Bacterial composition

Abstract

The present invention provides bacterial compositions. For example, provided herein are bacterial compositions having combinations of different bacterial strains formulated in a manner that maintains bacterial stability. The methods and materials provided herein can be used to deliver an effective and active dose of probiotic bacteria, to protect the probiotic bacteria during storage, and to achieve gastrointestinal bioavailability.

Description

[0001] Cross-referencing

[0002] This case asserts priority to U.S. Provisional Patent Application No. 62 / 099,410 (filed January 2, 2015, entitled "Bacterial Composition"), which is incorporated herein by reference in its entirety. Background Technology 1. Technical Field

[0004] This article relates to the field of bacterial compositions. For example, this article provides bacterial compositions with combinations of different strains, formulated in a manner that maintains bacterial stability.

[0005] 2. Background Information

[0006] Specific microorganisms that consume probiotic preparations can provide health benefits to mammals. Hundreds of different strains of bacteria exist in the human digestive system. Some of these different bacteria are believed to help maintain a healthy digestive tract and aid in the digestion of food.

[0007] Unfortunately, probiotic strains are extremely sensitive, and some strains may not survive commercial production, storage, or gastrointestinal transport after ingestion, where they are exposed to heat, moisture, bile, low pH, and digestive enzymes. Therefore, the probiotic strain count undergoes an undesirable reduction before the bacteria reach the intestines. Summary of the Invention

[0008] This document provides bacterial compositions. For example, it provides bacterial compositions of combinations of different strains, formulated in a manner that maintains bacterial stability. For example, the methods and materials provided herein can be used to deliver effective and active doses of probiotics to protect the probiotics during storage and to achieve gastrointestinal bioavailability.

[0009] As described herein, single bacterial compositions (e.g., capsules, tablets, or sacs) can be formulated to help maintain bacterial stability within the composition and to deliver bacteria to the gut after oral administration, comprising at least seven different bacterial strains, silica in the form of (e.g., silica powder), fructooligosaccharides, magnesium stearate, and a low-moisture filler (e.g., microcrystalline cellulose, such as MCC 112) such that the final composition has a water activity (A) of less than 0.3. w For example, at least about 1×10⁻⁶ can be used. 9 Lactobacillus acidophilus (CFU) Lactobacillus acidophilus Lactobacillus plantarum ( Lactobacillus plantarum ), Lactobacillus casei ( Lactobacillus casei Lactobacillus rhamnosus ( Lactobacillus rhamnosus ), Bifidobacterium longum ( Bifidobacterium longum ), Bifidobacterium lactis ( Bifidobacterium lactis ) and Bifidobacterium bifidum ( Bifidobacterium bifidum Each of the ingredients is formulated with silica, fructooligosaccharides, magnesium stearate, and fillers with low water content to form A. w Compositions with a concentration of less than 0.3% maintain at least about 80% bacterial viability for at least about 12 months, at least about 18 months, or at least about 24 months under standard storage conditions (e.g., room temperature at normal humidity).

[0010] As used herein, the term “about” when referring to a weight percent of a composition means ±10% of the reported weight percent. As used herein, the term “about” when referring to a measured characteristic of a composition means ±20% of the reported value.

[0011] The bacterial compositions provided herein can be coated and / or encapsulated to minimize or prevent moisture absorption and minimize the water activity of the final blend.

[0012] Generally, one aspect of this document provides a bacterial composition comprising *Lactobacillus acidophilus*, *Lactobacillus plantarum*, *Lactobacillus casei*, *Lactobacillus rhamnosus*, *Bifidobacterium longum*, *Bifidobacterium lactis*, and *Bifidobacterium bifidum*, wherein the bacterial composition, upon oral administration to a mammal, releases *Lactobacillus acidophilus*, *Lactobacillus plantarum*, *Lactobacillus casei*, *Lactobacillus rhamnosus*, *Bifidobacterium longum*, *Bifidobacterium lactis*, and *Bifidobacterium bifidum* into the mammalian intestine, and wherein the composition has a water activity of less than 0.3. For each gram of the composition, the composition may contain about 4 × 10⁻⁶. 9 Each settlement unit and approximately 50 × 10 9 Lactobacillus acidophilus between colony-forming units. The composition may contain approximately 5 × 10⁻⁶ Lactobacillus per gram of composition. 9 One settlement unit and approximately 60 × 10 9 Lactobacillus plantarum between colony-forming units. The composition may contain approximately 4 × 10⁻⁶ Lactobacillus plants per gram of composition. 9 Each settlement unit and approximately 50 × 10 9 Lactobacillus casei between colony-forming units. The composition may contain approximately 4 × 10⁻⁶ Lactobacillus per gram of composition. 9 Each settlement unit and approximately 50 × 10 9 Lactobacillus rhamnosus between colony-forming units. For each gram of composition, the composition may contain approximately 2 × 10⁻⁶ Lactobacillus rhamnosus. 9 Each settlement unit and approximately 40 × 10 9 Bifidobacterium longum between colony-forming units. The composition may contain approximately 4 × 10⁻⁶ per gram of composition. 9 Each settlement unit and approximately 50 × 10 9 Bifidobacterium lactis exists between colony-forming units. The composition may contain approximately 2 × 10⁻⁶ bacteria per gram of composition. 9Each settlement unit and approximately 40 × 10 9 The composition contains *Bifidobacterium bifidum* between colony-forming units. The composition may contain fructooligosaccharides. Per gram of composition, the composition may contain between about 0.5 mg and about 50 mg of fructooligosaccharides. The composition may contain silicon dioxide. Per gram of composition, the composition may contain between about 5 mg and about 20 mg of silicon dioxide. The composition may contain magnesium stearate. Per gram of composition, the composition may contain between about 10 mg and about 20 mg of magnesium stearate. Per gram of composition, the composition may contain more than 8 × 10⁻⁶ * ... 9 CFU of bacteria. The composition may contain more than 10 × 10⁻⁶ bacteria per gram of composition. 9 CFU of bacteria. The composition may contain more than 15 × 10⁻⁶ bacteria per gram of composition. 9 CFU of bacteria. After storage at room temperature and ambient humidity for approximately 12 months, the composition contains more than 10 × 10⁻⁶ bacteria per gram of composition. 9 The bacteria in the CFU remain viable. After storage at room temperature and ambient humidity for approximately 12 months, the composition contains more than 10 × 10⁻⁶ bacteria per gram of composition. 9 The bacteria in the CFU remain viable. *Lactobacillus acidophilus*, *Lactobacillus plantarum*, *Lactobacillus casei*, *Lactobacillus rhamnosus*, *Bifidobacterium longum*, *Bifidobacterium lactis*, and *Bifidobacterium bifidum* can be freeze-dried. It can be used in mammals, specifically humans. The bacterial composition may include a coating that prevents the release of *Lactobacillus acidophilus*, *Lactobacillus plantarum*, *Lactobacillus casei*, *Lactobacillus rhamnosus*, *Bifidobacterium longum*, *Bifidobacterium lactis*, and *Bifidobacterium bifidum* before the composition reaches the mammalian intestine after oral administration. The bacterial composition may comprise a capsule containing *Lactobacillus acidophilus*, *Lactobacillus plantarum*, *Lactobacillus casei*, *Lactobacillus rhamnosus*, *Bifidobacterium longum*, *Bifidobacterium lactis*, and *Bifidobacterium bifidum*. The composition may contain microcrystalline cellulose.

[0013] In another aspect, this article provides a method for manufacturing a bacterial composition. The method comprises, or substantially comprises, the following: (a) coating freeze-dried bacteria with silica to form coated freeze-dried bacteria; (b) adding MCC and fructooligosaccharides having a water activity of less than 0.2 to the coated freeze-dried bacteria to form a first mixture; (c) adding magnesium stearate and silica to the first mixture to form an admixture; and (d) encapsulating the admixture into a dosage form, thereby forming the bacterial composition. The freeze-dried bacteria may be a mixture of freeze-dried Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus rhamnosus, Bifidobacterium longum, Bifidobacterium lactis, and Bifidobacterium bifidum. For each gram of composition, the composition may contain (a) about 4 × 10⁻⁶ 9 Each settlement unit and approximately 50 × 10 9 (a) Lactobacillus acidophilus between colony-forming units; (b) Approximately 5 × 10 9One settlement unit and approximately 60 × 10 9 Lactobacillus plantarum between colony-forming units; (c) approximately 4 × 10 9 Each settlement unit and approximately 50 × 10 9 Lactobacillus casei between colony-forming units; (d) approximately 4 × 10 9 Each settlement unit and approximately 50 × 10 9 Lactobacillus rhamnosus among colony-forming units; (e) approximately 2 × 10 9 Each settlement unit and approximately 40 × 10 9 (f) Approximately 4 × 10⁻⁶ Bifidobacteria between colony-forming units; 9 Each settlement unit and approximately 50 × 10 9 Bifidobacterium lactis between colony-forming units; and (g) approximately 2 × 10 9 Each settlement unit and approximately 40 × 10 9 The composition contains *Bifidobacterium bifidum* between colony-forming units. Each gram of the composition may contain between about 0.5 mg and about 5.0 mg of fructooligosaccharides. Each gram of the composition may contain between about 5.0 mg and about 20.0 mg of silica. Each gram of the composition may contain more than 15 × 10⁻⁶ units of... 9 CFU of bacteria. After storage at room temperature and ambient humidity for approximately 12 months, the composition contains more than 10 × 10⁻⁶ bacteria per gram of composition. 9 CFU keeps the bacteria alive. It can be formulated as capsules.

[0014] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although similar or equivalent methods or materials may be used to practice this invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including definitions) shall prevail. Furthermore, materials, methods, and examples are illustrative only and are not intended to be restrictive.

[0015] Details of one or more specific embodiments of the present invention are set forth in the following description. Other features, objectives, and advantages of the invention will become apparent from the embodiments and from the claims. Detailed Implementation

[0016] This document provides bacterial compositions. For example, it provides bacterial compositions of different strains (e.g., combinations of at least six, seven, eight, nine, or ten different strains) formulated in a manner that maintains bacterial stability. The bacterial compositions provided herein may be in the form of powders, capsules, pills, tablets, chewing gum, lozenges, candies, or sachets. In some cases, the bacterial compositions provided herein may include a coating designed to prevent water absorption and minimize the water activity of the final blend to provide a formulation with good long-term stability.

[0017] For example, methacrylate coatings, hydroxypropyl methylcellulose phthalate coatings, cellulose acetate succinate coatings, hydroxypropyl methylcellulose acetate succinate coatings, polyvinyl acetate phthalate coatings, or cellulose acetate trimellitate sodium alginate coatings can be used to deliver the bacterial contents of a bacterial composition through the stomach. Such coatings can be manufactured and applied as described elsewhere (e.g., U.S. Patent Application Publication No. 2014 / 370091, Chinese Patent No. CN103976975, and Taiwan Patent No. TW201431561).

[0018] In some cases, the bacterial compositions provided herein may include any combination of at least six (e.g., at least seven, eight, nine, or ten) different strains. Examples of different strains that can be formulated into the bacterial compositions provided herein include (but are not limited to) *Lactobacillus acidophilus*, *Lactobacillus plantarum*, *Lactobacillus casei*, *Lactobacillus rhamnosus*, *Bifidobacterium longum*, *Bifidobacterium lactis*, *Bifidobacterium bifidum*, and *Bacillus coagulans*. Bacillus coagulans ), Lactobacillus paracasei ( Lactobacillus paracasei Lactobacillus johnsonii ( Lactobacillus johnsonii Lactobacillus loratadine () Lactobacillus reuteri Lactobacillus bulgaricus () Lactobacillus bulgaricus ), Bifidobacterium breve Bifidobacterium breve ), Lactobacillus brevis ( Lactobacillus brevis ), Lactococcus lactis ( Lactococcus lactis ) and thermophilic streptococci ( Streptococcus thermophilus In some cases, the bacterial compositions provided herein may include one or more bacterial species, such as *Saccharomyces boulardii*. Saccharomyces boulardii In some cases, the only strain present in a particular bacterial composition may be Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus rhamnosus, Bifidobacterium longum, Bifidobacterium lactis, and Bifidobacterium bifidum, or a combination of any six of these bacteria.

[0019] Unless otherwise stated, all bacterial quantities in this application are provided per gram of total bacterial composition based on the composition. In some cases, the bacterial compositions provided herein may contain at least about 1 × 10⁻⁶. 9 CFUs are included in various different strains in the bacterial composition. In some cases, the bacterial composition provided herein may contain about 1 × 10⁻⁶ CFUs. 8 With approximately 1×10 11 All species between 1 × 10⁶ bacteria (e.g., approximately 1 × 10⁶) 9 With approximately 4×10 10 (All species among bacteria). For example, the bacterial composition provided herein may include about 1 × 10⁻⁶ bacteria. 7 CFU and approximately 3×10 9 Lactobacillus acidophilus between CFU, approximately 1×10 7 CFU and approximately 4×10 9 Between CFU, Lactobacillus plantarum, approximately 1×10 7 CFU and approximately 3×10 9 Between CFU, Lactobacillus casei, approximately 1×10 7 CFU and approximately 3.2 × 10 9 Lactobacillus rhamnosus between CFU, approximately 1×10 7 CFU and approximately 1.6 × 10 9 Bifidobacterium longum between CFU, approximately 1×10 7 CFU and approximately 3.6 × 10 9 Bifidobacterium lactis between CFU, and approximately 1×10 7 CFU and approximately 1.6 × 10 9 Bifidobacteria between CFUs.

[0020] The bacterial strains included in the bacterial compositions provided herein can be obtained using any suitable method. For example, a large quantity of specific strains can be obtained using culture techniques. In some cases, the strains included in the bacterial compositions provided herein are commercially available. For example, *Lactobacillus acidophilus* is commercially available from DuPont Inc. (Madison, Wisconsin; catalog number LA-14 200B); *Lactobacillus plantarum* is commercially available from DuPont (Madison, Wisconsin; catalog number LP-115 400B); *Lactobacillus casei* is commercially available from DuPont (Madison, Wisconsin; catalog number LC-11 300B); *Lactobacillus rhamnosus* is commercially available from DuPont (Madison, Wisconsin; catalog number LR-32 200B); *Bifidobacterium longum* is commercially available from DuPont (Madison, Wisconsin; catalog number BL-05100B); and *Bifidobacterium lactis* is commercially available from DuPont (Madison, Wisconsin; catalog number BL-04). 450B), and Bifidobacterium bifidum is commercially available from DuPont (Madison, Wisconsin; catalog number BB-06 100B).

[0021] The bacterial compositions provided herein may include one or more other ingredients. For example, the bacterial compositions provided herein may include fructooligosaccharides. In some cases, the bacterial compositions provided herein may include fructooligosaccharides in amounts between about 0.5 mg and about 50 mg per bacterial composition (e.g., between about 1 mg and about 50 mg, between about 5 mg and about 50 mg, between about 10 mg and about 50 mg, between about 0.5 mg and about 25 mg, between about 0.5 mg and about 20 mg, between about 0.5 mg and about 15 mg, between about 0.5 mg and about 10 mg, or between about 5 mg and about 20 mg). For example, the bacterial compositions provided herein may contain *Lactobacillus acidophilus*, *Lactobacillus plantarum*, *Lactobacillus casei*, *Lactobacillus rhamnosus*, *Bifidobacterium longum*, *Bifidobacterium lactis*, and *Bifidobacterium bifidum*, and between about 5 mg and about 20 mg of fructooligosaccharides.

[0022] In some cases, the bacterial compositions provided herein may include between about 1 mg and about 50 mg per gram of the bacterial composition (e.g., between about 1 mg and about 20 mg, between about 2 mg and about 20 mg, or between about 2 mg and about 10 mg). For example, a 2-gram bacterial composition provided herein may contain between about 10 mg and about 100 mg of fructooligosaccharides.

[0023] Fructagaves that may be included in the bacterial compositions provided herein can be obtained using any suitable method. For example, fructagaves can be obtained, as described elsewhere (U.S. Patent Application Publication No. 2005 / 069627), by inulin degradation or by transfructization. In some cases, fructagaves may be commercially available from Agaviotica Corporation (Monterrey, NL, Mexico; Catalogue No. Fructagave PR-95).

[0024] In some cases, the bacterial compositions provided herein may include flow aids (e.g., silica). Examples of flow aids that may be included in the bacterial compositions provided herein include (but are not limited to) silica, stearic acid, calcium stearate, magnesium stearate, sodium stearate, glyceryl behenate (Compritol), liquid paraffin, Aerosil® (colloidal silica), starch and talc, DL-leucine and sodium lauryl sulfate. For example, the bacterial compositions provided herein may include a flow aid (e.g., silica) in amounts between about 2.00 mg and about 5.00 mg per bacterial composition (e.g., between about 0.005 mg and about 0.10 mg, between about 0.01 mg and about 0.02 mg, or between about 0.25 mg and about 0.50 mg). For example, the bacterial composition provided herein may contain Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus rhamnosus, Bifidobacterium longum, Bifidobacterium lactis and Bifidobacterium bifidum, and between about 0.005 mg and about 0.02 mg of silica.

[0025] In some cases, the bacterial compositions provided herein may include a flow aid (e.g., silica) of about 2.0 mg to about 5.0 mg per gram of the bacterial composition. For example, a 2-gram bacterial composition provided herein may contain a flow aid (e.g., silica) of about 4.0 mg to about 10.0 mg.

[0026] A flow aid (e.g., silica) that may be included in the bacterial compositions provided herein can be obtained using any suitable method. For example, silica can be obtained as described elsewhere (U.S. Patent Application Publication No. 2006 / 153764). In some cases, silica may be commercially available from Grace Davison, Inc. (Baltimore, MD; Catalogue No. Syloid 244).

[0027] In some cases, the bacterial compositions provided herein may include A wFillers with a concentration less than 0.2. Examples of such fillers include (but are not limited to) microcrystalline cellulose (MCC 112), rice maltodextrin, anhydrous lactose, mannitol, microcrystalline cellulose (MCC 302), microcrystalline cellulose (MCC 200 LM), microcrystalline cellulose (MCC 101), starch, xylitol, sorbitol, hydroxypropyl cellulose, gelatin, polyvinylpyrrolidone, and dicalcium phosphate. In some cases, the bacterial compositions provided herein may include A concentrations less than 0.2. w A filler (e.g., MCC 112) of approximately 300 mg to approximately 700 mg per bacterial composition. For example, the bacterial compositions provided herein may contain *Lactobacillus acidophilus*, *Lactobacillus plantarum*, *Lactobacillus casei*, *Lactobacillus rhamnosus*, *Bifidobacterium longum*, *Bifidobacterium lactis*, and *Bifidobacterium bifidum*, and a filler of approximately 300 mg to approximately 700 mg, A. w A filler with an A content of less than 0.2 (e.g., MCC 112). In some cases, the bacterial compositions provided herein may include between about 300 mg and about 700 mg per gram of bacterial composition, having an A content of less than 0.2. w The filler (e.g., MCC 112). For example, a 2-gram dose of the bacterial composition provided herein may contain between approximately 600 mg and approximately 1400 mg of A. w Fillers smaller than 0.2 (e.g., MCC 112). Therefore, the final composition A w It can be less than 0.3.

[0028] A can be obtained using any suitable method for use in the bacterial compositions provided herein. w Filler with a density less than 0.2. For example, A. w Fillers smaller than 0.2 can be obtained as described elsewhere (U.S. Patent Application Publication No. US20140322282, published October 30, 2014). In some cases, A w Fillers smaller than 0.2 are commercially available from Mingtai Chemical Co., Ltd., Taiwan, catalog number M112 D.

[0029] In some cases, the bacterial compositions provided herein may include magnesium stearate. For example, the bacterial compositions provided herein may include between 10 mg and 20 mg of magnesium stearate per bacterial composition. For example, the bacterial compositions provided herein may contain Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus rhamnosus, Bifidobacterium longum, Bifidobacterium lactis, and Bifidobacterium bifidum, and between 10 mg and 20 mg of magnesium stearate.

[0030] In some cases, the bacterial compositions provided herein may include between about 10 mg and about 20 mg of magnesium stearate per gram of bacterial composition. For example, a 2-gram bacterial composition provided herein may contain between about 20 mg and about 40 mg of magnesium stearate.

[0031] Magnesium stearate, which may be included in the bacterial compositions provided herein, can be obtained using any suitable method. For example, magnesium stearate may be obtained as described elsewhere (e.g., Chinese Patent No. CN103880645, dated June 25, 2014; Chinese Patent No. CN103524324, dated January 22, 2014; and Chinese Patent No. CN10319361, dated July 10, 2013). In some cases, magnesium stearate may be commercially available from Peter Greven Asia, Malaysia; catalog number Palmstar MGST 200. The bacterial compositions provided herein (e.g., capsules or tablets) may be formulated to have specific dosages. For example, the bacterial compositions provided herein may be in capsule or tablet form with a total weight between about 150 mg and about 800 mg (e.g., between about 200 mg and about 800 mg, between about 300 mg and about 800 mg, between about 350 mg and about 800 mg, between about 150 mg and about 700 mg, between about 150 mg and about 650 mg, or between about 350 mg and about 700 mg).

[0032] As described herein, single bacterial compositions (e.g., capsules or tablets) can be formulated in a manner that helps maintain bacterial stability within the composition to include at least six different strains and have an A value of less than 0.2. w The filler (e.g., MCC 112). In some cases, the single bacterial composition provided herein may be coated or placed in a capsule having the ability to deliver its contents into the mammalian gut after oral administration. For example, the bacterial composition provided herein may be designed to release its contents when the composition reaches a location in the gut where the pH is above about 6.8. In some cases, it includes at least seven different bacterial strains and has an A value of less than 0.2. w The bacterial composition of the filler (e.g., MCC 112) may include fructooligosaccharides, magnesium stearate, and / or silica. In some cases, the strains in the bacterial compositions provided herein may be lyophilized to form a dry powder containing live bacteria. In some cases, a single bacterial composition (e.g., capsules, tablets, or sacs) may be formulated to include at least seven different lyophilized strains with an A value of less than 0.2. w The fillers (e.g., MCC 112), fructooligosaccharides, magnesium stearate, and silicon dioxide.

[0033] In some cases, the bacterial compositions provided herein can maintain at least about 80% (e.g., at least about 90%, 95%, or 99%) of bacterial viability for at least 12 months (e.g., at least 40, 50, 60, 70, 80, or 90 days, or at least 3, 6, 9, or 12 months) under standard storage conditions (e.g., room temperature at normal humidity). In some cases, the bacterial compositions provided herein can maintain at least about 80% of bacterial viability for at least 18 months or at least 24 months.

[0034] The bacterial compositions provided herein may be administered to mammals (e.g., humans). In some cases, a person may be instructed to self-administer the bacterial compositions provided herein (e.g., capsules, tablets, or sacs) a certain number of times per unit of time (e.g., once, twice, three times, four times, five times, or more). For example, a person may be instructed to self-administer the bacterial compositions provided herein (e.g., the capsules of Example 1) once or twice daily.

[0035] The present invention is further described in the following embodiments, which do not limit the scope of the invention as set forth in the claims.

[0036] Example

[0037] Example 1 - Stability of the bacterial composition

[0038] Two bacterial compositions (Prototype Experiment 1 and Prototype Experiment 2) were prepared as follows. The final formulations were stored in multiple vials, as described below: (i) MCC 101 and MCC 112 were used as diluents in prototype testing.

[0039] (ii) Store the final capsules in PET bottles, HDPE bottles and sealed aluminum foil packs.

[0040] Prototype Experiment 1:

[0041] 270 mg of the above-mentioned mixture was filled into a single capsule.

[0042] 270mg of admixture potency = 24 / 1000 270 = 6.48 10 9 CFU / capsules

[0043] Prototype Experiment 2:

[0044] 270 mg of the above-mentioned mixture was filled into a single capsule.

[0045] 270mg of admixture potency = 24 / 1000 270 = 6.48 10 9 CFU / capsules

[0046] Prototype Experiment 3 was conducted as follows:

[0047] Use 270 mg of the above-mentioned blend to fill a single capsule.

[0048] 270mg of admixture potency = 24 / 1000 270 = 6.48 10 9 CFU / capsules

[0049] After the two bacterial compositions were prepared, their stability was tested (Table 1).

[0050] Table 1. Probiotic count (CFU / capsule) and water activity

[0051] These results demonstrate that the use of microcrystalline cellulose MCC 112 in bacterial compositions containing at least two strains improves stability better than MCC 101, and that the use of high-density polyethylene (HDPE) bottles or aluminum foil packaging improves stability better than the use of PET bottles.

[0052] Example 2 - Stability of the bacterial composition

[0053] The bacterial composition is prepared as follows.

[0054]

[0055] 400 mg of the above-mentioned blend was filled into a single DR capsule. The potency of the 400 mg blend was 20 mg per capsule. 10 9 CFU.

[0056] After the bacterial composition was prepared, its stability was tested (Table 2).

[0057] Table 2. Probiotic count (CFU / capsule) and water activity

[0058] These results demonstrate that the use of microcrystalline cellulose MCC 112 and high-density polyethylene (HDPE) bottles maintains the stability of bacterial compositions containing seven bacterial strains.

[0059] Example 3 - Bacterial Composition

[0060] The bacterial composition was prepared as follows (Table 3). In short, different CFUs of lyophilized bacteria from each of the following species—Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus rhamnosus, Bifidobacterium longum, Bifidobacterium lactis, and Bifidobacterium bifidum—were mixed with silica (silica; 2 mg) for two minutes to uniformly coat the dried bacteria. Silica adsorbs moisture, minimizing water activity to maintain the dryness of the lyophilized bacteria and improving the stability of the final formulation. MCC 112 (A) was then added. w Microcrystalline cellulose grade (700 mg) and fructooligosaccharides (50 mg) with a concentration less than 0.2 mg are added, and the mixture is then blended for another 20 minutes. After blending, additional silica (3 mg) is added as a gliding agent and magnesium stearate (15 mg) as a lubricant, and the mixture is blended for another three minutes. The final mixture is then inserted into a capsule (e.g., DR capsules). DR capsules are vegetarian capsules made using hydroxypropyl methylcellulose (HPMC) formulations, which help protect sensitive ingredients from the low pH environment of the stomach. By preventing early disintegration, the disintegration initiation time is typically about 45 minutes later than that of a typical immediate-release capsule (about 5 minutes), and the components are released in the alkaline pH of the intestine.

[0061] Table 3 Bacterial Compositions

[0062] Other implementation methods

[0063] It should be understood that although the invention has been described in conjunction with its embodiments, the foregoing description is intended to be illustrative and not to limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications fall within the scope of the following claims.

Claims

1. A bacterial composition comprising Lactobacillus acidophilus ( Lactobacillus acidophilus Lactobacillus plantarum ( Lactobacillus plantarum Lactobacillus casei ( Lactobacillus casei Lactobacillus rhamnosus ( Lactobacillus rhamnosus ), Bifidobacterium longum ( Bifidobacterium longum ), Bifidobacterium lactis ( Bifidobacterium lactis ) and Bifidobacterium bifidum ( Bifidobacterium bifidum The bacterial composition, after oral administration to a mammal, releases the Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus rhamnosus, Bifidobacterium longum, Bifidobacterium lactis, and Bifidobacterium bifidum into the intestine of the mammal, and the composition has a water activity of less than 0.

3.

2. The composition of claim 1, wherein, with respect to each gram of the composition, the composition comprises about 4 × 10⁻⁶. 9 Each settlement unit and approximately 50 × 10 9 Lactobacillus acidophilus between colony-forming units.

3. The composition of claim 1 or 2, wherein, with respect to each gram of the composition, the composition comprises about 5 × 10⁻⁶. 9 One settlement forming unit and approximately 60 × 10 9 Lactobacillus plantarum between colony-forming units.

4. The composition according to any one of claims 1-3, wherein, with respect to each gram of the composition, the composition comprises about 4 × 10⁻⁶ 9 Each settlement unit and approximately 50 × 10 9 Lactobacillus casei between colony-forming units.

5. The composition according to any one of claims 1-4, wherein, with respect to each gram of the composition, the composition comprises about 4 × 10⁻⁶. 9 Each settlement unit and approximately 50 × 10 9 Lactobacillus rhamnosus among the colony-forming units.

6. The composition according to any one of claims 1-5, wherein, with respect to each gram of the composition, the composition comprises about 2 × 10⁻⁶ 9 Each settlement unit and approximately 40 × 10 9 Bifidobacteria longiformis between colony-forming units.

7. The composition according to any one of claims 1-6, wherein, for every gram of the composition, the composition comprises about 4 × 10⁻⁶ 9 Each settlement unit and approximately 50 × 10 9 Bifidobacterium lactis between colony-forming units.

8. The composition according to any one of claims 1-7, wherein, with respect to each gram of the composition, the composition comprises about 2 × 10⁻⁶ 9 Each settlement unit and approximately 40 × 10 9 Bifidobacteria between colony-forming units.

9. The composition of any one of claims 1-8, wherein the composition comprises fructooligosaccharides.

10. The composition of any one of claims 1-9, wherein, for each gram of the composition, the composition comprises between about 0.5 mg and about 50 mg of fructooligosaccharides.

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