Magnesium oxide composition

Abstract

To suppress changes in bacterial flora caused by magnesium oxide preparations (1); and to provide methods that can improve the stability such as the survival rate of lactic acid bacteria, when magnesium oxide and lactic acid bacteria coexist for a certain period of time and the stability such as the survival rate of lactic acid bacteria is reduced (2), and can improve the survival rate of lactic acid bacteria and accurately measure the number of viable lactic acid bacteria when magnesium oxide and lactic acid bacteria coexist and the stability such as the survival rate of lactic acid bacteria is reduced (3).SOLUTION: The above problems are solved by (1) Lactobacillus acidophilus or Lactobacillus gasseri, lactic acid bacteria such as Bifidobacterium, (2) phosphate (preferably anhydrous calcium hydrogen phosphate, and the like), and (3) a mixture of one or more phosphates (preferably a mixture of potassium dihydrogen phosphate and anhydrous sodium monohydrogen phosphate, and the like).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to (1) a composition for inhibiting changes in bacterial flora caused by magnesium oxide, (2) a composition or method for improving the stability of lactic acid bacteria, such as the survival rate, which decreases when magnesium oxide and lactic acid bacteria coexist for a certain period of time, and (3) a method for accurately measuring the viable cell count of lactic acid bacteria by improving the survival rate of lactic acid bacteria. [Background technology]

[0002] When administered into the body, magnesium oxide becomes a poorly absorbed carbonate, which absorbs water from the intestinal wall. It stimulates the intestinal wall, enhances peristalsis, and softens the contents of the intestinal tract. Magnesium oxide preparations are therefore used as a remedy for constipation (Patent Document 1).

[0003] However, it is not known that magnesium oxide preparations change the intestinal bacterial flora, and it is not known at all that lactic acid bacteria such as Lactobacillus acidophilus and Lactobacillus gasseri can suppress changes in the bacterial flora caused by magnesium oxide preparations. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Laid-Open No. 2012-031150 Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention aims to (1) suppress changes in the bacterial flora in the body (preferably in the intestines) caused by a magnesium oxide preparation (particularly, improve disturbances in the bacterial flora). Furthermore, during the investigation of (1), the present inventors discovered that the coexistence of magnesium oxide and lactic acid bacteria reduces the stability of the lactic acid bacteria, such as their survival rate, and continued to investigate this novel problem. Specifically, the present invention also aims to (2) improve the stability of the lactic acid bacteria, such as their survival rate, when the stability decreases after the coexistence of magnesium oxide and lactic acid bacteria for a certain period of time, and (3) provide a method for improving the survival rate of lactic acid bacteria when magnesium oxide and lactic acid bacteria coexist, and for accurately measuring the viable cell count of lactic acid bacteria. [Means for solving the problem]

[0006] As a result of intensive research to solve the above problems, the present inventors have surprisingly found that (1) lactic acid bacteria such as Lactobacillus acidophilus or Lactobacillus gasseri can suppress changes in bacterial flora caused by magnesium oxide preparations (improving bacterial flora disturbances). Furthermore, (2) when magnesium oxide and lactic acid bacteria coexist for a certain period of time, the stability of the lactic acid bacteria, such as their survival rate, which decreases, can be improved by using an excipient or a phosphate (preferably, anhydrous calcium hydrogen phosphate, etc.), and (3) when magnesium oxide and lactic acid bacteria coexist, a mixture of two or more phosphates (preferably, a mixture of potassium dihydrogen phosphate and anhydrous sodium monohydrogen phosphate, etc.) can improve the survival rate of lactic acid bacteria and accurately measure the viable cell count of lactic acid bacteria. Continuing their research, the present inventors have now completed the present invention.

[0007] That is, the present invention relates to the following. [1] A composition for inhibiting changes in bacterial flora, comprising magnesium oxide and lactic acid bacteria. [2] The composition according to [1], wherein the lactic acid bacteria are one or more bacteria selected from the group consisting of Lactobacillus acidophilus, Lactobacillus gasseri, Lactobacillus bulgaricus, Lactobacillus casei, Streptococcus faecium, and Streptococcus faecalis. [3] The composition according to [1], characterized in that the lactic acid bacterium is Lactobacillus acidophilus KS-13 (accession number: NITE BP-819). [4] The composition according to [1], wherein the lactic acid bacteria are one or more bacteria selected from the group consisting of Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium infantis, and Bifidobacterium breve. [5] The composition according to [1], characterized in that the lactic acid bacterium is Bifidobacterium bifidum G9-1 (Accession number: NITE BP-817). [6] When the composition is liquid, the lactic acid bacteria is present in an amount of 10 4 ~10 10 cfu / mL, or When the composition is solid, the lactic acid bacteria is present in an amount of 10 5 ~10 10 The composition according to any one of [1] to [5], characterized in that it contains cfu / g. [7] The composition according to any one of [1] to [6], further comprising one or more phosphates selected from the group consisting of anhydrous calcium hydrogen phosphate, calcium phosphate, anhydrous calcium phosphate, calcium hydrogen phosphate, calcium hydrogen phosphate dihydrate, and calcium dihydrogen phosphate monohydrate. [8] The composition according to [7], wherein the phosphate is anhydrous calcium hydrogen phosphate. [9] The composition according to [7] or [8], wherein the mass ratio of the phosphate to the magnesium oxide is 100:1 to 1:100.

[10] The composition according to any one of [1] to [9], characterized in that the magnesium oxide content is 94.3 mass % or less based on the total mass of the composition.

[11] The composition according to any one of [1] to

[10] , further comprising an excipient.

[12] The composition according to any one of [1] to

[11] , further comprising a desiccant.

[13] The composition according to any one of [1] to

[12] , further characterized in that the stability of lactic acid bacteria is improved.

[14] The composition according to any one of [1] to

[13] , further characterized by improving bowel movements.

[15] A method for measuring the viable cell count of lactic acid bacteria, characterized by measuring in the presence of a diluent or buffer solution containing a mixture of one or more phosphates selected from the group consisting of potassium dihydrogen phosphate, dipotassium phosphate, tripotassium phosphate, dipotassium hydrogen phosphate, monosodium phosphate (monohydrate), monosodium phosphate (anhydrous), anhydrous sodium monohydrogen phosphate, disodium phosphate (dihydrate), disodium phosphate (heptahydrate) and trisodium phosphate (anhydrous) in a range of 0.0001 to 1 g / mL, in the presence of lactic acid bacteria and magnesium oxide.

[16] The method for measuring the viable cell count of lactic acid bacteria according to

[15] , characterized in that the mixture of phosphates contains potassium dihydrogen phosphate and anhydrous sodium monohydrogen phosphate.

[17] The method for measuring the viable cell count of lactic acid bacteria according to

[16] , characterized in that in the mixture of phosphates, the mass ratio of potassium dihydrogen phosphate to anhydrous sodium monohydrogen phosphate is 0.1:100 to 100:0.1. [Effects of the Invention]

[0008] The present invention can provide (1) a composition capable of inhibiting changes in bacterial flora caused by magnesium oxide (hereinafter also referred to as a "composition for inhibiting changes in bacterial flora"), (2) a method for improving the stability of lactic acid bacteria, such as the survival rate, which decreases when magnesium oxide and lactic acid bacteria coexist for a certain period of time, and (3) a method for accurately measuring the viable cell count of lactic acid bacteria when magnesium oxide and lactic acid bacteria coexist. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 is a schematic diagram of a spray drying apparatus. [Figure 2] Figure 2 shows the percentage change in total fecal wet weight from Day 0 to Day 2. * p<0.05 vs. Control by Steel [Figure 3] Figure 3 shows a graph quantifying the difference in gut microbiota composition from the normal group. * p<0.05 vs NN by Student's t-test, ## p<0.01 vs NC by Tukey-Kramer, †† p<0.01 vs NM by Tukey-Kramer. [Figure 4] Figure 4 shows the Firmicutes occupancy (n=7, mean SE). [Figure 5] Figure 5 shows the Bacteroidetes occupancy (n = 7, mean SE). * p<0.05 vs. Control by Student's t-test, ## p<0.01 vs. Control by Dunnett. [Figure 6]FIG. 6 is a graph showing the relationship between the blending ratio of magnesium oxide:phosphate and the viable cell count of bifidobacteria (Bifidobacterium bifidum G9-1) after storage for one month. [Figure 7] FIG. 7 is a graph showing the relationship between the blending ratio of magnesium oxide:phosphate and the viable cell count of bifidobacteria (Bifidobacterium bifidum G9-1) after storage for one month. [Figure 8] FIG. 8 is a graph showing the change in survival rate of lactic acid bacteria (Lactobacillus acidophilus KS-1) over the number of months of storage in magnesium oxide compositions (preparations) with and without a desiccant. [Figure 9] FIG. 9 is a graph showing the change in moisture content in magnesium oxide compositions (preparations) with and without a desiccant over the number of months of storage. [Figure 10] FIG. 10 is a graph showing the relationship between the magnesium oxide concentration and the influence of different diluents and buffer solutions on the viable cell count of lactic acid bacteria (Lactobacillus acidophilus KS-13). [Figure 11] FIG. 11 is a graph showing the effect of different diluents and buffer solutions on the viable cell count of bifidobacteria (Bifidobacterium bifidum G9-1) and the relationship with magnesium oxide concentration. [Figure 12] 12 is a graph showing the relationship between the amount of excipient (glucose) added and the survival rate of magnesium oxide compositions containing lactic acid bacteria (Lactobacillus acidophilus KS-13). Error bars indicate standard error, and * = p<0.05 (by Dunnett's test). [Figure 13] 13 is a graph showing the relationship between the amount of excipient (lactose hydrate) added and the survival rate of magnesium oxide compositions containing lactic acid bacteria (Lactobacillus acidophilus KS-13). Error bars indicate standard error, and * = p<0.05 (by Dunnett's test). [Figure 14]14 is a graph showing the relationship between the amount of excipient (dried cornstarch) added and the survival rate of magnesium oxide compositions containing lactic acid bacteria (Lactobacillus acidophilus KS-13). Error bars indicate standard error, and * = p<0.05 (by Dunnett's test). DETAILED DESCRIPTION OF THE INVENTION

[0010] [Composition for inhibiting changes in bacterial flora] In the present invention, "suppression of changes in bacterial flora" generally refers to the ability to suppress changes in bacterial flora (particularly improve disturbances in bacterial flora) when the bacterial flora in an animal's body (intestine) is changed by magnesium oxide or a preparation containing magnesium oxide, preferably the ability to suppress the changes in the bacterial flora by lactic acid bacteria, and more preferably the ability to suppress the changes in the bacterial flora by lactic acid bacteria such as Lactobacillus acidophilus or Lactobacillus gasseri, or lactic acid bacteria (bifidobacteria) such as Bifidobacterium bifidum.

[0011] Specific examples of the inhibition of changes in the bacterial flora (improvement of the bacterial flora), as will be explained in the Examples below, include, but are not limited to, the fact that in an analysis based on the intestinal bacterial flora composition (unweighted Unifrac Distance), when there is a constipation-induced group and a normal group (non-constipation-induced group) in terms of composition and there is a certain distance between them, the constipation-induced group administered with the composition of the present invention will be closer to the normal group (the distance will be shorter) than the constipation-induced group not administered with anything. Another preferred example of the improvement of the bacterial flora according to the present invention may include, but is not limited to, a decrease in the number of bacteria belonging to the phylum Firmicutes, known as so-called "fat bacteria," or an increase in the number of bacteria belonging to the phylum Bacteroidetes, known as so-called "skinny bacteria," from the viewpoint of maintaining health and preventing obesity.

[0012] Furthermore, as used herein, "intestine" generally refers to the inside or surface of the intestinal tract, such as the cecum, large intestine, small intestine, jejunum, ileum, duodenum, or colon, within an animal's body, and is preferably, but not limited to, the large intestine, small intestine, or cecum. Furthermore, animals having an intestine (intestinal tract) are preferably mammals, and examples of such mammals include humans, mice, rats, rabbits, dogs, cats, cows, horses, pigs, and monkeys, with humans, mice, and rats being more preferred, and humans being even more preferred, but not limited to these animals.

[0013] [Magnesium oxide] The magnesium oxide contained in the composition of the present invention or used in the method of the present invention can be a commercially available product, and magnesium oxide itself or a commercially available magnesium oxide preparation can be used. Preferably, in the composition of the present invention, magnesium oxide is contained in an amount, for example, but not limited to, greater than 0% by mass, greater than 0.01% by mass, greater than 1% by mass, greater than 10% by mass, or greater than 25% by mass relative to the entire composition. Furthermore, in the composition of the present invention, magnesium oxide is contained in an amount, for example, but not limited to, 94.3% by mass or less, 90% by mass or less, 85% by mass or less, or 80% by mass or less, more preferably 50 to 94.3% by mass, and particularly preferably 50 to 80% by mass, relative to the entire composition. When a magnesium oxide preparation is used, the amount of magnesium oxide in the composition (preparation) is preferably equal to or less than the above-mentioned ratio. When the magnesium oxide content is within the above-mentioned range, the effect of inhibiting bacterial flora change of the present invention is preferably achieved.

[0014] [Improved stability of lactic acid bacteria] In the present invention, "improving the stability of lactic acid bacteria" refers to the improvement of the stability of lactic acid bacteria, such as the survival rate of lactic acid bacteria during storage, which may normally decrease when magnesium oxide and lactic acid bacteria coexist for a certain period of time, by the composition of the present invention. Here, the "certain period" is not particularly limited, but examples include storage in a hospital, pharmacy, or the like, where magnesium oxide and lactic acid bacteria coexist for one week to three years, preferably one month to six months, and more preferably one month to three months. The storage temperature is typically room temperature (e.g., about 1 to 30°C) or ordinary temperature (e.g., about 15 to 25°C), but may vary depending on the storage environment. For example, the temperature may be higher in summer or warm seasons and lower in winter or cold seasons. In another preferred embodiment, the certain period is one minute to ten days, and in yet another preferred embodiment, it may be one hour or less.

[0015] [Improvement of bowel movements] In the present invention, "improved bowel movements" generally refers to the fact that when a composition containing magnesium oxide and lactic acid bacteria of the present invention is administered to a subject, the subject's bowel movements are improved significantly, preferably synergistically, compared to administration of magnesium oxide alone or lactic acid bacteria alone. A specific indicator of improved bowel movements is, for example, an increase in fecal weight (wet weight) compared to a non-subject administration group. Preferred subjects for which bowel movements are improved include, but are not limited to, animals having the intestines described above.

[0016] Preferably, the composition of the present invention further contains one or more phosphates selected from the group consisting of anhydrous calcium hydrogen phosphate, calcium phosphate, anhydrous calcium phosphate, calcium hydrogen phosphate, calcium hydrogen phosphate dihydrate, and calcium dihydrogen phosphate monohydrate, and more preferably, the inclusion of anhydrous calcium hydrogen phosphate improves the stability of the lactic acid bacteria, such as their survival rate.

[0017] In the composition of the present invention, anhydrous calcium hydrogen phosphate is preferably included when the stability, such as the survival rate of lactic acid bacteria, is improved. Commercially available anhydrous calcium hydrogen phosphate (e.g., Fujicalin (Fuji Chemical Industry Co., Ltd.)) can be used, and examples thereof include, but are not limited to, spherical granules obtained by spray-drying anhydrous calcium hydrogen phosphate. Preferably, in the present invention, the mass ratio of anhydrous calcium hydrogen phosphate to magnesium oxide in the composition is 100:1 to 1:100, more preferably 20:1 to 1:20, but is not limited thereto. Another preferred example is, in the present invention, the mass ratio of anhydrous calcium hydrogen phosphate to magnesium oxide in the composition is 5:0.25 to 5:5, more preferably 5:0.5 to 5:5, and particularly preferably 5:1 to 5:3, but is not limited thereto. Alternatively, in the present invention, the mass ratio of anhydrous calcium hydrogen phosphate to lactic acid bacteria in the composition is preferably, but not limited to, 1:5 to 1:100, more preferably 1:10 to 1:100, and particularly preferably 1:5 to 1:100. The mass ratio of the other one or more phosphates (total amount) to magnesium oxide may be the same as that of anhydrous calcium hydrogen phosphate described above. Although the details of why the stability of lactic acid bacteria, such as their survival rate, decreases when they coexist with magnesium oxide preparations are unknown, it is possible that the presence of the magnesium oxide preparation causes part of the composition (preparation) to become alkaline, which may affect the lactic acid bacteria.

[0018] [Excipients] The composition of the present invention preferably further contains an excipient. The inclusion of an excipient is preferred because it can reduce the amount of magnesium oxide in the formulation or composition. Examples of preferred excipients include sugars or polysaccharides such as fructose, glucose, glucose hydrate, lactose, anhydrous lactose, lactose hydrate, sucrose, powdered sugar, pullulan, pectin, dextrin, alginic acid, carrageenan, and gum arabic; sugar alcohols such as mannitol, isomalt, inositol, xylitol, sorbitol, maltitol, and lactitol; starches such as corn starch, potato starch, wheat starch, rice starch, pregelatinized starch, partially pregelatinized starch, and hydroxypropyl starch; celluloses such as powdered cellulose, crystalline cellulose, microcrystalline cellulose, hydroxypropyl cellulose, low-substituted hydroxypropyl cellulose, croscarmellose sodium, and carmellose calcium; anhydrous calcium hydrogen phosphate, calcium hydrogen phosphate hydrate, potassium dihydrogen phosphate, and dipotassium phosphate. Examples of such additives include, but are not limited to, phosphates such as sodium hydrogen carbonate and tricalcium phosphate; carbonates such as calcium carbonate, magnesium carbonate and ammonium carbonate; citric acids such as calcium citrate, citric acid hydrate, sodium citrate hydrate, disodium citrate and anhydrous citric acid; povidone; crospovidone; silicic acid or a salt thereof such as light anhydrous silicic acid, magnesium aluminosilicate, calcium silicate and magnesium silicate; amino acids such as aspartic acid, glycine and glutamine; acetates such as potassium acetate and calcium acetate; metal oxides such as zinc oxide and titanium oxide; diatomaceous earth; cinnamon powder; tragacanth; casein; agar; cyclodextrin; paraffin; gypsum; talc; lecithin; gelatin; shellac; zein; powdered reduced maltose syrup; sodium chloride; sulfates; lactates; tartaric acid or a salt thereof; and stearic acid or a salt thereof. In addition to the above, excipients listed in the Pharmaceutical Additives Dictionary 2016 (edited by the Japan Pharmaceutical Additives Association) can also be used as appropriate. In the present invention, among the above, for example, sugars, polysaccharides, starches, and celluloses can be preferably used, and in particular, corn starch, crystalline cellulose, glucose, and lactose hydrate can be preferably used, but are not limited to these. The excipient may be solid or liquid, but is preferably solid.

[0019] In the composition of the present invention, the excipient is preferably contained in an amount of 0 to 50% by mass, more preferably 0 to 20% by mass, relative to the total mass of the composition, but is not limited to these ranges. For example, when the content of the excipient is within the above range, the magnesium oxide content in the composition can be reduced, which preferably results in improved survival rate of the lactic acid bacteria of the present invention, improved stability during storage, or an effect of inhibiting changes in the intestinal flora when administered.

[0020] Water activity (Aw) represents the vapor pressure (energy state of water) of the surface water of a substance. Generally, the lower the water activity, the higher the stability of live bacteria in the composition. In the present invention, excipients having various water activity values ​​can be used without any particular limitation, but for example, when an excipient with a water activity value higher than that of magnesium oxide is used, the excipient is preferably contained in an amount of 0 to 50% by mass, more preferably 0 to 20% by mass, relative to the total composition. Also, when an excipient with a water activity value lower than that of magnesium oxide is used, the excipient is preferably contained in an amount of 0 to 50% by mass, more preferably 0 to 20% by mass, relative to the total composition, but is not limited to these ranges. The water activity is preferably measured in accordance with, for example, the Japanese Pharmacopoeia 3.05 Adsorption-Desorption Isotherm Measurement Method and Water Activity Measurement Method, and an instrument for measuring water activity, such as the LabMaster-aw NEO manufactured by Novacina, can be suitably used.

[0021] [Desiccant] The composition of the present invention preferably further contains a desiccant. Preferred examples of the desiccant include silica gel (a desiccant mainly composed of SiO), Chiburet (a porous amorphous material mainly composed of AlO·SiO·nHO), calcium chloride, etc., and a specific example of the desiccant that can be preferably used is Chiburet (AS-W1510) manufactured by Tokai Chemical Industry Co., Ltd., but is not limited to these.

[0022] Preferably, the desiccant content of the composition of the present invention is 0.1 to 20% by mass, more preferably 1 to 10% by mass, and even more preferably 4 to 5% by mass, based on the total mass of the composition, but is not limited thereto. When the content of the desiccant is within the above range, an effect of improving the survival rate of the preferred lactic acid bacteria of the present invention during storage or an effect of improving the intestinal flora when administered can be obtained.

[0023] Other components in the composition of the present invention, the production method, lactic acid bacteria, etc. will be described in detail below. [Lactic acid bacteria such as Lactobacillus and Bifidobacteria] The lactic acid bacteria contained in the composition of the present invention or used in the method of the present invention may be bifidobacteria or non-bifidobacteria, and examples thereof include lactobacilli of the genus Lactobacillus, such as Lactobacillus acidophilus, L. casei, L. gasseri, L. plantarum, L. delbrueckii subsp. bulgaricus, L. delbrueckii subsp. lactis, L. fermentum, L. helveticus, L. johnsonii, L. paracasei subsp. paracasei, L. reuteri, L. rhamnosus, L. salivarius, and L. brevis; bacteria of the genus Leuconostoc, such as Leuconostoc mesenteroides; Streptococcus (Enterococcus) faecalis, Streptococcus (Enterococcus) faecium, Streptococcus (Enterococcus) hirae, and Streptococcus Examples of lactic acid bacteria include, but are not limited to, the genus Streptococcus such as Lactococcus thermophilus (currently classified as the genus Enterococcus), the genus Lactococcus such as Lactococcus lactis and L. cremoris, the genus Tetragenococcus such as Tetragenococcus halophilus, the genus Pediococcus such as Pediococcus acidilactici and P. pentosaceus, and the genus Oenococcus such as Oenococcus oeni. In this specification, unless otherwise specified, bacteria are classified according to current taxonomy, but according to the old classification, the genus Streptococcus may include the genera Streptococcus and Enterococcus.

[0024] In the present invention, when the lactic acid bacteria are lactic acid bacteria other than bifidobacteria, for example, the genus Lactobacillus is preferred, and more preferably Lactobacillus acidophilus, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus paracasei subsp. paracasei, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus salivarius, or Lactobacillus brevis, and particularly preferably Lactobacillus acidophilus KS-13. KS-13) and the like are preferred, but are not limited to these. It should be noted that Lactobacillus acidophilus KS-13 is an old classification, and the current classification and certificate of authenticity state that it is Lactobacillus gasseri KS-13. However, for convenience, this specification may use the old classification, which is the name that has been used for a long time.

[0025] Furthermore, in the present invention, when the lactic acid bacteria are bifidobacteria, preferred examples of bifidobacteria include Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium adolescentis, Bifidobacterium pseudolongum, and Bifidobacterium thermophilum, and more preferably Bifidobacterium bifidum or Bifidobacterium bifidum is used, with Bifidobacterium bifidum being particularly preferred. G9-1 (Bifidobacterium bifidum G9-1, accession number: NITE BP-817) is preferred, but is not limited thereto.

[0026] Other preferred examples of lactic acid bacteria other than Lactobacillus or Bifidobacteria include, for example, Streptococcus, particularly Streptococcus faecalis, and more preferably Streptococcus faecalis 129 BIO3B or Clostridium, but are not limited to these.

[0027] The above-mentioned lactic acid bacteria may be a single species or a mixture of multiple species. In the present invention, the composition preferably contains 1 to 5 species of lactic acid bacteria. More preferably, the composition contains 1 to 3 species of lactic acid bacteria. Particularly preferably, the composition contains 1 or 2 species of lactic acid bacteria, and most preferably, the composition contains 1 species of lactic acid bacteria.

[0028] In the composition of the present invention, the proportion of lactic acid bacteria is, for example, about 10 when the composition is liquid. 4 ~10 10 cfu / mL, preferably about 10 6 ~10 9 cfu / mL, etc., and when the composition is solid, for example, 10 lactic acid bacteria per whole composition. 5 ~10 10 cfu / g, preferably 10 6 ~10 9 These may include, but are not limited to, cfu / g, etc.

[0029] The composition of the present invention may further contain bacteria other than the above-mentioned lactic acid bacteria. Preferred examples of such bacteria other than lactic acid bacteria include, but are not limited to, butyric acid-producing bacilli such as Clostridium butyricum.

[0030] [Method for obtaining bacterial cells] The above-mentioned lactic acid bacteria cells such as Lactobacillus and Bifidobacteria can be easily obtained from organizations such as ATCC (registered trademark) or IFO, the Japan Bifidobacteria Center (a foundation), the National Institute of Technology and Evaluation (NITE) Patent Microorganisms Depositary, etc. Commercially available cells can also be used as appropriate. Note that the cells before culturing may be stored in a frozen state.

[0031] For example, Lactobacillus gasseri KS-13 has been internationally deposited with the National Patent Microorganisms Depositary (NPMD), National Institute of Technology and Evaluation (Address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan, Postal Code 292-0818) on September 17, 2009 (original deposit date: September 17, 2009) under the accession number NITE BP-819. In this specification, for convenience, the above-mentioned Lactobacillus gasseri KS-13 (accession number NITE BP-819) is sometimes referred to as Lactobacillus acidophilus KS-13, which is an older classification, but the two are the same bacterium.

[0032] Furthermore, for example, Bifidobacterium bifidum G9-1 has been internationally deposited with the National Patent Microorganisms Depositary (NPMD), National Institute of Technology and Evaluation (Address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan, Postal Code 292-0818) on September 30, 2009 (original deposit date: September 17, 2009) under the accession number NITE BP-817.

[0033] [Cultivation of lactic acid bacteria such as Lactobacillus and Bifidobacteria] Lactic acid bacteria are usually inoculated into a medium for culture. The basic composition of the medium used to culture these bacteria may be based on known media for culturing lactic acid bacteria or bifidobacteria, such as MRS medium, LBS medium, and Rogasa medium, which are general-purpose, highly nutritious growth media. Furthermore, media for anaerobic bacteria can also be preferably used, for example, GAM liquid media such as GAM bouillon and modified GAM bouillon, but are not limited to these.

[0034] The medium used in the present invention may contain, but is not limited to, a carbon source, a nitrogen source, amino acids, vitamins, minerals, animal and plant proteins or extracts and decomposition products thereof, inorganic salts, buffers, surfactants, antibiotics, stabilizers, water, or any combination thereof. Commercially available products can be obtained as components of the medium and used as appropriate.

[0035] Examples of nitrogen sources include animal or plant peptones, ammonium salts such as ammonium nitrate, ammonium sulfate, ammonium chloride, and ammonium acetate, and hydrates thereof, and ammonia. Examples of peptones that can be preferably used include, but are not limited to, soybean peptone, proteose peptone, casein peptone, myocardium peptone, and meat peptone. The nitrogen source content in the medium may be, for example, 0.1 to 1% by mass, or 0.1 to 0.5% by mass, based on the total mass of the medium.

[0036] Examples of carbon sources include monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, and polysaccharides. Examples of monosaccharides include glucose, xylose, arabinose, mannose, galactose, and any combination thereof. Examples of disaccharides include maltose, cellobiose, trehalose, sucrose, lactulose, lactose, and any combination thereof. The content of the carbon source in the medium may be, for example, 0.1 to 1% by mass or 0.1 to 0.5% by mass based on the total mass of the medium, but is not limited thereto.

[0037] The medium used in the present invention preferably contains components such as amino acids and vitamins as growth factors. Examples of amino acids include, but are not limited to, alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, pyrrolysine, proline, glutamine, arginine, serine, threonine, selenocysteine, valine, tryptophan, tyrosine, or any combination or salt thereof. These amino acids are usually L-type. The content of amino acids in the medium may be, for example, 0.01 to 0.1% by mass or 0.01 to 0.05% by mass based on the total mass of the medium, but is not limited thereto.

[0038] The vitamins preferably used include, but are not limited to, vitamins such as vitamins A, B, C, D, E, and K, or derivatives or salts thereof, biotin, riboflavin, thiamine, or any combination thereof. The content of the vitamins in the medium may be, for example, but is not limited to, 0.01 to 0.1% by mass or 0.01 to 0.05% by mass based on the total mass of the medium.

[0039] In addition, preferred minerals include, but are not limited to, magnesium, potassium, calcium, phosphorus, zinc, iron, etc. The mineral content in the medium may be, for example, 0.01 to 0.1% by mass or 0.01 to 0.05% by mass based on the total mass of the medium, but is not limited to these.

[0040] As the animal or plant protein or its extract or degradation product, for example, a plant extract, meat extract, liver extract, or yeast extract can be preferably used. The content of such an extract or degradation product in the medium may be, for example, 0.1 to 1% by mass or 0.1 to 0.5% by mass based on the total mass of the medium, but is not limited thereto.

[0041] Examples of inorganic salts include, but are not limited to, phosphates, sodium chloride, sodium nitrate, potassium nitrate, manganese sulfate hydrate, magnesium sulfate hydrate, etc. The content of inorganic salts in the medium may be, for example, but is not limited to, 0.01 to 0.1% by mass or 0.01 to 0.05% by mass based on the total mass of the medium.

[0042] Examples of buffering agents include, but are not limited to, PBS, HBSS, HEPES, HANKS, etc. Examples of surfactants that can be preferably used include, but are not limited to, polysorbates such as polysorbate 20, polysorbate 60, polysorbate 65, and polysorbate 80, macrogol, sodium lauryl sulfate, etc. Examples of antibiotics include, but are not limited to, penicillin, mycin-based antibiotics such as streptomycin and kanamycin, etc.

[0043] Other medium components or additives may include, but are not limited to, carbonate, bicarbonate, albumin, insulin, transferrin, selenium, hormones, cytokines, L-cysteine ​​hydrochloride, sodium thioglycolate, hemin, soluble starch, digested serum powder, vitamins, short-chain fatty acids, and the like. The medium can be prepared by mixing the above-listed components and heat sterilizing them using a high-pressure steam sterilizer.

[0044] The medium is preferably neutral (for example, pH 6 to 8, pH 7 to 8), and a known pH adjuster or the above-mentioned buffer may be used to achieve neutrality.

[0045] When inoculating lactic acid bacteria and / or bifidobacteria into a medium, the ratio of bacteria to the medium is, for example, 10 1 ~10 8 cfu / mL, and 5 ~10 8 The concentration may be, but is not limited to, cfu / mL. The medium for inoculation (seed culture) and the medium for growth (main culture) may be the same or different.

[0046] The culture temperature is, for example, preferably 25 to 45° C., and more preferably 36 to 38° C. The culture time is, for example, preferably 4 to 72 hours, and more preferably 12 to 24 hours. Within these culture temperature and culture time ranges, lactic acid bacteria and / or bifidobacteria tend to grow easily. In order to culture lactic acid bacteria and / or bifidobacteria under anaerobic conditions, an anaerobic box or an anaerobic chamber may be used. Commercially available anaerobic boxes or anaerobic chambers may be used.

[0047] The form of the bacterial cells used in the present invention is not particularly limited, and any form, such as live bacterial cells, wet bacterial cells, or dried bacterial cells, can be used. Furthermore, processed bacterial products obtained by further processing live bacterial cells, wet bacterial cells, or dried bacterial cells may also be used. The term "processed bacterial product" refers to lactic acid bacteria that have been subjected to some kind of treatment, and the treatment is not particularly limited. Specific examples of the processed product include a solution in which the bacterial cells have been disrupted by ultrasonication or the like, a culture solution or culture supernatant of the bacterial cells, and a solid residue obtained by separating the same using solid-liquid separation means such as filtration or centrifugation. Other examples of the processed product include a solution in which the cell walls have been removed using enzymes or mechanical means, and protein complexes (e.g., proteins, lipoproteins, glycoproteins) or peptide complexes (e.g., peptides, glycopeptides) obtained by trichloroacetic acid treatment or salting-out treatment. Furthermore, the processed product also includes concentrates, dilutions, or dried products thereof. Methods for obtaining unprocessed products from bacterial cells after harvesting them from a culture solution have been well established in the art, and these methods may be used in the present invention. Furthermore, the processed product of the present invention also includes a solution of the bacterial cells disrupted by ultrasonication or the like, a culture solution or culture supernatant of the cells, and the like, which has been further treated, for example, by separation using various types of chromatography.

[0048] Killed bacterial cells are also included in the processed bacterial products of the present invention. Killed bacterial cells can be obtained, for example, by enzyme treatment, heat treatment, treatment with drugs such as antibiotics, treatment with chemicals such as formalin, or treatment with radiation such as gamma rays. These techniques have been well established in the past, and such techniques may be used in the present invention.

[0049] Next, a preferred method for producing dried or wet bacterial cells will be described. The bacterial cells are dispersed in a solvent to form a bacterial cell solution. The solvent for dispersing the bacterial cells to form a bacterial cell solution may be any known solvent used in the art, but water or a buffer solution such as PBS is preferred. Ethanol or the like may also be added as desired. The bacterial cell solution may also be a suspension, and the solvent may be the same as those described above. A suspending agent, such as sodium alginate, may also be used when suspending the bacterial cells.

[0050] Furthermore, additives generally used in the art, such as antistatic agents, may be added to the bacterial cell liquid in the usual blending ratios according to known techniques. Antistatic agents include, for example, finely divided or non-finely divided talc, colloidal silica, modified silica, precipitated silica, and the like.

[0051] Furthermore, the composition may be sterilized. Sterilization is preferably carried out by, for example, filtration through a filter, but other known sterilization methods, for example, heat methods such as moist heat sterilization, dry heat sterilization, and high-frequency sterilization, gas methods such as ethylene oxide gas sterilization and hydrogen peroxide sterilization, and radiation methods such as gamma ray irradiation sterilization and electron beam irradiation sterilization, may also be used.

[0052] The above bacterial cell liquid can be subjected to a drying operation using a spray dryer to produce dried bacterial cells. The spray dryer is preferably equipped with an atomizer capable of forming single-micron spray droplets. By forming spray droplets with a very small particle size, the surface area per unit mass of the spray droplets increases, allowing for efficient contact with the drying hot air, improving productivity. Here, single-micron droplets preferably refer to spray droplets with a particle size of 1 to 10 μm, rounded off to the first decimal place.

[0053] Examples of spray dryers include those in which the atomizing device is a rotary atomizer (rotating disk), a pressure nozzle, or a two-fluid or four-fluid nozzle that utilizes the force of compressed gas. The spray dryer may be any of the above types of spray dryer as long as it can form single-micron spray droplets, but it is preferable to use a spray dryer with a four-fluid nozzle.

[0054] In a spray drying device with a four-fluid nozzle, for example, the four-fluid nozzle is preferably structured such that the gas flow path and the liquid flow path are arranged as one system, with two systems symmetrically arranged at the nozzle edge, and a slope that forms the fluid flow surface is formed at the nozzle edge. Furthermore, an external mixing type device is preferred, in which compressed gas and liquid converge from both sides toward the collision focus at the tip of the nozzle edge. This type enables long-term spraying without nozzle clogging.

[0055] A spray drying apparatus with a four-channel nozzle will be described in more detail with reference to Figure 1. At the nozzle edge of the four-channel nozzle, the bacterial liquid gushing out from liquid channel 3 or 4 is thinly stretched on fluid flow surface 5 by the high-speed gas flow of compressed gas from gas channel 1 or 2, and the stretched liquid is atomized by shock waves generated at collision focus 6 at the tip of the nozzle edge, forming single-micron spray droplets 7.

[0056] The compressed gas may be, for example, air, carbon dioxide gas, nitrogen gas, argon gas, or other inert gas. In particular, when spray-drying a substance that is easily oxidized, it is preferable to use an inert gas such as carbon dioxide gas, nitrogen gas, or argon gas. The pressure of compressed gas is usually about 1 to 15 kgf / cm 2 , preferably about 3 to 8 kg weight / cm 2 The amount of gas in the nozzle is usually about 1 to 100 L / min, and preferably about 10 to 20 L / min per 1 mm of the nozzle edge.

[0057] Typically, the sprayed droplets are then exposed to hot drying air in a drying chamber to evaporate the water and obtain a dried bacterial cell product. The inlet temperature of the drying chamber is typically about 2 to 400°C, preferably about 5 to 250°C, and more preferably about 5 to 150°C. Even if the inlet temperature is as high as about 200 to 400°C, the temperature inside the drying chamber does not rise significantly due to the heat of vaporization caused by the evaporation of water, and by shortening the residence time in the drying chamber, it is possible to suppress to some extent the death or damage of live bacteria. The outlet temperature is typically about 0 to 120°C, preferably about 5 to 90°C, and more preferably about 5 to 70°C.

[0058] As described above, reducing the particle size of the dried bacterial cell product has the advantage of increasing the viability and providing a composition (preparation) with a high viability. That is, to obtain a single-micron dried bacterial cell product, it is preferable to spray single-micron droplets. Reducing the particle size of the spray droplets increases the surface area per unit mass of the spray droplets, which allows for efficient contact with the drying warm air and minimizes the death or damage of the bacterial cells due to the heat of the drying warm air. As a result, a dried bacterial cell product with an increased viability and a high viable cell count can be obtained.

[0059] Wet bacterial cells can be obtained by methods known in the art, such as collecting bacterial cells from the culture medium by centrifugation, washing them with a phosphate buffer solution, and then centrifuging them again to obtain bacterial cells, which can then be frozen and stored.

[0060] [Composition] The composition of the present invention can be easily produced by mixing magnesium oxide, lactic acid bacteria, and other ingredients by methods known in the art. The other ingredients are not particularly limited as long as they achieve the effects of the present invention. The composition of the present invention can be used in the form of a pharmaceutical product, quasi-drug, food or drink, feed, etc. Such pharmaceutical products containing the agent of the present invention are also a preferred embodiment of the present invention.

[0061] The composition of the present invention may contain known additives commonly used in the art, such as water, solvents, pH adjusters, humectants, flavoring agents, sweeteners, thickeners, flavoring agents, gelling agents, solubilizers, coloring agents, preservatives, surfactants, suspending agents, emulsifiers, binders, disintegrants, lubricants, and stabilizers, but these are not limited to these. Preferred examples of binders include, but are not limited to, hydroxypropyl cellulose, polyvinylpyrrolidone, xanthan gum, etc. Preferred examples of disintegrants include, but are not limited to, low-substituted hydroxypropyl cellulose, carmellose calcium, partially pregelatinized starch, croscarmellose sodium, crospovidone, carboxymethyl starch, etc.

[0062] Lactic acid bacteria are generally anaerobic and susceptible to air or oxygen in a dry state, and are also susceptible to high temperatures and humidity. Therefore, when formulating the composition, it is preferable to treat the bacteria in the presence of an inert gas or in a vacuum at low temperatures.

[0063] The composition of the present invention may be administered to humans or non-human animals. The administration form of the present invention is not particularly limited, but examples include oral administration and parenteral administration (intravenous administration, transdermal administration, topical ocular administration, etc.). Examples of the dosage form of the present invention for oral administration include tablets, capsules, granules, powders, etc., while examples of parenteral administration include enemas, suppositories, and other inserts. The dosage can be appropriately selected depending on the dosage form, symptoms, age, body weight, and other factors of the patient. For example, in the case of oral administration, the dosage can be 0.05 to 5000 mg, preferably 0.1 to 2000 mg, and particularly preferably 1 to 1000 mg per kg of body weight per day, administered once or in divided doses, but is not limited thereto.

[0064] [Method for measuring viable lactic acid bacteria count] The present invention includes a method for measuring the viable cell count of lactic acid bacteria in the presence of lactic acid bacteria and magnesium oxide, i.e., in the presence of both, in the presence of a diluent or buffer solution containing a mixture of one or more phosphates selected from the group consisting of potassium dihydrogen phosphate, dipotassium phosphate, tripotassium phosphate, dipotassium hydrogen phosphate, monosodium phosphate (monohydrate), monosodium phosphate (anhydrous), anhydrous sodium monohydrogen phosphate, disodium phosphate (dihydrate), disodium phosphate (heptahydrate), and trisodium phosphate (anhydrous) in a total concentration range of 0.0001 to 10 g / mL, preferably 0.001 to 1 g / mL, more preferably 0.002 to 0.5 g / mL, and even more preferably 0.0348 to 0.1389 g / mL. In the present invention, when the mixture of one or more phosphates is within the above range, the survival rate of the lactic acid bacteria is further improved, allowing for more accurate measurement of the viable cell count of the lactic acid bacteria.

[0065] In the above method, the mixture of one or more phosphate salts in the diluent or buffer preferably contains potassium dihydrogen phosphate and anhydrous sodium monohydrogen phosphate, with the potassium dihydrogen phosphate and anhydrous sodium monohydrogen phosphate being present in a total concentration of 0.0001 to 1 g / mL, more preferably 0.001 to 0.5 g / mL, more preferably 0.002 to 0.3 g / mL, and even more preferably 0.0348 to 0.1389 g / mL. In the present invention, when the mixture of potassium dihydrogen phosphate and anhydrous sodium monohydrogen phosphate is present in the above range, the survival rate of lactic acid bacteria is further improved, and the viable cell count of lactic acid bacteria can be measured more accurately.

[0066] Furthermore, when the phosphate mixture in the diluent or buffer solution contains both potassium dihydrogen phosphate and anhydrous sodium monohydrogen phosphate, the mass ratio of potassium dihydrogen phosphate to anhydrous sodium monohydrogen phosphate is, for example, preferably 0.1:100 to 100:0.1, more preferably 1:10 to 10:1, even more preferably 1:2 to 2:1, and particularly preferably 16.0:18.8. In the present invention, a mass ratio of potassium dihydrogen phosphate to anhydrous sodium monohydrogen phosphate within the above range is preferred because it further improves the survival rate of lactic acid bacteria and enables more accurate measurement of the viable cell count of lactic acid bacteria.

[0067] Lactic acid bacteria preferably used in the method for measuring the viable cell count of lactic acid bacteria of the present invention include the lactic acid bacteria that can be used in the composition of the present invention described above, and preferably Bifidobacterium bifidum. Other preferred lactic acid bacteria include, but are not limited to, Lactobacillus acidophilus or Lactobacillus gasseri. The viable cell count of lactic acid bacteria is measured according to a known method, such as the method (quantitative method) described in the section on bifidobacteria or lactomin in the Japanese Pharmacopoeia Extraordinary Pharmaceutical Standards (extraordinary regulations). [Example]

[0068] Next, the present invention will be explained in more detail by way of test examples and examples, but the present invention is not limited to these examples in any way, and many modifications can be made by those skilled in the art within the technical concept of the present invention.

[0069] The compounds used in the test examples and examples of the present invention are readily available as commercially available products, and these can be used. The following abbreviations may be used below: MgO: Magnesium oxide LA or Lac: Lactic acid bacteria Lactobacillus Acidophilus KS-13 (this is the old classification name; in the new classification, it is Lactobacillus gasseri KS-13, accession number NITE BP-819. Both are the same bacterium) Bif: Bifidobacterium bifidum G9-1 (Accession number: NITE BP-817) CE-2 powdered feed: CE-2 CaHPO4: anhydrous calcium hydrogen phosphate

[0070] [Example 1] Confirmation of the effect of the composition of the present invention in suppressing changes in bacterial flora Test method: Thirty-two male 6-week-old Slc:SD rats were purchased as test animals from Japan SLC Co., Ltd. From the acclimation period, all 32 rats were given free access to gamma-ray sterilized (30 kGy) CE-2 powdered feed (Lot No: E2020-FU) manufactured by Japan CLEA Co., Ltd., and tap water as drinking water using a water bottle (manufactured by Japan CLEA Co., Ltd.). After 5 days of acclimation, the rats were divided into four groups (8 rats each) - Normal group, Control group, MgO group, and MgO+LA group - so that the weights of the groups were uniform. During the test period, the MgO and MgO+LA groups were fed CE-2 containing magnesium oxide (Magmit Granules 83%, Kyowa Chemical Industry Co., Ltd.) at a dose of 400 mg / kg. In addition, rats in the three groups other than the Normal group were given loperamide (Sigma-Aldrich) subcutaneously at a dose of 5.0 mg / kg twice daily for four days from Day 0 to Day 3 to induce constipation.

[0071] The normal group received the same volume of saline (Otsuka Pharmaceutical Factory, Inc.) as a vehicle, administered subcutaneously twice a day for four days from Day 0 to Day 3. In addition, the MgO+LA group had a 3.3×10 8 LA was orally administered at a concentration of cfu / mL / head three times a day for four days from Day 0 to Day 3. The three groups other than the MgO + LA group received the same volume of vehicle buffer solution (Nacalai Tesque) three times a day for four days from Day 0 to Day 3. The total fecal weight was measured daily during the test period to assess fecal quality. The total fecal weight was measured as the weight of all feces excreted over a 24-hour period from 9:00 AM to 9:00 AM the following day (Figure 1). Fresh feces for bacterial flora analysis were collected on the morning of the final day of the test (Day 4), flash-frozen in liquid nitrogen immediately after collection, and stored at -80°C until use in the assay.

[0072] Microbial community analysis method: For microbiota analysis, comprehensive 16S rRNA sequencing was performed using next-generation sequencing (NGS). DNA was extracted from feces using the bead-phenol method, and sequencing of the V3-V4 region of the 16S rRNA gene was performed using the Miseq platform according to the method described in Fadrosh DW et al., Microbiome 2014, 2: 6. The sequence read data obtained from Miseq was then analyzed using the QIIME pipeline. Read synthesis was performed using Fastq-join, and quality filtering (QV ≥ 25) was performed using USEARCH v6.1. Chimeric reads were removed from the filtered read data, and the resulting read data was used for microbiota analysis. Five thousand reads per sample were randomly extracted, and operational taxonomic units (OTUs) were created using USEARCH with a homology ratio of 97%. Representative sequences from the OTUs were subjected to a homology search using UCLUST, and each read was identified to the bacterial phylum level. The dissimilarity of the gut microbiota composition (unweighted unifrac distance) for each sample was calculated using the statistical analysis software R (https: / / www.r-project.org / ), and the obtained values ​​were plotted using principal coordinate analysis (PCoA). Principal coordinate analysis graphically represents a similarity matrix between p elements (individuals, variables, objects, etc.), and in the present invention, it is used to confirm changes and improvements in the microbiota.

[0073] Test results (total fecal wet weight): The rate of change in total fecal wet weight on Day 2 after the start of the test tended to increase in the MgO and LA groups compared to the control group. On the other hand, the MgO + LA group had a significantly higher value than the control group, and a significant increase in fecal weight was observed at an earlier stage, indicating a synergistic improvement in bowel movements due to MgO and LA (Figure 2).

[0074] Test results (improvement of intestinal flora (1): composition of intestinal flora) We analyzed the gut microbiota profile based on the gut microbiota composition (unweighted unifrac distance). The distance (dissimilarity) between the Normal group and each group in the principal coordinate analysis of gut microbiota composition was quantified and plotted as shown in Figure 3. Specifically, the distance between the Normal group and the MgO group and the Normal group and the MgO+LA group was significantly smaller than the distance (dissimilarity) between the Normal group and the Control group in the principal coordinate analysis of gut microbiota composition. Therefore, the disruption of the gut microbiota due to constipation was improved in the MgO and MgO+LA groups. In particular, the distance between the Normal group and the MgO+LA group was significantly smaller than the distance between the Normal group and the MgO group. This indicates that the MgO+LA group, which received magnesium oxide and lactic acid bacteria, suppressed changes in the gut microbiota more effectively than the MgO group, which received only magnesium oxide. This indicates that the disruption of the gut microbiota was improved (Figure 3).

[0075] Test results (improvement of bacterial flora (2): type of bacteria) When considering changes in the intestinal flora from the perspectives of maintaining health and preventing obesity, it is preferable to suppress the increase of bacteria in the phylum Firmicutes, which are known as "fat bacteria." Furthermore, it is preferable to suppress the decrease of bacteria in the phylum Bacteroidetes, which are known as "skinny bacteria." When comparing bacterial species (phyla), the control group showed a tendency to increase the number of bacteria in the phylum Firmicutes compared to the normal group, but this increase was suppressed in the MgO group and the MgO + LA group (Figure 4). On the other hand, while the Bacteroidetes bacteria were significantly reduced in the control group (constipation-induced group), the MgO group showed a tendency to suppress the reduction compared to the control, and the MgO+LA group showed an even more significant suppression of the reduction compared to the MgO group (Figure 5). Therefore, it can be said that the combination of magnesium oxide and lactic acid bacteria of the present invention suppressed changes in the intestinal flora (improved the disturbance of the flora).

[0076] [Example 2] Survival rate of lactic acid bacteria (bifidobacteria) after storage for a certain period Four types of mixed powders containing bifidobacterium (Bifidobacterium bifidum G9-1) powder with the blending ratios shown in Table 1, magnesium oxide, and anhydrous calcium hydrogen phosphate were prepared, and 45 g of each mixed powder was filled into glass bottles and stored at 40°C and 75% relative humidity for two months. The viable bacterial counts in the mixed powders were then measured at the start of storage and after one and two months of storage. Figure 6 shows the survival rate of bacteria after one month of storage relative to the viable bacterial count at the start. The viable lactic acid bacteria count was measured according to the method (quantitative method) described in the section on bifidobacteria in the Japanese Pharmacopoeia Extra-Pharmacopoeia (extra-pharmacy regulations). The highest survival rate of bifidobacteria was observed when the ratio of magnesium oxide to anhydrous calcium hydrogen phosphate (MgO:CaHPO4) was 5:1 (B in Table 1). However, when the ratio (MgO:CaHPO4) was 5:3 (C in Table 1) and 5:5 (D in Table 1), bifidobacteria also showed a higher survival rate than when no anhydrous calcium hydrogen phosphate was contained (A in Table 1). That is, even when the combination of magnesium oxide and lactic acid bacteria reduces the survival rate of the lactic acid bacteria, the reduction in the survival rate of the lactic acid bacteria can be suppressed by using a phosphate (for example, anhydrous calcium hydrogen phosphate) in combination.

[0077] [Table 1]

[0078] Furthermore, taking into consideration the results shown in Figure 6 above, an investigation was conducted between a case in which no magnesium oxide was included (Table 1, A) and a case in which the blending ratio of magnesium oxide and anhydrous calcium hydrogen phosphate (MgO:CaHPO4) was 5:1 (Table 1, B). As shown in Table 2, three types of mixed powders were prepared with different blending ratios for different amounts of anhydrous calcium hydrogen phosphate, and 45 g of each mixed powder was filled into glass bottles and stored at 40°C and 75% (relative humidity) for one month. The viable bacterial counts in the mixed powders were then measured at the start of storage and after one month of storage. The results of the bacterial survival rate after each month of storage relative to the viable bacterial count at the start are shown in Figure 7. Bifidobacteria showed a high survival rate when the ratio of magnesium oxide to anhydrous calcium hydrogen phosphate (MgO:CaHPO4) was 5:0.25 (E in Table 2) and when the ratio (MgO:CaHPO4) was 5:0.5 (F in Table 2).

[0079] [Table 2]

[0080] As a result, when anhydrous calcium hydrogen phosphate was added to a mixed powder containing bifidobacteria (Bifidobacterium bifidum G9-1) and magnesium oxide, the survival rate of the bifidobacteria after one month of storage increased compared to when anhydrous calcium hydrogen phosphate was not added. In particular, a greater effect in improving the survival rate was observed when the ratio of magnesium oxide to anhydrous calcium hydrogen phosphate was 5:1 to 5:3. Furthermore, an effect in improving the survival rate of bifidobacteria was also observed when the ratio of magnesium oxide to anhydrous calcium hydrogen phosphate was 5:0.25 to 5:0.5.

[0081] [Example 3] Further examination of the effect of adding a desiccant on survival rate A mixed powder containing 1% Lactobacillus acidophilus K-13 (KS-13) bacterial powder and 99% magnesium oxide was prepared and filled into glass bottles at 45 g each. The mixture was prepared with and without a desiccant (Tokai Chemical Industry Co., Ltd., Shiburet: AS-W1510) and stored at 40°C and 75% relative humidity for two months. The viable cell counts in the mixed powder were measured at the start of storage and after one and two months, and the survival rate relative to the initial storage time was calculated. The moisture content was also measured according to the Japanese Pharmacopoeia General Test Method, Loss on Drying Test (1 g, 105°C, 4 hours). The survival rate results are shown in Figure 8, and the moisture content results in the composition (formulation) are shown in Figure 9. The viable cell count of lactic acid bacteria was measured according to the method (quantitative method) specified in the Japanese Pharmacopoeia Extra-Pharmacopoeial Drug Standards (extra-pharmacy regulations) for Lactomin.

[0082] As a result, the addition of a desiccant to the bottle significantly improved the survival rate of lactic acid bacteria. Normally, there is a relationship between the presence of a desiccant and the moisture content in the formulation or composition, but in this experiment, there was no significant difference in the moisture content between the formulation or composition with and without a desiccant. Therefore, it is thought that factors other than the moisture content in the formulation or composition contributed to the improvement in the survival rate of lactic acid bacteria observed when a desiccant was added.

[0083] [Example 4] Measurement of the viable cell count of magnesium oxide and lactic acid bacteria using the viable cell count measurement method of the present invention Test method: 1 g of bacterial powder (live bacteria) containing the lactic acid bacteria Lactobacillus acidophilus KS-13 and 1 g, 2 g, or 3 g of magnesium oxide (Kyowa Chemical Industry, fine granules) were uniformly mixed, and a buffer solution (composition shown in Table 3) was added to make a 50 mL solution, which was then thoroughly suspended to prepare the sample stock solution. 1 mL of the sample stock solution was accurately measured and added to 9 mL of separately accurately dispensed dilution solution (potassium dihydrogen phosphate: 4.5 g, sodium monohydrogen phosphate anhydrous: 6.0 g, polysorbate 80: 0.5 g, L-cysteine ​​hydrochloride monohydrate: 0.5 g, agar: 1.0 g) repeatedly (10-fold dilution method) until the concentration was such that 1 mL contained approximately 20 to 300 live bacteria, which was used as the sample solution. One mL of the sample solution was mixed with 10 mL of agar medium (beef liver infusion: 1000 mL, casein peptone: 10 g, glucose: 10 g, polysorbate 80: 1 g, L-cysteine: 0.5 g, agar: 15 g) kept at approximately 50°C and plated on a petri dish. After solidification, the mixture was anaerobically cultured at 37°C for 24 to 72 hours. The colonies were counted, and the bacterial count was calculated. A control was prepared by adding 0 g of magnesium oxide to a 50 mL plate containing diluent (potassium dihydrogen phosphate: 4.5 g, sodium monohydrogen phosphate anhydrous: 6.0 g, polysorbate 80: 0.5 g, L-cysteine ​​hydrochloride monohydrate: 0.5 g, agar: 1.0 g). The bacterial count was then measured in the same manner. The viable lactic acid bacteria count was measured according to the method (quantitative method) described in the section on Lactomin in the Japanese Pharmacopoeia Extraordinary Pharmaceutical Standards (extraordinary regulations).

[0084] [Table 3]

[0085] (Results) As shown in Figure 10, in the case of a conventional dilution solution (listed in Table 3), the viable cell count of Lactobacillus acidophilus KS-13 decreased as the final concentration of magnesium oxide increased (Lac + MgO (dilution solution)). However, by using buffer solutions 1 to 3 of the present invention shown in Table 3, the decrease in the viable cell count of lactic acid bacteria due to magnesium oxide could be suppressed (Lac + MgO (buffer solutions 1 to 3)). In particular, when the magnesium oxide concentration exceeded 4%, the decrease in the viable cell rate could be significantly suppressed (Lac + MgO (buffer solutions 1 to 3)).

[0086] From the above, it has become clear that (i) the stability of lactic acid bacteria, such as their survival rate, which is reduced by magnesium oxide, can be improved by using one or more specific phosphate salts (preferably a mixture of potassium dihydrogen phosphate and anhydrous sodium monohydrogen phosphate), and (ii) the effect of magnesium oxide on lactic acid bacteria can be further reduced by increasing the concentration of one or more phosphate salts (preferably a mixture of potassium dihydrogen phosphate and anhydrous sodium monohydrogen phosphate) in the solution.

[0087] [Example 5] Measurement of viable counts of magnesium oxide and bifidobacteria using the method of the present invention (Test method) 1 g of bacterial powder containing the bifidobacterium Bifidobacterium bifidum G9-1 and 1 g, 2 g, or 3 g of magnesium oxide (Kyowa Chemical Industry, fine granules) were uniformly mixed, and a buffer solution (composition shown in Table 4) was added to the mixture to make a total volume of 50 mL, and the mixture was thoroughly suspended to prepare a sample stock solution. 1 mL of the sample stock solution was accurately measured and added to 9 mL of separately accurately dispensed dilution solution (potassium dihydrogen phosphate: 4.5 g, sodium monohydrogen phosphate anhydrous: 6.0 g, polysorbate 80: 0.5 g, L-cysteine ​​hydrochloride monohydrate: 0.5 g, agar: 1.0 g) repeatedly (10-fold dilution method) until the concentration was such that 1 mL contained approximately 20 to 300 live bacteria, which was used as the sample solution. 1 mL of the sample solution was mixed with 10 mL of bifidobacteria test agar medium (beef liver infusion: 1000 mL, casein peptone: 10 g, glucose: 10 g, polysorbate 80: 1 g, L-cystine: 0.5 g, agar: 15 g) kept at approximately 50°C, and the mixture was spread on a petri dish. After solidification, the mixture was cultured anaerobically at 37°C for 24 to 72 hours, and the number of colonies was counted to determine the number of bacteria and the recovery rate compared to the control. As a control, 0 g of magnesium oxide was used, and a diluent (4.5 g of potassium dihydrogen phosphate, 6.0 g of anhydrous sodium monohydrogen phosphate, 0.5 g of polysorbate 80, 0.5 g of L-cysteine ​​hydrochloride monohydrate, 1.0 g of agar) was added to make 50 mL, and the number of bacteria was measured in the same manner. The viable lactic acid bacteria count was measured according to the method (quantitative method) described in the section on bifidobacteria in the Japanese Pharmacopoeia Extra-Pharmacopoeia Pharmaceutical Standards (extra-pharmacopoeial regulations).

[0088] [Table 4]

[0089] (Results) As shown in Figure 11, in the conventional dilution solution (listed in Table 4), a decrease in the viable cell count of bifidobacterium (Bifidobacterium bifidum G9-1) was observed as the final concentration of magnesium oxide increased (Bif + MgO (dilution solution)). However, it was confirmed that the decrease in the viable cell count of bifidobacterium due to magnesium oxide could be suppressed by using buffer solutions 2 and 3 shown in Table 4 (Bif + MgO (buffer solutions 2 and 3)). In particular, when the magnesium oxide concentration exceeded 4%, the decrease in the viable cell rate was significantly suppressed (Bif + MgO (buffer solutions 2 and 3)).

[0090] From the above, it has become clear that (i) the stability of bifidobacteria, such as their viability, which is reduced by magnesium oxide, can be improved by using one or more specific phosphate salts (preferably a mixture of potassium dihydrogen phosphate and anhydrous sodium monohydrogen phosphate), and (ii) the effect of magnesium oxide on the viability of bifidobacteria can be further reduced by increasing the concentration of one or more phosphate salts (preferably a mixture of potassium dihydrogen phosphate and anhydrous sodium monohydrogen phosphate) in the solution.

[0091] [Example 6] Examination of the effect of adding excipients on survival rate The effects of adding an excipient to the magnesium oxide composition of the present invention to reduce the magnesium oxide content in the composition on the survival rate of lactic acid bacteria (viable bacteria) and the water activity value of the entire composition were examined. The glucose and lactose hydrate used in Experimental Examples 1 and 2 have higher water activity values ​​(water activity values: 0.397 and 0.284) than magnesium oxide (water activity value: 0.116). Therefore, when an excipient is added to a composition, the water activity value of the entire composition becomes higher compared to when no excipient is added. The dried cornstarch used in Experimental Example 3 has a lower water activity (water activity: 0.026) than magnesium oxide (water activity: 0.116). Therefore, when an excipient is added to a composition, the water activity of the entire composition is lower than when no excipient is added.

[0092] Experimental example 1: When the excipient is glucose (water activity: 0.397) Four types of mixed powders containing Lactobacillus acidophilus KS-13 powder with the composition shown in Table 5, magnesium oxide, and glucose (manufactured by Nippon Shokuhin Kako Co., Ltd., product name: Nisshoku Medicarose) were prepared, and 45g each was filled into glass bottles. After storage at 40 ° C and 75% (relative humidity) for two months, the viable cell count in the mixed powder was measured at the start of storage and after two months of storage. The ratio of the viable cell count after two months to the viable cell count at the start (survival rate) was calculated and is shown in Figure 12 and Table 5 below.

[0093] [Table 5]

[0094] The results showed that in compositions (compositions Nos. 2-4) in which an excipient was added to a mixed powder containing the lactic acid bacteria Lactobacillus acidophilus KS-13 and magnesium oxide, the survival rate of the lactic acid bacteria (viable bacteria) after two months of storage was improved by approximately 17.9 to 30.1% compared to the composition (composition No. 1) in which no excipient was added. In other words, the use of an excipient improved the survival rate of the lactic acid bacteria (viable bacteria) when the blending rate of magnesium oxide in the composition was approximately 94.3% or less.

[0095] Furthermore, the water activity values ​​were measured at 25°C according to the Japanese Pharmacopoeia 3.05 Adsorption-Desorption Isotherm Measurement Method and Water Activity Measurement Method, and the results are shown in Table 5 above (instrument used for measurement: water activity measuring device (Novacina LabMaster-aw NEO)). Generally, a composition with a higher water activity value is expected to have lower stability. However, in the present invention, when glucose was used as an excipient, the higher the water activity value in the composition, the higher the stability in terms of bacterial survival rate, etc.

[0096] Experimental Example 2: When the excipient is lactose hydrate (water activity: 0.284) Four types of mixed powders containing Lactobacillus acidophilus KS-13 powder, magnesium oxide, and the excipient lactose hydrate (manufactured by DFE Pharma, product name: Pharmatose 200M) were prepared, with the compositions shown in Table 6 below. Each was filled into a glass bottle at 45 g, and stored at 40°C and 75% relative humidity for two months. The viable cell counts in the mixed powders were measured at the start of storage and after two months of storage. The ratio of the viable cell count after two months to the viable cell count at the start (survival rate) was calculated, and is shown in Figure 13 and Table 6 below.

[0097] [Table 6]

[0098] The results showed that in compositions (compositions Nos. 6-8) in which excipients were added to a mixed powder containing Lactobacillus acidophilus KS-13 and magnesium oxide, the survival rate of lactic acid bacteria (viable bacteria) after two months of storage was improved by approximately 13.0 to approximately 22.9% compared to a composition (composition No. 5) in which no excipient was added. In other words, the use of excipients improved the survival rate of lactic acid bacteria (viable bacteria) when the blending rate of magnesium oxide in the composition was approximately 94.3% or less.

[0099] Furthermore, the water activity was measured using the same method as in Experimental Example 1, and the results are shown in Table 6 above. It is generally expected that a composition with a higher water activity will have lower stability. In the present invention, when lactose hydrate was used as an excipient, the survival rate improved when the magnesium oxide content in the composition was 94.3% compared to when no excipient was added, which was an expected trend. When the magnesium oxide content in the composition was 84.3% or less, the survival rate improved compared to when no excipient was added, despite the increased water activity. In other words, regardless of the water activity value of the excipient, in the present invention, it was found that the survival rate of lactic acid bacteria (viable bacteria) can be improved by setting the amount of magnesium oxide in the composition to a certain amount or less (preferably, approximately 94.3% or less), preferably by using an excipient in the composition.

[0100] Experimental Example 3: When the excipient is dry cornstarch (water activity: 0.026) In Experimental Examples 1 and 2, an excipient with a water activity value higher than that of magnesium oxide was used, whereas in Experimental Example 3, an excipient with a water activity value lower than that of magnesium oxide was used. Four types of mixed powders containing Lactobacillus acidophilus KS-13 powder, magnesium oxide, and the excipient Matsutani dried cornstarch (manufactured by Matsutani Chemical Industry Co., Ltd.) were prepared, with the compositions shown in Table 7 below. Each was filled into a glass bottle at 45 g, and stored at 40°C and 75% relative humidity for two months. The viable cell counts in the mixed powders were measured at the start of storage and after two months of storage. The ratio of the viable cell count after two months to the viable cell count at the start (survival rate) was calculated, and is shown in Figure 14 and Table 7 below.

[0101] [Table 7]

[0102] The results showed that in compositions (compositions Nos. 10-12) in which an excipient was added to a mixed powder containing the lactic acid bacteria Lactobacillus acidophilus KS-13 and magnesium oxide, the survival rate of the lactic acid bacteria (viable bacteria) after two months of storage was improved by approximately 14.4 to 24.2% compared to a composition (composition No. 9) in which no excipient was added. In other words, the use of an excipient improved the survival rate of the lactic acid bacteria (viable bacteria) when the blending rate of magnesium oxide in the composition was approximately 94.3% or less.

[0103] Furthermore, the water activity values ​​measured in the same manner as in Experimental Examples 1 and 2 are shown in Table 7. Generally, a composition with a lower water activity value is expected to have higher stability, but in the present invention, even when dry cornstarch is used as an excipient, it was found that a composition with a lower water activity value (a larger amount of excipient added) can improve the survival rate of bacteria.

[0104] Summary of Test Example 6 (Experimental Examples 1 to 3): The results of Experimental Examples 1 to 3 above demonstrate that the survival rate of bacteria can be improved by adjusting the amount of magnesium oxide in the composition to a certain level or less (preferably, about 94.3% or less), and preferably by using an excipient in the composition. Furthermore, it was found that the effect of improving the survival rate of bacteria is not particularly affected by the water activity of the excipient or the water activity of the entire composition. [Industrial Applicability]

[0105] According to the present invention, (1) changes in bacterial flora caused by magnesium oxide preparations can be suppressed. Furthermore, (2) when magnesium oxide and lactic acid bacteria coexist for a certain period of time and the stability of the lactic acid bacteria, such as the survival rate, decreases, the stability can be improved. Furthermore, (3) when magnesium oxide and lactic acid bacteria coexist and the stability of the lactic acid bacteria, such as the survival rate, decreases, the survival rate of the lactic acid bacteria can be improved, allowing accurate measurement of the viable cell count of the lactic acid bacteria. Therefore, the composition or method of the present invention is useful in fields such as pharmaceuticals and foods.

Claims

1. A composition for inhibiting a change in bacterial flora in which the storage stability of lactic acid bacteria is improved, Contains magnesium oxide, lactic acid bacteria and excipients, the lactic acid bacterium is Lactobacillus acidophilus KS-13 (accession number: NITE BP-819); A composition for inhibiting bacterial flora change, characterized in that magnesium oxide is contained in an amount of 79.4% by mass or more and 94.3% by mass or less relative to the total amount of the composition.

2. When the composition is liquid, the lactic acid bacteria is present in an amount of 10 ４ ~10 １０ cfu / mL, or When the composition is solid, the lactic acid bacteria is present in an amount of 10 ５ ~10 １０ The composition of claim 1, characterized in that it contains 0.5 cfu / g of the microbial organism.

3. 3. The composition according to claim 1, further comprising one or more phosphates selected from the group consisting of anhydrous calcium hydrogen phosphate, calcium phosphate, anhydrous calcium phosphate, calcium hydrogen phosphate, calcium hydrogen phosphate dihydrate, and calcium dihydrogen phosphate monohydrate.

4. 4. The composition according to claim 3, wherein the phosphate is anhydrous calcium hydrogen phosphate.

5. 5. The composition according to claim 3, wherein the mass ratio of phosphate to magnesium oxide is from 100:1 to 1:

100.

6. The composition according to any one of claims 1 to 5, further comprising a drying agent.

7. The composition according to any one of claims 1 to 6, further characterized by improving bowel movements.

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