention may also be incorporated into chewing gum. The chewing gum preferably includes a flavor and may be hard chewing gum or soft chewing gum. Where necessary, the flavored hard or soft chewing gum may include a sweetener, which may be an artificial or natural sweetener or both. Procedures for manufacturing gum are known to those of ordinary skill in the art.

(29) Gum is traditionally made using four ingredients: a gum base, such as a resin obtained from pine trees; a natural sweetener, such as sugar, or an artificial sweetener; a softener, such as glycerin; and flavoring. The raw materials for the gum are mixed with a mixer while kept at a constant temperature during the prescribed processing time. Mixed gum materials are then sent to a hopper and extruded by twin screws to make a gum sheet. The inside and outside of the gum sheets are powdered to prevent sticking to the matching during rolling and packaging. During the extruding process, the gum sheet thickness is controlled with a roller. After extrusion, the gum sheet is scored and cut for proper sizing, passed through a cooling tunnel, and stacked on trays for packaging. Where the gum is a hard chewing gum, such as a chewing gum tablet, the gum is coated between the cutting and cooling steps. Specially designed machines are available for each of the steps; thus, gum can be made using a mixing machine, an extruding machine, a forming machine, a cooling machine, and a stacking machine.

(30) Gum may also be prepared using the direct compression procedure described in U.S. Patent Publication No. 2004/0013767 to Norman and Amin, which is incorporated by reference herein. Under this procedure, a gum base, granulating agent, processing aid, and one or more lubricants are mixed and subjected to direct compression on a traditional tabletting machine. Sweeteners, colorings, and flavorings may also be added to the mixture. A commercially available mixture of polyol(s) and/or sugars in a gum base is sold commercially as PHARMAGUM (SPI Polyols, Inc., New Castle, Del.). The direct compression procedure is particularly useful for preparing chewing gum tablets.

(31) In both of the foregoing procedures, the probiotic is added during the initial mixing stage prior to extrusion or direct compression.

(32) In addition to the foregoing, the probiotic compositions of the present invention may be prepared as a powder that is intended to be dissolved in a liquid, such as water, milk, juice, and yogurt. It is understood that the individual liquids may be mixed together where appropriate. For example, the probiotic formulation may be combined with fruit juice and yogurt or milk and yogurt to make probiotic yogurt shakes. The probiotic formulation may also be combined with milk and ice cream to make probiotic milk shakes. Flavorings for the probiotic liquid formulations contemplated under the invention are known to those of ordinary skill in the art.

(33) Examples of excipients that may be used to formulate appropriate dosage forms include binders, disintegrants, lubricants, coatings, plasticizers, compression agents, wet granulation agents, and sweeteners, all of which are known to those of ordinary skill in the art to which the invention pertains. All of the following examples are provided by way of illustration and not limitation. Binders are used where appropriate to help the dosage form ingredients still together. Examples of binders include carbopol, povidone, and xanthan gum. Lubricants are generally always used in the manufacture of dosage forms by direct compression in order to prevent the compacted powder mass from sticking to the equipment during the tabletting or encapsulation process. Examples of lubricants include calcium stearate, magnesium stearate, stearic acid, sodium stearyl fumerate, and vegetable based fatty acids. Disintegrants aid in the break up of the compacted mass when placed in a fluid environment. Examples of disintegrants include sodium croscarmellose, crospovidone, gellan gum, hydroxypropyl cellulose, starch, and sodium starch glycolate. Coatings are used to control the solubility of the drug. Examples of coatings include carrageenan, cellulose acetate phthalate, ethylcelulose, gellan gum, matodextrin, methacrylates, methylcellulose, microcrystalline cellulose, and shellac. Plasticizers are used to control the release rate of the drug from the dosage form. Examples of plasticizers include citrate esters, dibutyl sebacate, diethyl phthalate, polyvinylacetate phthalate, and triacetin. Compression agents include calcium carbonate, dextrose, fructose, guar gum, honey, lactose, maltodextrin, maltose, mannitol, microcrystalline cellulose, molasses, sorbitol, starch, and sucrose. Wet granulation agents include calcium carbonate, lactose, maltodextrin, mannitol, microcrystalline cellulose, povidone, and starch. Sweeteners include aspartame, dextrose, fructose, honey, lactose, maltodextrin, maltose, mannitol, molasses, monoammonium glycyrrhizinate, sorbitol, sucralose, and sucrose. Excipients that are generally used in the manufacture of chewable tablets include by way of illustration and not limitation, dextrose, fructose, guar gum, lactose, maltodextrin, maltose, mannitol, microcrystalline cellulose, and sorbitol. As is evident from the foregoing list, many of the same ingredients may be used for various different purposes in various different dosage forms.

(34) In each of the probiotic compositions described above, the probiotic should be present in a range of approximately 10.sup.6 colonies per dosage unit to approximately 10.sup.10 colonies per dosage unit, although higher counts are also acceptable. Where the probiotic composition is to be administered to an adult human being, i.e., a human being over the age of 16, the probiotic is preferably in the range of at least 10.sup.9, more preferably 10.sup.10 or even higher. Where the probiotic composition is to be administered to a child, i.e., a human being under the age of 16, the probiotic is preferably in the range of approximately 10.sup.6 to approximately 10.sup.8. The Examples set forth various studies to determine the conditions at which the probiotic compositions of the present invention have the longest duration. As is shown in Example 10, and as would be expected, the storage conditions play a significant difference in the duration of the probiotic compositions, c.f., the stability of the probiotic compositions at 25 C./60% RH, 30 C./65% RH, and 40 C./75% RH (Tables 16-18, 20-22 and 24-26). Further, the presence of a desiccant in the storage package may also make a difference under certain storage conditions, although, this is not always the case; cf., Tables 12, 13, 16-18. Examples of desiccants that can be used with the present invention include without limitation, silica gel (silicon dioxide), indicating silica gel (silica gel washed with cobalt chloride), montmorillonite clay, calcium oxide, calcium sulfate, activated alumina beads, and molecular sieve (e.g., aluminosilicate materials or synthetic compounds such as clays, porous glass, microporous charcoal, or active carbon).

(35) The probiotic compositions of the present invention have widespread utility in the manufacture of various consumable commercial products that upon ingestion by a human or an animal will regulate their digestive systems by ensuring that the digestive tract is populated by live probiotic species. Because it is essential that probiotic species are viable upon ingestion, the enhanced stability of the probiotic species in the probiotic compositions of the present invention ensure that the probiotic compositions have the capability to reduce the number of pathogenic species in the colon. Examples of pathogenic bacteria that may be reduced through ingestion of the probiotic compositions of the present invention include without limitation, Campylobacter jejuni, E. coli, S. aureaus, Vibrio cholera, bacteroides, clostridia, klebsiella, listeria, proteus, salmonella, shigella, and veilloniella. An example of pathogenic yeast that may be reduced though ingestion of the probiotic compositions of the present invention include without limitation C. albicans. As previously noted, beneficial probiotic species, such as lactobacilli and bifidobacteria keep those potentially disease causing pathogens under control preventing disease-related dysfunctions.

(36) All patent and non-patent publications mentioned herein, both supra and infra, are incorporated by reference in their entireties.

(37) An advantage of the probiotic formulations of the presenting invention is that they are a pleasant-tasting product that may be consumed without water. Further, the probiotic formulations of the present invention are stable at room temperature and thus, do not need special conditions in order to improve or sustain the shelf life of the product.

EXPERIMENTAL

(38) The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the compositions of the invention. The examples are intended as non-limiting examples of the invention.

(39) The following examples will use, unless otherwise indicated, conventional techniques of pharmaceutical formulation, medicinal chemistry and the like, which are within the skill of the art. Such techniques are explained fully in the literature. Preparation of various types of pharmaceutical formulations are described, for example, in REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY, 20.sup.th edition (Lippincott Williams & Wilkins, 2000) and Ansel et al., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 6.sup.th Ed. (Media, Pa.: Williams & Wilkins, 1995).

(40) In the examples that follow, efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but experimental error and deviations should be taken into account when conducting the described experiments. Unless indicated otherwise, parts are parts by weight, temperature is degrees centigrade and pressure is at or near atmospheric. All components were obtained commercially unless otherwise indicated.

(41) In each of the stability tests set forth in the examples, five tablets per pull were tested for L. acidophilus stability testing with L. acidophilus alone; five tablets per pull were tested for B. animalis subsp. lactis stability testing with B. animalis subsp. lactis alone, and five tablets per pull were tested when L. acidophilus and B. animalis subsp. lactis were tested as a combined assay; and six tablets per pull were tested for samples where L. acidophilus and B. animalis subsp. lactis were individually assayed from the combined product. For the stability testing, weekly, biweekly, or monthly pulls were carried out until a reduction in assay count below 110.sup.6 was observed (exceptions to this procedure are noted in the examples).

Example 1

Manufacture of L. Acidophilus Tablets

(42) A batch of twenty L. acidophilus probiotic tablets were manufactured using the ingredients set forth in Table 1.

(43) TABLE-US-00001 TABLE 1 Ingredients mg/tab % w/w L. acidophilus 50.0 10.0 100 billion colonies/gram PHARMABURST C1 440.0 88.0 (carrier) Magnesium stearate 10.0 2.00 (lubricant) 0 0 Total 500.0 100.00

(44) The L. acidophilus tablets were prepared by direct compression on a single punch machine using standard concave tooling. Hardness of the tablets was 8-10 kp (kilopond=kilogram/N=force exerted by one kg of mass at Earth gravity). The compressed tablets were put into 60 cc high-density polyethylene (HDPE) bottles and subjected to the controlled temperature of 25 C./60% RH. The product was subjected to microbiological stability testing at the following time intervals: 0 time, 1 month, 2 months, 4.5 months, 6 months, and 8.5 months. The results of the stability test are set forth in Table 2.

(45) TABLE-US-00002 TABLE 2 L. acidophilus Stability Time Periods (Presence of Anaerobe, Gram Positive Rod) Tested Total Count/gm 0 Time 1.08 10.sup.10 1 month 6.3 10.sup.9 2 months 5.8 10.sup.9 4.5 months 3.2 10.sup.8 6 months 8.1 10.sup.5 8.5 months 5.2 10.sup.5

Example 2

Manufacture of L. Acidophilus Tablets with Color, Flavor, and Sweetener

(46) L. acidophilus probiotic tablets were prepared using the ingredients set forth in Table 3. Prior to tabletting, the L. acidophilus raw material was tested and found to have a microbial count of 1.810.sup.11 colonies/gram.

(47) TABLE-US-00003 TABLE 3 Orange Bubble Gum Pineapple Flavored Tablets Flavored Tablets Flavored Tablets 800 Tablets 600 Tablets 700 Tablets Ingredients mg/tab % w/w mg/tab % w/w mg/tab % w/w L. acidophilus 50.0 8.3333 50.0 8.3333 50.0 8.3333 PHARMABURST C1 539.3 89.8833 537.1 89.5167 535.9 89.3167 (carrier) Sucralose 0.9 0.1500 0.9 0.1500 0.9 0.1500 (ChemPoint.com, Seattle, WA) (sweetener) Spray dried natural and 0.6 0.1000 1.8 0.3000 3.0 0.2000 artificial flavor FD&C Colorant 1.2 0.2000 1.2 0.2000 1.2 0.2000 Magnesium Stearate 8.0 1.3333 9.0 1.5000 9.0 1.5000 (lubricant) Total 600.0 100.0000 600.0 100.0000 600.0 100.0000 *Pink, Yellow, and Orange FD&C colorants were selected in amounts suitable to match a color to the selected flavor.

(48) The orange and bubble gum flavored tablets were prepared by direct compression on a rotary machine using a round standard concave tooling and the pineapple flavored tablets were prepared using a single punch press capsule-shaped tooling. The hardness of all of the tablets was in the range of 7-9 kp and disintegration times were all approximately 22 seconds.

(49) The tablets of Table 3 were stored at 25 C./60% RH for stability testing without a desiccant, with a set of three tablets of each batch set aside as reserve samples for each flavor dosage form; the reserve samples were stored at 4 C. Table 4 sets forth the results of the monthly stability tests on the dosage forms of Table 3 over an 11-month period and Table 5 sets forth the results of the stability test of the reserve samples at month 13; five tablets were selected for each stability test pull. The stability of the L. acidophilus in the dosage forms was determined by measuring the presence of the gram-positive rod of L. acidophilus.

(50) TABLE-US-00004 TABLE 4 L. acidophilus Stability (Presence of Anaerobic Gram-Positive Rod) Total Count/gram Orange Bubble Gum Pineapple Time Periods at Flavored Flavored Flavored 25 C./60% RH Tablets Tablets Tablets 0 Time 9.45 10.sup.9 7.6 10.sup.9 9.15 10.sup.9 1 Month 7.45 10.sup.9 7.1 10.sup.9 8.25 10.sup.9 2 Month 8.05 10.sup.9 7.7 10.sup.9 7.05 10.sup.9 3 Month 3.70 10.sup.9 4.15 10.sup.9 2.55 10.sup.9 4 Month 1.90 10.sup.9 1.05 10.sup.9 1.05 10.sup.9 5 Month 2.95 10.sup.8 2.75 10.sup.8 1.0 10.sup.8 6 Month 2.85 10.sup.8 2.25 10.sup.8 6.5 10.sup.7 7 Month 8.5 10.sup.7 7.0 10.sup.7 3.5 10.sup.7 8 Month 1.4 10.sup.7 1.7 10.sup.7 9.5 10.sup.6 9 Month 8.5 10.sup.5 1.0 10.sup.6 4.95 10.sup.5 10 Month 5.55 10.sup.5 4.20 10.sup.5 1.65 10.sup.5 11 Months 7.54 10.sup.4 6.90 10.sup.4 6.2 10.sup.4

(51) TABLE-US-00005 TABLE 5 L. acidophilus Stability (Presence of Anaerobe, Gram Positive Rod) Total Count/gram Orange Bubble Gum Pineapple Time Period Flavored Flavored Flavored at 4 C. Tablets Tablets Tablets 13 Months 7.53 10.sup.9 4.65 10.sup.9 7.55 10.sup.9

(52) As indicated in Table 4, at ten months, the total count of gram-positive rods of L. acidophilus in each flavor batch was less than 110.sup.6; by contrast, the samples stored at 4 C. showed no reduction in microbial count at 13 months.

Example 3

Manufacture of B. Animalis Subsp. Lactis BB 12 Tablets

(53) A batch of thirty B. animalis subsp. lactis strain BB 12 (hereinafter referred to as B. animalis subsp. lactis) probiotic tablets were manufactured using the ingredients set forth in Table 6.

(54) TABLE-US-00006 TABLE 6 Ingredients mg/tab % w/w B. animalis subsp. lactis 50.0 10.0 100 billion colonies/gram PHARMABURST C1 442.50 88.50 (carrier) Magnesium stearate 7.50 1.50 (lubricant) 0 0 Total 500.0 100.00

(55) The B. animalis subsp. lactis tablets were prepared by direct compression on a rotary machine using 7/16 standard concave tooling. Hardness of the tablets was in the range of 10-12 kp. The compressed tablets were put into 60 cc HDPE bottles without a desiccant and exposed to the controlled temperature of 25 C./60% RH. Five tablets per pull were subjected to microbiological stability testing at the following time intervals: 0 time, 1 month, 2 months, and 3 months. The results of the stability test are set forth in Table 7.

(56) TABLE-US-00007 TABLE 7 B. animalis subsp. lactis Stability Time Period at (Presence of Anaerobe, Gram Positive Rod) 25 C./60% RH Total Count/gram 0 Time.sup. 3.8 10.sup.9 1 month.sup. 3.9 10.sup.6 2 months 2.1 10.sup.5 3 months 1.9 10.sup.5

(57) The results of the stability testing were below 110.sup.6 at the two-month pull, but were continued for an additional month.

Example 4

Manufacture of L. Acidophilus Tablets with Color, Flavor, and Sweetener

(58) Probiotic tablets (2000 tablets per flavor batch) containing both L. acidophilus and B. animalis subsp. lactis probiotic were prepared using the ingredients set forth in Table 8.

(59) TABLE-US-00008 TABLE 8 Orange Bubblegum Pineapple Flavored Tablets Flavored Tablets Flavored Tablets 2000 Tablets 2000 Tablets 2000 Tablets Ingredients mg/tab % w/w mg/tab % w/w mg/tab % w/w L. acidophilus 50.00 7.69 50.00 7.69 50.00 7.69 100 billion colonies/gram B. animalis subsp. lactis 50.00 7.69 50.00 7.69 50.00 7.69 100 billion colonies/gram PHARMABURST C1 537.3 82.66 536.10 82.48 534.90 82.29 (carrier) Sucralose 0.90 0.14 0.90 0.14 0.90 0.14 (sweetener) Spray dried natural & artificial 0.60 0.09 1.80 0.28 3.00 0.46 flavor FD & C Colorant 1.20 0.18 1.20 0.18 1.20 0.18 Magnesium Stearate 10.0 1.54 10.00 1.54 10.00 1.54 (lubricant) Total 650.0 100.0 650.00 100.00 650.00 100.00

(60) The tablets were prepared by direct compression on a rotary machine using a round standard concave tooling. The hardness of the tablets was in the range of 8-10 kp. The compressed tablets were packaged into 180 cc HDPE bottles of 100 tablets each without a desiccant and exposed to the controlled temperature of 25 C./60% RH. The product was subjected to monthly microbiological stability testing over a period of 12 months or until a reduction in the assay count below 110.sup.6 was observed. Six tablets per pull were analyzed at 0 time and 1 month for both L. acidophilus and B. animalis subsp. lactis and five tablets per pull were tested for L. acidophilus and B. animalis subsp. lactis independently for the remaining time periods. The results of the stability test are set forth in Tables 9 and 10, respectively.

(61) TABLE-US-00009 TABLE 9 L. acidophilus and B. animalis subsp. lactis Stability (Presence of Anaerobe, Gram Positive Rod) Total Count/gram Orange Bubble Gum Pineapple Time Period at Flavored Flavored Flavored 25 C./60% RH Tablets Tablets Tablets 0 Time 4.2 10.sup.9 3.35 10.sup.9 4.45 10.sup.9 1 Month 4.3 10.sup.9 3.8 10.sup.9 4.2 10.sup.9

(62) TABLE-US-00010 TABLE 10 Stability (Presence of Anaerobe, Gram Positive Rod) Total Count/gram Orange Bubble Gum Pineapple Time Period at Flavored Tablets Flavored Tablets Flavored Tablets 25 C./60% RH Lacto Bifido Lacto Bifido Lacto Bifido 2 Months 1.19 10.sup.9 1.18 10.sup.9 1.08 10.sup.9 1.02 10.sup.9 1.18 10.sup.9 1.19 10.sup.9 3 Months 1.8 10.sup.9 2.15 10.sup.9 1.95 10.sup.9 1.65 10.sup.9 2.35 10.sup.9 2.0 10.sup.9 4 Months 5.6 10.sup.8 6.7 10.sup.8 7.75 10.sup.8 7.2 10.sup.8 6.15 10.sup.8 7.05 10.sup.8 5 Months 6.95 10.sup.7 6.9 10.sup.7 5.85 10.sup.7 6.15 10.sup.7 5.55 10.sup.7 4.0 10.sup.7 6 Months 5.8 10.sup.6 4.2 10.sup.6 3.15 10.sup.6 2.05 10.sup.6 3.9 10.sup.6 3.45 10.sup.6 7 Months 2.05 10.sup.6 3.0 10.sup.6 2.86 10.sup.6 1.4 10.sup.6 1.95 10.sup.5 2.1 10.sup.5 8 Months 2.8 10.sup.5 2.95 10.sup.5 2.6 10.sup.5 2.5 10.sup.5 4.15 10.sup.4 4.25 10.sup.4

(63) As noted in Table 10, after 8 months, the orange flavored tablets and the bubble gum flavored tablets yielded less than 1.010.sup.6 total counts/gram of gram positive rods and after 7 months, the pineapple flavored tablets yielded less than 110.sup.6 total counts/gram of gram positive rods; accordingly, the stability study was discontinued after the eight-month pull.

Example 5

Composite Packaging without Desiccant

(64) The three flavor batches of Example 4 were tested for stability in a composite packaging of 100 tablets (33 of each flavor plus one additional tablet randomly selected) without a desiccant by placing the composite packaging in a 180 cc HDPE bottle at the controlled temperature of 25 C./60% RH and testing the tablets for microbiological stability each month for a period of 12 months or until an assay count below 110.sup.6 was observed. Five tablets per pull (randomly selected from each flavor) were analyzed for total count of L. acidophilus and B. animalis subsp. lactis combined at 1 month and six tablets per pull (randomly selected from each flavor) were analyzed for L. acidophilus and B. animalis subsp. lactis independently for the remaining time periods (in this experiment, the typical six tablet per pull per for the combined pulled probiotic and five tablet per pull for the independently pulled probiotic was reversed). The results of the stability tests are set forth in Tables 11 and 12, respectively.

(65) TABLE-US-00011 TABLE 11 Stability Time Period at (Presence of Anaerobe, Gram Positive Rod) 25 C./60% RH Total Count/gram 1 Month 4.55 10.sup.9

(66) TABLE-US-00012 TABLE 12 Stability (Presence of Anaerobe, Gram Positive Rod) Time Period at Total Count/gram 25 C./60% RH L. acidophilus B. animalis subsp. lactis 2 Months 4.55 10.sup.9 5.50 10.sup.9 3 Months 1.5 10.sup.9 1.75 10.sup.9 4 Months 3.2 10.sup.8 3.3 10.sup.8 5 Months 5.35 10.sup.6 9.1 10.sup.6 6 Months 3.5 10.sup.5 1.9 10.sup.5 7 Months 1.3 10.sup.5 1.75 10.sup.5

(67) The results of the stability testing were below 110.sup.6 after six months, but were continued for an additional month.

Example 6

Composite Packaging with Desiccant

(68) The three flavor batches of Example 6 were tested for stability in a composite packaging of 100 tablets (33 of each flavor plus one additional tablet randomly selected) with a desiccant by placing the composite packaging in a 180 cc HDPE bottle at the controlled temperature of 25 C./60% RH and testing the tablets for microbiological stability at 3 months, 6 months, 9 months, and 12 months, or until an assay count below 110.sup.6 was observed. The tablets were analyzed for L. acidophilus and B. animalis subsp. lactis independently. The results of the stability test are set forth in Table 13.

(69) TABLE-US-00013 TABLE 13 Stability (Presence of Anaerobe, Gram Positive Rod) Time Period at Total Count/gram 25 C./60% RH L. acidophilus B. animalis subsp. lactis 3 Months 1.34 10.sup.9 1.32 10.sup.9 6 Months 2.65 10.sup.5 2.05 10.sup.5

(70) The results of the stability testing were below 110.sup.6 after six months; accordingly, the study was discontinued after the six-month pull.

Example 7

Manufacture of Probiotic Dosage Form with L. Acidophilus, B. Animalis Subsp. Lactis, Lactitol (Prebiotic), and OPC from Grape Seed Extract (Antioxidant)

(71) Batches of orange flavored tablets were manufactured with PHARMABURST C1, one of two lactitol prebiotics, i.e., CM 50 at 7.7% or FINLAC DC (Xyrofin Oy Corp., Helsinki, Finland) at 39.7% with a corresponding decrease in PHARMABURST C1, and OPC from grape seed extract. The batches were used to test the following parameters: (1) the organoleptic characteristics of lactitol and OPC containing products; (2) the effect of the prebiotic lactitol on probiotic stability; and (3) the effect of OPC on probiotic stability. The ingredients set forth in manufacturing the tablets for each batch are set forth in Table 14; the heading for each batch specifies the lactitol sweetener used and the number of tablets per batch.

(72) TABLE-US-00014 TABLE 14 Batch No. 1 Batch No. 2 Batch No. 3 Batch No. 4 7.7% Lactitol 39.7% Lactitol 7.7% Lactitol 39.7% Lactitol (CM 50) (FINLAC DC) (CM 50) (FINLAC DC) 800 Tablets 800 Tablets 1500 Tablets 1500 Tablets Ingredients mg/tab % w/w mg/tab % w/w mg/tab % w/w mg/tab % w/w L. acidophilus 50.00 7.69 50.00 7.69 50.00 7.69 50.00 7.69 B. animalis subsp. 50.00 7.69 50.00 7.69 50.00 7.69 50.00 7.69 lactis OPC from grape seed 10.00 1.54 10.00 1.54 extract (antioxidant) PHARMABURST 477.60 73.48 269.30 41.43 487.60 75.02 279.30 42.97 C1 (carrier) Lactitol 50.00 7.69 258.30 39.74 50.00 7.69 258.30 39.74 (prebiotic) Sucralose 0.60 0.09 0.60 0.09 0.60 0.09 0.60 0.09 (sweetener) Spray dried natural & 0.60 0.09 0.60 0.09 0.60 0.09 0.60 0.09 artificial orange flavor FD & C yellow 1.20 0.18 1.20 0.18 1.20 0.18 1.20 0.18 No. 6 Al lake Magnesium Stearate 10.00 1.54 10.00 1.54 10.00 1.54 10.00 1.54 (lubricant) Total 650.00 100.00 650.00 100.00 650.00 100.00 650.00 100.00

(73) The tablets of Table 14 were made by direct compression on a rotary machine using a round standard concave tooling. The hardness of the tables was in the range of 8-10 kp. One hundred of the compressed tablets from each batch were packaged into 180 cc HDPE bottles and stability tested at a controlled temperature until a reduction in the assay count was observed. Table 15 provides the parameters for the stability testing of Batches 1 to 4 from Table 14 and includes for each testing lot: the controlled temperature used, the time periods of testing, and whether or not a desiccant was used. All samples were also tested at 0 time in addition to the time periods specified in Table 15.

(74) TABLE-US-00015 TABLE 15 Testing Storage Time Periods for Des- Batch No. Lot Condition Sampling iccant Batch No. 1 Test Lot 1 25 C./60% RH 3, 6, 9, 10 months Yes Batch No. 1 Test Lot 2 25 C./60% RH 3, 6, 9, 10 months No Batch No. 1 Test Lot 3 30 C./65% RH monthly Yes Batch No. 1 Test Lot 4 30 C./65% RH monthly No Batch No. 2 Test Lot 1 25 C./60% RH 3, 6, 9, 10 months Yes Batch No. 2 Test Lot 2 25 C./60% RH 3, 6, 9, 10 months No Batch No. 2 Test Lot 3 30 C./65% RH monthly Yes Batch No. 2 Test Lot 4 30 C./65% RH monthly No Batch No. 3 Test Lot 1 25 C./60% RH 3, 6, 9, 10 months Yes Batch No. 3 Test Lot 2 25 C./60% RH 3, 6, 9, 10 months No Batch No. 3 Test Lot 3 30 C./65% RH monthly Yes Batch No. 3 Test Lot 4 30 C./65% RH monthly No Batch No. 3 Test Lot 5 40 C./75% RH 2, 4, and 6 weeks Yes Batch No. 3 Test Lot 6 40 C./75% RH 2, 4, and 6 weeks No Batch No. 4 Test Lot 1 25 C./60% RH 3, 6, 9, 10 months Yes Batch No. 4 Test Lot 2 25 C./60% RH 3, 6, 9, 10 months No Batch No. 4 Test Lot 3 30 C./65% RH monthly Yes Batch No. 4 Test Lot 4 30 C./65% RH monthly No Batch No. 4 Test Lot 5 40 C./75% RH 2, 4, and 6 weeks Yes Batch No. 4 Test Lot 6 40 C./75% RH 2, 4, and 6 weeks No

(75) The results of the stability count for the testing lots of Table 15 are set forth in Table 16.

(76) TABLE-US-00016 TABLE 16 BATCH NOS. 3 AND 4, TEST LOTS 5 AND 6 BI-WEEKLY PULLS AT 40 C./75% RH WITH OR WITHOUT DESICCANT Stability (Presence of Anaerobe, Gram Positive Rod) Total Count/gram Batch 3 Batch 4 Time Periods at 7.7% Lactitol/No OPC 39.7% Lactitol/No OPC 40 C./75% RH Lacto Bifido Lacto Bifido 0 Time 3.95 10.sup.9 4.30 10.sup.9 3.30 10.sup.9 2.80 10.sup.9 Test Lot 5 4.65 10.sup.8 6.75 10.sup.8 4.9 10.sup.8 6.4 10.sup.8 2 weeks (desiccant) Test Lot 6 5.65 10.sup.8 6.75 10.sup.8 4.05 10.sup.8 5.25 10.sup.8 2 weeks (no desiccant) 4 weeks 1.87 10.sup.6 1.81 10.sup.7 2.3 10.sup.7 2.8 10.sup.7 Test Lot 5 (desiccant) Test Lot 6 2.18 10.sup.8 3.20 10.sup.8 7.4 10.sup.7 9.35 10.sup.7 4 weeks (no desiccant) Test Lot 5 3.75 10.sup.5 5.6 10.sup.5 1.0 10.sup.5 1.9 10.sup.5 6 weeks (desiccant) Test Lot 6 1.01 10.sup.7 1.14 10.sup.7 1.35 10.sup.5 1.45 10.sup.5 6 weeks (no desiccant)

(77) At six weeks, Test Lot 6 from Batch 3 (7.7% lactitol, no OPC, no desiccant) had stability readings above 110.sup.7, indicating that the 7.7% lactitol (i.e., CM 50) and the absence of a desiccant has a positive effect on the probiotic stability of the dosage form when compared to comparable dosage forms prepared with 39.7% lactitol (i.e., FINLAC) and/or are stored with a desiccant. At 6 weeks, the results for Test Lot 5 from Batch 3 and Test Lots 5 and 6 from Batch 4 were all below 110.sup.6.

(78) TABLE-US-00017 TABLE 17 BATCH NOS. 1, 2, 3 AND 4, TEST LOTS 3 AND 4 MONTHLY PULLS AT 30 C./65% RH WITH OR WITHOUT DESICCANT Stability (Presence of Anaerobe, Gram Positive Rod) Total Count/gram Batch No 1 Batch No. 2 Batch No. 3 Batch No. 4 7.7% Lactitol 39.7% Lactitol 7.7% Lactitol 39.7% Lactitol Time Periods at OPC OPC no OPC no OPC 30 C./65% RH Lacto Bifido Lacto Bifido Lacto Bifido Lacto Bifido 0 Time 1.85 10.sup.9 1.25 10.sup.9 1.75 10.sup.9 1.80 10.sup.9 3.95 10.sup.9 4.30 10.sup.9 3.30 10.sup.9 2.80 10.sup.9 1 month 6.2 10.sup.8 6.1 10.sup.8 6.85 10.sup.8 5.2 10.sup.8 1.16 10.sup.9 1.15 10.sup.9 1.06 10.sup.9 1.08 10.sup.9 Test Lot 3 (desiccant) 1 month 7.6 10.sup.8 6.15 10.sup.8 6.9 10.sup.8 7.75 10.sup.8 8.45 10.sup.8 6.75 10.sup.8 1.01 10.sup.9 9.5 10.sup.8 Test Lot 4 (no desiccant) 2 months 1.75 10.sup.7 4.0 10.sup.7 1.45 10.sup.7 6.45 10.sup.7 6.2 10.sup.7 9.6 10.sup.7 Test Lot 3 (desiccant) 2 months 1.17 10.sup.8 1.69 10.sup.8 Test Lot 4 (no desiccant) 3 months 7.95 10.sup.6 1.9 10.sup.6 5.85 10.sup.6 4.1 10.sup.6 7.7 10.sup.4 1.2 10.sup.5 4.75 10.sup.6 9.05 10.sup.6 Test Lot 3 (desiccant) 3 months 6.0 10.sup.6 5.0 10.sup.6 6.85 10.sup.6 4.55 10.sup.6 1.50 10.sup.6 1.26 10.sup.6 1.25 10.sup.6 1.9 10.sup.6 Test Lot 4 (no desiccant) 4 months 5.45 10.sup.5 4.25 10.sup.5 3.7 10.sup.5 4.8 10.sup.5 3.4 10.sup.5 2.9 10.sup.5 5.35 10.sup.5 3.2 10.sup.5 Test Lot 3 (desiccant) 4 months 3.3 10.sup.6 3.65 10.sup.6 4.1 10.sup.6 4.1 10.sup.6 2.8 10.sup.6 2.05 10.sup.6 2.5 10.sup.6 2.0 10.sup.6 Test Lot 4 (no desiccant) 5 months 4.35 10.sup.5 1.95 10.sup.5 2.9 10.sup.5 2.95 10.sup.5 4.2 10.sup.5 2.5 10.sup.5 1.7 10.sup.5 2.45 10.sup.5 Test Lot 3 (desiccant) 5 months 4.8 10.sup.5 5.95 10.sup.5 2.25 10.sup.5 2.35 10.sup.5 2.5 10.sup.5 2.5 10.sup.5 2.1 10.sup.5 3.1 10.sup.5 Test Lot 4 (no desiccant) 6 months 8.35 10.sup.4 8.35 10.sup.4 1.60 10.sup.5 1.76 10.sup.5 1.29 10.sup.5 1.26 10.sup.5 1.43 10.sup.5 1.67 10.sup.5 Test Lot 3 (desiccant) 6 months 1.55 10.sup.5 1.54 10.sup.5 1.04 10.sup.5 1.01 10.sup.5 1.26 10.sup.5 1.14 10.sup.5 1.37 10.sup.5 1.29 10.sup.5 Test Lot 4 (no desiccant)

(79) After five months, the results of all test lots are below 110.sup.6. The stability studies for Batch No. 1, Test Lots 3 and 4 (with and without a desiccant) were discontinued after the six-month pull and the stability studies for the remaining test lots were discontinued after the five-month pull.

(80) The results of Table 17 show that Test Lot 4 from Batch 1 (7.7% lactitol, OPC, no desiccant) had slightly increased probiotic stability over the remaining test lots.

(81) TABLE-US-00018 TABLE 18 BATCH NOS. 1, 2, 3 AND 4, TEST LOTS 1 AND 2 MONTHLY PULLS AT 25 C./60% RH WITH OR WITHOUT DESICCANT Stability (Presence of Anaerobe, Gram Positive Rod) Total Count/gram Batch No 1 Batch No. 2 Batch No. 3 Batch No. 4 7.7% Lactitol 39.7% Lactitol 7.7% Lactitol 39.7% Lactitol Time Periods at OPC OPC no OPC no OPC 25 C./60% RH Lacto Bifido Lacto Bifido Lacto Bifido Lacto Bifido 0 Time 1.85 10.sup.9 1.25 10.sup.9 1.75 10.sup.9 1.80 10.sup.9 3.95 10.sup.9 4.30 10.sup.9 3.30 10.sup.9 2.80 10.sup.9 3 months 2.32 10.sup.7 2.62 10.sup.7 2.24 10.sup.7 1.68 10.sup.7 1.86 10.sup.7 1.60 10.sup.7 1.86 10.sup.7 1.71 10.sup.7 Test Lot 1 (desiccant) 3 months 2.65 10.sup.7 2.22 10.sup.7 2.43 10.sup.7 1.92 10.sup.7 1.16 10.sup.7 2.00 10.sup.7 1.06 10.sup.7 1.24 10.sup.7 Test Lot 2 (no desiccant) 6 months 8.35 10.sup.5 8.35 10.sup.5 1.60 10.sup.5 1.76 10.sup.5 1.29 10.sup.5 1.26 10.sup.5 1.43 10.sup.5 1.67 10.sup.5 Test Lot 1 (desiccant) 6 months 1.55 10.sup.5 1.54 10.sup.5 1.04 10.sup.5 1.01 10.sup.5 1.26 10.sup.5 1.14 10.sup.5 1.37 10.sup.5 1.29 10.sup.5 Test Lot 2 (no desiccant) 9 months 0 0 2.3 10.sup.3 0 6.1 10.sup.6 6.6 10.sup.6 1.9 10.sup.6 7.7 10.sup.5 Test Lot 1 (desiccant) 9 months 0 0 2.0 10.sup.1 0 0 0 0 0 Test Lot 2 (no desiccant) 10 months 0 0 3.15 10.sup.2 0 3.3 10.sup.6 3.9 10.sup.6 1.06 10.sup.6 4.3 10.sup.5 Test Lot 1 (desiccant) 10 months 0 0 0 0 Test Lot 2 (no desiccant)

(82) The results of Table 18 show that up to 3 months, all Test Lots tested relatively consistently, with Batch 1, Test Lots 1 and 2 (7.7% lactitol, OPC, with and without desiccant), having the highest counts. At 6 months, Batch No. 1, Test Lot 1 (7.7% lactitol, OPC, with desiccant) had the highest number of viable colonies of both L. acidophilus and B. animalis subsp. lactis. At nine months, however, Batch No. 1, Test Lot 1, did not have any positive colonies, while Test Lots 3 (no OPC, desiccant) of both Batch Nos. 3 and 4 still had viable colonies of both bacterial species at ten months. At nine and ten months, Test Lot 3 of Batch 3 (7.7% lactitol, no OPC, desiccant) had a significantly higher count of viable species than did Test Lot 3 of Batch 4 (39.7% lactitol, no OPC, desiccant).

Example 8

Manufacture of Probiotic Dosage Form with L. Acidophilus, B. Animalis Subsp. Lactis, Vitamin A Palmitate, and Encapsulated Zinc Oxide

(83) A batch of 500 tablets of a mango-flavored combination probiotic dosage form containing L. acidophilus, B. animalis subsp. lactis, vitamin A, and zinc oxide was prepared with the ingredients set forth in Table 19.

(84) TABLE-US-00019 TABLE 19 Mango Flavored Tablets 500 Tablets Ingredients mg/tab % w/w L. acidophilus 50.00 7.46 100 billion colonies/gram B. animalis subsp. lactis 50.00 7.46 100 billion colonies/gram Encapsulated Zinc Oxide 10.00 1.49 Vitamin A Palmitate 6.80 1.01 PHARMABURST C1 539.10 80.46 (carrier) Sucralose 0.90 0.13 (sweetener) P11040 Artificial Mango 2.00 0.30 Flavor S.D. Powder FD & C Colorant 1.20 0.18 Magnesium Stearate 10.0 1.49 (lubricant) 0 0 Total 670.0 100.00

(85) The tablets were prepared by direct compression on a rotary machine using a round standard concave tooling. The hardness of the tablets was in the range of 8-12 kp. The mango flavored tablets were stored with a desiccant and subjected to stability testing for L. acidophilus and B. animalis subsp. lactis independently at varying time points under three different storage conditions: 40 C./75% RH (Table 20); 35 C./65% RH (Table 21); and 25 C./60% RH (Table 22).

(86) TABLE-US-00020 TABLE 20 Stability (Presence of Anaerobe, Gram Positive Rod) Time Period at Total Count/gram 40 C./75% RH L. acidophilus B. animalis subsp. lactis 0 time.sup. 4.6 10.sup.9 5.45 10.sup.9 2 weeks 6.55 10.sup.8 7.5 10.sup.8 1 month 2.25 10.sup.6 3.65 10.sup.6

(87) TABLE-US-00021 TABLE 21 Stability (Presence of Anaerobe, Gram Positive Rod) Time Period at Total Count/gram 30 C./65% RH L. acidophilus B. animalis subsp. lactis 0 time.sup. 4.6 10.sup.9 5.45 10.sup.9 1 month.sup. 3.6 10.sup.9 3.4 10.sup.9 2 months 2.45 10.sup.8 3.00 10.sup.8 3 months 2.26 10.sup.8 2.05 10.sup.8 8 months 3.8 10.sup.5 2.6 10.sup.6 9 months 3.1 10.sup.5 1.58 10.sup.6

(88) TABLE-US-00022 TABLE 22 Stability (Presence of Anaerobe, Gram Positive Rod) Time Periods at Total Count/gram 25 C./60% RH L. acidophilus B. animalis subsp. lactis 0 time.sup. 4.6 10.sup.9 5.45 10.sup.9 3 months 2.17 10.sup.8 2.34 10.sup.8 6 months 1.91 10.sup.7 1.87 10.sup.7

(89) Tables 21 and 22 show that the samples tested at 30 C./65% RH and 25 C./60% RH had very high stability counts at 3 months time and the samples tested at 25 C./60% RH had high stability counts at 6 months time indicating that the presence of vitamin A and zinc oxide may have a positive effect on the stability of the probiotic dosage form under these conditions during a 3 to 6 month time period. Comparatively, the values of the 3-month counts for the samples of Tables 21 and 22 are significantly higher than the values of the 3-month counts for the samples of Tables 17 and 18.

Example 9

Manufacture of Probiotic Dosage Form with L. Acidophilus, B. Animalis Subsp. Lactis, and OPC from Pine Bark Source

(90) A batch of 600 tablets of an orange-flavored combination probiotic dosage form containing L. acidophilus, B. animalis subsp. lactis, and OPC from pine bark was prepared with the ingredients set forth in Table 23.

(91) TABLE-US-00023 TABLE 23 Orange-Flavored Tablets 600 Tablets Ingredients mg/tab % w/w L. acidophilus 50.00 7.69 100 billion colonies/gram B. animalis subsp. lactis 50.00 7.69 100 billion colonies/gram OPC from Pine Bark 10.00 1.54 (antioxidant) Lactitol (FINLAC DC) 50.00 7.69 (prebiotic) PHARMABURST C1 477.60 73.48 (carrier) Sucralose 0.60 0.09 (sweetener) Spray dried natural & 0.60 0.09 artificial orange flavor FD & C Yellow No. 6 1.20 0.18 Aluminum Lake Magnesium Stearate 10.0 1.54 (lubricant) 0 0 Total 650.0 100.00

(92) The tablets were prepared by direct compression on a rotary machine using a round standard concave tooling. The hardness of the tablets was in the range of 8-12 kp. The tablets as prepared were stored with a desiccant and subjected to stability testing for L. acidophilus and B. animalis subsp. lactis as set forth in Tables 24, 25, and 26.

(93) TABLE-US-00024 TABLE 24 Stability (Presence of Anaerobe, Gram Positive Rod) Time Periods at Total Count/gram 40 C./75% RH L. acidophilus B. animalis subsp. lactis 0 time.sup. 1.06 10.sup.9 1.08 10.sup.9 2 weeks 1.06 10.sup.8 7.2 10.sup.7 1 month.sup. 1.9 10.sup.6 1.1 10.sup.6 2 months 2.2 10.sup.5 2.35 10.sup.5

(94) TABLE-US-00025 TABLE 25 Stability (Presence of Anaerobe, Gram Positive Rod) Time Periods at Total Count/gram 30 C./65% RH L. acidophilus B. animalis subsp. lactis 0 time.sup. 1.06 10.sup.9 1.08 10.sup.9 1 month.sup. 4.3 10.sup.8 4.65 10.sup.8 2 months 5.25 10.sup.8 5.8 10.sup.8 3 months 2.4 10.sup.8 2.35 10.sup.8 4 months 4.0 10.sup.6 2.8 10.sup.6 6 months 0 0

(95) TABLE-US-00026 TABLE 26 Stability (Presence of Anaerobe, Gram Positive Rod) Time Periods at Total Count/gram 25 C./60% RH L. acidophilus B. animalis subsp. lactis 0 time.sup. 1.06 10.sup.9 1.08 10.sup.9 3 months 8.0 10.sup.8 7.75 10.sup.8 6 months 1.7 10.sup.8 1.5 10.sup.8 9 months 9.4 10.sup.7 3.1 10.sup.7

(96) Table 25 shows a high percentage of live colonies of both bacterial species at 30 C./65% RH at 6 months and Table 26 shows a very high percentage of live colonies of both bacterial species at 25 C./60% RH at 9 months, indicating that perhaps the OPC from pine bark enhances the stability of the probiotic dosage forms under the specified conditions. Comparatively, the values of the 3-month counts for the samples of Tables 25 and 26 (OPC from pine bark, FINLAC lactitol, desiccant) are significantly higher than the values of the 3-month counts for the samples of Batch 2, Test Lot 2 (OPC from grape seed, FINLAC lactitol, desiccant) in Tables 17 and 18.

Example 10

Stability Assessment of L. Acidophilus Together with Mannitol and/or Sorbitol

(97) Two batches of 200 probiotic tablets were prepared with L. acidophilus and mannitol (Batch A) and L. acidophilus and sorbitol (Batch B) Table 27 sets forth the ingredients that were used to prepare the tablets.

(98) TABLE-US-00027 TABLE 27 Batch A Batch B Mannitol Sorbitol 200 Tablets 200 Tablets Ingredients mg/tab % w/w mg/tab % w/w L. acidophilus 50.0 10.00 50.00 10.00 Mannitol 440.0 88.00 Sorbitol 440.0 88.00 Magnesium Stearate 10.0 2.00 10.00 2.00 (lubricant) 0 0 0 0 Total 500.00 100.00 500.00 100.00 Tablet Hardness 5-7 kp 24-27 kp

(99) Tablets were made by direct compression on a rotary machine using a round standard concave tooling. As shown in Tablet 27, the hardness of the tablets varied dramatically due to the characteristics of mannitol and sorbitol. Table 28 shows the results of the stability assay for L. acidophilus at 40 C./75% RH without a desiccant at three time points.

(100) TABLE-US-00028 TABLE 28 L. acidophilus Stability (Presence of Anaerobe, Gram Positive Rod) Total Count/gm Time Periods at Batch A Batch B 40 C./75% RH Mannitol Sorbitol 0 time.sup. 2.65 10.sup.8 4.1 10.sup.8 3 weeks 0 0 1 month 0 0

(101) The results of Table 28 show that the probiotic cultures in the tablets failed to survive storage at 40 C./75% RH indicating that-mannitol and sorbitol alone have no effect on the stability of L. acidophilus at 40 C./75% RH.

(102) As a control for the testing of Batches A and B, probiotic formulations were prepared with the carriers PHARMABURST C1 (Batch C; SPI Polyols, New Castle, Del.) and STARCH 1500 (Batch D; Colorcon, Inc., West Point, Pa.). The ingredients for the control formulations are set forth in Table 29.

(103) TABLE-US-00029 TABLE 29 Batch C Batch D STARCH 1500 PHARMABURST 200 Tablets 200 Tablets Ingredients mg/tab % w/w mg/tab % w/w L. acidophilus 50.0 10.00 50.00 10.00 PHARMABURST C1 440.0 88.00 (carrier) STARCH 1500 (carrier) 440.0 88.00 Magnesium Stearate 10.0 2.00 10.00 2.00 (lubricant) 0 0 0 0 Total 500.00 100.00 500.00 100.00 Tablet Hardness 2-3 Kp 8-10 Kp

(104) Tablets were made by direct compression on a rotary machine using a round standard concave tooling. The hardness of the tablets varied with the characteristics of each formulation. The control batches were stored for two weeks at 40 C./75% RH without a desiccant and tested for L. acidophilus at two time periods, which are set forth in Table 30.

(105) TABLE-US-00030 TABLE 30 L. acidophilus Stability (Presence of Anaerobe, Gram Positive Rod) Total Count/gram Time Periods at Batch C Batch D 25 C./60% RH STARCH 1500 PHARMABURST 0 time.sup. 4.45 10.sup.8 3.0 10.sup.9 2 weeks 8.2 10.sup.3 2.65 10.sup.3 1 month 0 0

(106) The results of Table 30 show that the presence of PHARMABURST alone provides a limited increase in the increase of the probiotic dosage from 0 time to 2 weeks at 25 C./60% RH.

Example 11

Testing for Pharmaburst, Lactitol, and OPC Synergism on Probiotic Stability

(107) To determine if lactitol and OPCs have a synergistic effect with the PHARMABURST carrier of Tables 29 and 30, thus enhancing stability of the L. acidophilus probiotic, four batches of tablets were prepared and tested under the harsh storage conditions of 40 C./75% RH. The following parallel formulations were manufactured: (1) PHARMABURST C1 (abbreviated as PHRMBRST in Tables 31 and 32) and lactitol with magnesium stearate (Batch E); (2) PHARMABURST C1 and lactitol with calcium stearate (Batch F); (3) PHARMABURST C1 and pine bark OPC with lactitol (Batch G); and (4) PHARMABURST C1 and pine bark OPC without lactitol (Batch H). All parallel formulations were placed on accelerated stability testing according to the formulations set forth in Table 31.

(108) TABLE-US-00031 TABLE 31 Batch E Batch F Batch G Batch H PHRMBRST, PHRMBRST, PHRMBRST, PHRMBRST Lactitol, and Lactitol, and Lactitol, OPC, OPC, and Mg Stearate Ca Stearate and Mg Stearate Mg Stearate 200 Tablets 200 Tablets 200 Tablets 200 Tablets Ingredients mg/tab % w/w mg/tab % w/w mg/tab % w/w mg/tab % w/w L. acidophilus 50.00 10.00 50.00 10.00 50.00 10.00 50.00 10.00 PHARMABURST C1 390.00 78.00 390.00 78.00 384.00 76.40 434.00 86.80 (carrier) Lactitol 50.00 10.00 50.00 10.00 50.00 10.00 (prebiotic) OPC from Pine Bark 6.00 1.20 6.00 1.20 (antioxidant) Calcium Stearate 10.00 2.00 Magnesium Stearate 10.00 2.00 10.00 2.00 10.00 2.00 Total 500.00 100.00 500.00 100.00 500.00 100.00 500.00 100.00 Hardness 8-10 kp 8-10 kp 8-10 kp 8-10 kp

(109) Tablets were made by direct compression on a rotary machine using a round standard concave tooling. The hardness of the tablets varied with the characteristics of each formulation. All samples were stored for one month at 40 C./75% RH without a desiccant and tested for L. acidophilus at the time periods specified in Table 32.

(110) TABLE-US-00032 TABLE 32 L. acidophilus Stability (Presence of Anaerobe, Gram Positive Rod) Total Count/gram Batch E Batch F Batch G Batch H PHRMBRST, PHRMBRST, PHRMBRST, PHRMBRST, Time Periods at Lactitol, and Lactitol, and Lactitol, OPC, and OPC, and 40 C./75% RH Mg Stearate Ca Stearate Mg Stearate Ca Stearate 0 time.sup. 5.6 10.sup.9 4.5 10.sup.9 2.2 10.sup.9 3.7 10.sup.9 2 weeks 1.59 10.sup.8 3.55 10.sup.7 1.61 10.sup.7 4.95 10.sup.7 1 month 0 0 0 0

Example 12

The Effect of Lactitol and Pharmaburst on L. Acidophilus Stability at 40 C./75% RH

(111) To test if a synergistic reaction is occurring between the lactitol and PHARMABURST to result in increased stability of L. acidophilus, three additional batches of tablets (Batches I, J, and K) were formulated as set forth in Table 33; the stability results for the tablets in the three batches at 40 C./75% RH are set forth in Table 34.

(112) TABLE-US-00033 TABLE 33 Batch K Batch I Batch J Lactitol and Lactitol PHARMABURST PHARMABURST, 200 Tablets 200 Tablets 200 Tablets Ingredients mg/tab % w/w mg/tab % w/w mg/tab % w/w L. acidophilus 50.00 10.00 50.00 10.00 50.00 10.00 PHARMABURST C1 440.00 88.00 40.00 8.00 (carrier) Lactitol 440.00 88.00 400.00 80.00 (prebiotic) Magnesium Stearate 10.00 2.00 10.00 2.00 10.00 2.00 Total 500.00 100.00 500.00 100.00 500.00 100.00

(113) TABLE-US-00034 TABLE 34 L. acidophilus Stability (Presence of Anaerobe, Gram Positive Rod) Time Total Count/gram Periods Batch K at 40 C./ Batch I Batch J Lactitol and 75% RH Lactitol PHARMABURST PHARMABURST 0 time.sup. 4.5 10.sup.8 4.4 10.sup.8 4.1 10.sup.8 2 weeks 0 1.62 10.sup.3 2.3 10.sup.2

(114) The results of Table 34 show that lactitol alone shows no viable L. acidophilus colonies after two weeks at 40 C./75% RH whereas PHARMABURST alone and lactitol with even a relatively low weight percent of PHARMABURST did show viable colonies at 2 weeks time under the harsh storage conditions of 40 C./75% RH.