Method for protecting useful microorganisms

Abstract

The present invention addresses the problem of providing a composition containing a useful microorganism and provided in the form of a food or beverage product or a composition for preparing a food or beverage product, wherein the useful microorganism has and maintains stability against decomposition by gastric fluid when the composition containing the useful microorganism is ingested.　The present invention provides a composition containing a useful microorganism having stability against decomposition by gastric fluid by combining (A) dried probiotic cells or a dried probiotic cell formulation of a useful microorganism and (B) a polycationic polymer powder selected from chitosan, chitosan oligosaccharide, chitin, polylysine, and protamine. This composition containing a useful microorganism can be applied to a variety of compositions containing a useful microorganism because the composition can be prepared as a powder mixture of dried probiotic cells or a dried probiotic cell formulation of the useful microorganism and a polycationic polymer powder.

Description

[Technical field]

[0001] The present invention relates to providing a composition containing useful microorganisms such as probiotics that has been imparted stability to degradation in gastric fluid, and in particular to providing a composition containing useful microorganisms that is provided in the form of a food or drink or a composition for preparing a food or drink, and that has been imparted and maintained stability to the useful microorganisms against the decomposition action by gastric fluid when the composition is ingested, i.e., stability against degradation in gastric fluid.Furthermore, the present invention relates to a method for protecting the useful microorganisms in environments that are harsh for the survival of the useful microorganisms, such as temperature, humidity, salt concentration, etc. in addition to pH. [Background technology]

[0002] "Probiotics" was defined in 1989 by British microbiologist Fuller as "live microorganisms that have beneficial effects on humans by improving the balance of intestinal flora," and the concept of probiotics products is defined and provided as "products that contain live microorganisms and microbial metabolites that benefit the body by acting on the normal bacterial flora in the body, especially in the intestinal tract, and improving that balance." Many beneficial effects of "probiotics" have been reported for humans, including intestinal regulation, maintenance and improvement of intestinal flora, inhibition of the proliferation of harmful bacteria and viruses, immune activation, and inhibition of the production of carcinogens, and they are widely used in medicines, foods, etc.

[0003] In order to ingest probiotics in the form of food or the like and to exert their useful functions, the probiotic product must be stable during production, distribution, and storage, and at the same time, the ingested probiotics must be stable against degradation by gastric juice before reaching the intestines, i.e., stable against degradation in gastric juice. In order for probiotics to exert their effective functions, it is important to overcome the hurdle of degradation.

[0004] A wide variety of microorganisms (bacteria, yeasts, and fungi) are used in probiotics, including lactic acid-producing bacteria such as Lactobacillus, Enterococcus, and Lactococcus, Bifidobacterium, yeast (Saccharomyces), fungi (Aspergillus oryzae), and acid-forming spore bacteria (Bacillus and Clostridium). In foods and beverages, however, lactic acid-producing bacteria such as Lactobacillus, Enterococcus, and Lactococcus are predominant.

[0005] For products using live lactic acid bacteria, various methods have been disclosed so far as methods for imparting storage stability and gastric acid resistance to the live bacteria (lactic acid bacteria) in the products. For example, cited reference 1 discloses a method for providing a lactic acid bacteria tablet having storage stability of the live bacteria and resistance to gastric acid by mixing a live lactic acid bacteria powder with an additive and coating the produced tablet with a fat-soluble substance such as hydroxymethylcellulose, hydroxypropylmethylcellulose phthalate, cellulose acetate phthalate, or methacrylic acid copolymer. cited reference 2 discloses a method for preventing decomposition in gastric juice by forming a granulated material such as a powder of bacteria such as lactic acid bacteria or bifidobacteria into a granulated material having two layers: (A) a layer containing at least one of wheat, soybean, rice, collagen, hydroalcohol-soluble protein derived from gelatin, or zein, oils and fats, and an excipient, and (B) a layer containing at least one of wheat, soybean, rice, collagen, hydroalcohol-soluble protein derived from gelatin, or zein, so that when the granulated material is ingested, it does not dissolve in the stomach but dissolves only in the intestinal tract.

[0006] Furthermore, cited document 3 discloses a method for enhancing the effect of lactic acid bacteria by encapsulating lactic acid bacteria cells in a fat-soluble capsule formed by a shell containing gelatin and a fat-solubilizing agent in a capsule such as a soft capsule. cited document 4 discloses a method for protecting lactic acid bacteria from gastric acid when ingested by (a) coating lactic acid bacteria with sodium alginate to form a primary coating, and (b) coating the primary coating with one or more controlled-release coating bases selected from corn protein extract, hydroxypropyl methylcellulose phthalate, and shellac to form a secondary coating. cited documents 5 and 6 disclose methods for preventing the death of lactic acid bacteria by gastric acid when ingested by mixing live lactic acid bacteria with an oil and fat composition such as chocolate.

[0007] Furthermore, Cited Document 7 discloses that a probiotic preparation stabilized against the decomposition action of gastric juice is provided by containing dried and stabilized microorganisms (probiotics) and a gastric juice decomposition-resistant substance consisting of sodium alginate, hydroxypropylmethylcellulose and its derivatives, methacrylic acid derivatives, galactomannan, and mixtures thereof as matrix components of the microorganisms (probiotics) and the gastric juice decomposition-resistant substance.

[0008] On the other hand, in the food sector, such as in foods for people with swallowing difficulties, attempts have been made to add probiotics as one of the nutritional components. For example, in Reference 8, a nutritional product for persons with dysphagia is prepared as a nutritional product containing an aqueous solution of a food-grade biopolymer such as a microbial hydrocolloid such as xanthan gum, gellan gum, or curdlan, and the like, which is improved to promote safe swallowing for persons with dysphagia, and which contains probiotics as one of the nutritional components of the nutritional product. In References 9 and 10, a thickener formulation for persons with dysphagia is prepared by blending carrageenan, starch, and a nutritional agent and adjusting the viscosity of the nutritional formulation, thereby minimizing the increase in viscosity of the nutritional composition at the refrigerated storage temperature of the food and providing a consistent viscosity of the nutritional composition during a long storage period and at different temperatures. In preparing the thickener formulation, it is disclosed that probiotics are blended as one of the components such as carbohydrates, proteins, fats, phytonutrients, vitamins, and minerals to be blended as nutritional components. However, although it has been shown that these nutritional products for persons with dysphagia and thickener formulations for persons with dysphagia contain probiotics as one of the components, no particular consideration has been given to the stability of the probiotics, i.e., the stability of the probiotics in products containing them, or the stability of the probiotics against the decomposition action of gastric juices, which becomes a problem after ingestion of the nutritional products or formulations.

[0009] As described above, in order to utilize the useful functions of probiotics in probiotic products, important issues include the stability of probiotics during distribution and storage, as well as the maintenance of the stability of probiotics against the decomposition action by gastric juice when the probiotic composition is ingested, and various methods for solving the issues have been disclosed. Most of these conventional techniques for protecting probiotics from gastric acid are established by preventing contact between the probiotic bacteria and gastric acid by coating or the like. However, there have been no reports suggesting that a substance that inhibits the growth of microorganisms can contribute to solving the issues. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] Japanese Patent Application Publication No. 4-41434 [Patent Document 2] Japanese Patent Application Publication No. 5-186335 [Patent Document 3] Japanese Patent Application Publication No. 11-199494 [Patent Document 4] Special Publication No. 2002-505251: WO1999 / 020745) [Patent Document 5] WO2016 / 194366 [Patent Document 6] Patent No. 6181254 [Patent Document 7] WO1997 / 16198 [Patent Document 8] Special Publication No. 2015-505851 [Patent Document 9] Special Publication No. 2013-508416 [Patent Document 10] JP 2015-091808 A Summary of the Invention [Problem to be solved by the invention]

[0011] The object of the present invention is to provide a composition containing useful microorganisms such as probiotics that has been imparted stability to degradation in gastric fluid, and in particular, to provide a composition containing useful microorganisms that is provided in the form of a food or drink or a composition for preparing a food or drink, and that has been imparted and maintained stability of the useful microorganisms against the decomposition action by gastric fluid when the composition is ingested, i.e., stability resistant to degradation in gastric fluid. Another object of the present invention is to provide a method for protecting the useful microorganisms under environments that are harsh for the survival of the useful microorganisms, such as temperature, humidity, salt concentration, etc. [Means for solving the problem]

[0012] Means for Solving the Problems The present inventors have conducted intensive research into the provision of a probiotic composition that can be applied to various probiotic compositions and that has resistance to degradation in gastric fluid. As a result, they have found that a probiotic composition that has resistance to degradation in gastric fluid can be provided by blending (A) dried probiotic cells or a dried probiotic cell preparation with (B) a powder of a polycationic polymer selected from chitosan, chitosan oligosaccharide, chitin, polylysine, and protamine, and have thus completed the present invention.

[0013] That is, the present invention comprises a composition containing a useful microorganism having stability against degradation in gastric juice, characterized in that (A) dried cells or a dried cell preparation of a useful microorganism is blended with (B) a powder of a polycationic polymer selected from chitosan, chitosan oligosaccharide, chitin, polylysine, and protamine. The composition containing the useful microorganism of the present invention can be produced by preparing a powder mixture of the dried cells or a dried cell preparation of the useful microorganism and the powder of the polycationic polymer, and blending the powder mixture, and therefore can be applied to various compositions, and a useful microorganism preparation having storage stability and resistance to degradation in gastric juice can be provided.

[0014] The composition containing useful microorganisms of the present invention can be applied to compositions containing various useful microorganisms. In particular, it is possible to provide a composition containing useful microorganisms that retains storage stability and resistance to degradation in gastric juice as a food or beverage product or a composition for preparing a food or beverage product, which is prepared by blending a powder mixture prepared by mixing (A) dried cells or a dried cell preparation of useful microorganisms with (B) a powder of a polycationic polymer selected from chitosan, chitosan oligosaccharide, chitin, polylysine, and protamine so that the useful microorganisms and the polycationic polymer coexist in a contact state.

[0015] In the composition containing useful microorganisms of the present invention, the chitosan to be blended as the polycationic polymer (B) in the dried cells or dried cell preparation of useful microorganisms (A) can be blended as a chitosan polymer, such as a low molecular weight chitosan polymer having a molecular weight of 50,000 to 100,000, a medium molecular weight chitosan polymer having a molecular weight of 100,000 to 1,000,000, or a high molecular weight chitosan polymer having a molecular weight of 1,000,000 or more.

[0016] In the composition containing useful microorganisms of the present invention, the mixing ratio of the polycationic polymer (B) to the dried cells or dried cell preparation of the useful microorganism (A) can be in the range of 0.2 to 2500 parts by weight of the polycationic polymer (B) per 1 part by weight of the dried cells of the useful microorganism (A). In particular, when preparing foods and beverages or compositions for preparing foods and beverages, the polycationic polymer (B) is preferably mixed in the range of 0.2 to 2500 parts by weight, and the mixture can be added to the raw materials of the foods and beverages or compositions for preparing foods and beverages to produce a composition containing useful microorganisms.

[0017] In the composition containing the useful microorganisms of the present invention, a powder mixture prepared by mixing the useful microorganisms and the polycationic polymer so that they coexist in a contact state is further granulated, and the composition can be used in the form of a granulated product of the powder mixture.

[0018] The present invention encompasses a method for producing a composition containing useful microorganisms that has been imparted stability against degradation in gastric juice, the method comprising the steps of: (1) mixing (A) dried cells or a dried cell preparation of useful microorganisms with (B) a powder of a polycationic polymer selected from chitosan, chitosan oligosaccharide, chitin, polylysine, and protamine in an amount of 0.2 to 2500 parts by weight of the powder of the polycationic polymer (B) per 1 part by weight of the dry cells in the dried cells or dried cell preparation of useful microorganisms (A) to prepare a powder mixture in which the useful microorganisms and the polycationic polymer coexist in a contact state; and (2) blending the powder mixture with a raw material for the composition containing useful microorganisms.

[0019] In the method for producing a composition containing useful microorganisms of the present invention, the powder mixture obtained in step (1) can be further subjected to a granulation step to prepare the powder mixture in the form of a granulated product, and the method can be carried out.

[0020] The present invention encompasses a method for producing a composition containing useful microorganisms of the present invention, the method for producing a composition containing useful microorganisms being a method for producing a food or beverage comprising blending a powder mixture or a granulated product thereof prepared by mixing and processing useful microorganisms with a polycationic polymer into raw materials for producing a food or beverage, or a method for producing a composition for preparing a food or beverage comprising blending the powder mixture or a granulated product thereof into raw materials for a composition for preparing a food or beverage.

[0021] The present invention also encompasses an invention of a method for imparting storage stability and resistance to degradation by gastric juice to the useful microorganisms in a composition containing the useful microorganisms, by mixing and processing (A) dried cells or a dried cell preparation of the useful microorganisms and (B) a polycationic polymer so that the useful microorganisms and the polycationic polymer coexist in a contact state to prepare a powder mixture.

[0022] Furthermore, the present invention encompasses a method for imparting resistance to gastric juice decomposition stability to useful microorganisms in a composition containing useful microorganisms, by mixing (A) dried cells or a dried cell preparation of useful microorganisms with (B) a powder of a polycationic polymer selected from chitosan, chitosan oligosaccharide, chitin, polylysine, and protamine so that the useful microorganisms and the powder of the polycationic polymer coexist in a contact state to prepare a powder mixture, or by further granulating the powder mixture.

[0023] Specifically, the present invention comprises the following inventions: [1] A composition containing useful microorganisms having stability against degradation in gastric juice, characterized in that it comprises (A) a dried cell or a dried cell preparation of useful microorganisms and (B) a powder of a polycationic polymer selected from chitosan, chitosan oligosaccharide, chitin, polylysine, and protamine. [2] A composition containing useful microorganisms according to the above item [1], characterized in that the composition containing useful microorganisms is a food or drink, or a composition for preparing a food or drink. [3] A composition containing useful microorganisms according to the above [1] or [2], characterized in that the polycationic polymer (B) is blended in an amount ranging from 0.2 to 2,500 parts by weight per 1 part by weight of the dried cells of the useful microorganisms (A) or the dried cells of the useful microorganisms in the dried cell preparation. [4] A composition containing useful microorganisms according to any one of [1] to [3] above, characterized in that the chitosan blended as the polycationic polymer (B) is a low molecular weight chitosan polymer having a molecular weight of 50,000 to 100,000, a medium molecular weight chitosan polymer having a molecular weight of 100,000 to 1,000,000, or a high molecular weight chitosan polymer having a molecular weight of 1,000,000 or more. [5] A composition containing useful microorganisms according to any one of [1] to [4] above, which is prepared by mixing and processing dried useful microorganism cells or a dried microorganism cell preparation with a powder of a polycationic polymer to prepare a powder mixture, and then granulating the mixture. [6] A method for producing a composition containing useful microorganisms that has been provided with stability against degradation in gastric juice, comprising the steps of: (1) mixing (A) dried cells or a dried cell preparation of useful microorganisms with (B) a powder of a polycationic polymer selected from chitosan, chitosan oligosaccharide, chitin, polylysine, and protamine in an amount of 0.2 to 2,500 parts by weight of the powder of the polycationic polymer (B) per 1 part by weight of the dry cells in the dried cells or dried cell preparation of useful microorganisms (A) to prepare a powder mixture in which the useful microorganisms and the polycationic polymer coexist in a contact state; and (2) blending the powder mixture with a raw material for the composition containing useful microorganisms. [7] A method for producing a composition containing useful microorganisms according to the above [6], characterized in that the powder mixture obtained in step (1) is further subjected to a granulation step. [8] A method for producing a composition containing useful microorganisms according to [6] or [7] above, characterized in that the method for producing a composition containing useful microorganisms is a method for producing a food or beverage comprising blending a powder mixture or a granulated product thereof prepared by mixing and processing useful microorganisms with a polycationic polymer into raw materials for producing a food or beverage, or a method for producing a composition for preparing a food or beverage comprising blending the powder mixture or a granulated product thereof into raw materials for the composition for preparing a food or beverage. [9] A method for imparting resistance to gastric juice decomposition stability to useful microorganisms in a composition containing useful microorganisms, comprising mixing (A) dried cells or a dried cell preparation of useful microorganisms with (B) a powder of a polycationic polymer selected from chitosan, chitosan oligosaccharide, chitin, polylysine, and protamine so that the useful microorganisms and the powder of the polycationic polymer coexist in a contact state to prepare a powder mixture, or further granulating the powder mixture. Effect of the Invention

[0024] The present invention is applied to compositions containing various useful microorganisms to provide compositions containing useful microorganisms that are imparted with resistance to degradation in gastric fluid when ingested from the oral cavity, and in particular, to compositions containing useful microorganisms provided in the form of foods and beverages or compositions for preparing foods and beverages, the present invention provides compositions containing useful microorganisms that are imparted and retained with storage stability during distribution and storage, and stability against degradation by gastric fluid when the composition containing the useful microorganisms is ingested, i.e., storage stability and resistance to degradation in gastric fluid. In the present invention, the powder mixture or granulated product thereof used to impart storage stability and resistance to degradation in gastric fluid to the composition containing useful microorganisms can be prepared as a mixture of (A) dried cells or dried cell preparation of useful microorganisms and (B) a polycationic polymer, so that there are no restrictions on the materials used or the product form when applied to various useful microbial products such as foods and beverages, and the present invention can be applied to the preparation of a wide range of useful microbial products to provide useful microbial products that have the functions exerted by the useful microorganisms, such as effective storage stability and resistance to degradation in gastric fluid. Furthermore, the method of the present invention for imparting resistance to gastric fluid degradation stability to useful microorganisms can be expected to be useful not only in low pH environments such as gastric fluid, but also in environments that are generally harsh for the survival of useful microorganisms, such as high pH environments, high temperature environments, low temperature environments, high salt concentration environments, and dry environments. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] The present invention provides a composition containing useful microorganisms that is resistant to degradation by gastric juice by mixing (A) dried cells or a dried cell preparation of useful microorganisms with (B) a powder of a polycationic polymer selected from chitosan, chitosan oligosaccharide, chitin, polylysine, and protamine so that the useful microorganisms and the polycationic polymer coexist in a contact state to prepare a powder mixture, and then blending the powder mixture with a composition containing useful microorganisms to provide a composition containing useful microorganisms that is resistant to degradation by gastric juice.

[0026] In the present invention, the useful microorganisms contained in the composition containing useful microorganisms include probiotics, which are defined as "live microorganisms that have beneficial effects on humans by improving the balance of intestinal flora," as well as microorganisms belonging to the genera Escherichia, Corynebacterium, Bacillus, Saccharomyces, and Candida, which are used for fermentation production of useful compounds including amino acids, nucleic acids, and proteins. Specific examples of probiotics include microorganisms belonging to the genera Bifidobacterium, Lactobacillus, Enterococcus, and Lactococcus, but other than these, microorganisms useful not only for humans but also for livestock and poultry can be used as probiotics in the present invention.

[0027] Examples of microorganisms belonging to these genera include the following: Bifidobacterium genus microorganisms include Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium adolescentis, and Bifidobacterium catenulatum. Lactobacillus genus microorganisms include Lactobacillus acidophilus, Lactobacillus casei, and Lactobacillus spp. Examples of microorganisms belonging to the genus Enterococcus include Lactobacillus gasseri, Lactobacillus plantarum, Lactobacillus bulgaricus, Lactobacillus lactis, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus paracasei, and Lactobacillus brevis. Examples of microorganisms belonging to the genus Enterococcus include Enterococcus faecalis and Enterococcus faecium. Examples of microorganisms belonging to the genus Lactococcus include Lactococcus plantarum.Examples of useful microorganisms in the present invention include microorganisms belonging to Lactococcus plantarum, Lactococcus raffinolactis, etc. The useful microorganisms in the present invention may be any of the above-mentioned microorganisms, but probiotic microorganisms classified into the genera Lactobacillus, Enterococcus, and Lactococcus are more preferably used, and microorganisms belonging to the genera Lactobacillus and Enterococcus are even more preferably used.

[0028] The useful microorganisms used in the present invention, particularly in probiotics, may be dead bacteria regardless of their original definition, but are preferably bacterial cells in a proliferative state or bacterial cells that can transition to a proliferative state, i.e., live bacteria. The useful microorganisms may be isolated from wild organisms and cultured according to a known method, and the culture solution obtained may be used as is, or a product prepared from the culture solution, or a commercially available product may be used. In the composition containing the useful microorganisms of the present invention, the probiotics are mixed as dried bacterial cells or dried bacterial cell preparations, and as the dried bacterial cells, dried bacterial cells obtained by subjecting the useful microorganisms to a drying process such as freeze-drying are preferably used. The dried bacterial cells may also be used as a dried bacterial cell preparation containing excipients such as starch.

[0029] The polycationic polymer used in the present invention includes polycationic polymers selected from putrescine, cadaverine, spermidine, spermine, agmatine, chitosan, chitosan oligosaccharide, chitin, polylysine, and protamine, and preferably chitosan, chitosan oligosaccharide, chitin, polylysine, and protamine. These polycationic polymers are substances that often exhibit a bacteriostatic effect. Chitosan used as the polycationic polymer in the present invention is a linear polysaccharide consisting of poly-β1→4-glucosamine, and can be obtained industrially by deacetylating chitin obtained mainly from the exoskeleton of crustaceans such as crabs and shrimps by boiling in concentrated alkali. Chitosan is treated under harsh conditions such as boiling in concentrated alkali, and depending on the treatment conditions, the poly-β1→4-glucosamine structure may also have a chain cut, and the molecular weight may range from several hundred thousand to more than one million. For example, there are low molecular weight chitosans with a molecular weight of 50,000 to 100,000, medium molecular weight chitosans with a molecular weight of 100,000 to 1,000,000, and high molecular weight chitosans with a molecular weight of 1,000,000 or more, and all of these are commercially available.

[0030] Chitosan oligosaccharide is another name for oligo-N-acetylglucosamine, and has a structure in which 1 to 6 N-acetylglucosamines are linked together. It can be produced by treating deacetylated chitosan with hydrochloric acid, dissolving it, and enzymatically decomposing it with chitosanase. Chitosan oligosaccharide itself can be obtained from a commercial source. Chitin itself is another name for poly-β1-4-N-acetylglucosamine, and is a linear nitrogen-containing mucopolysaccharide polymer that constitutes the exoskeleton of arthropods and crustaceans, and is a raw material compound for chitosan and chitosan oligosaccharide. Chitin itself can be obtained from a commercial source.

[0031] Polylysine, another name for ε-poly-L-lysine, is a cationic polymer that has a peptide bond between the ε-amino group and the carboxyl group present in the side chain of the L-lysine residue. Polylysine is produced by fermentation using the bacterium Streptomyces albulus, and polylysine itself can be obtained commercially. Protamine is the main component of protein extracted from fish testes (milt of fish of the Salmonidae and Clupeidae families) and is used as a food additive. Protamine products can be obtained commercially.

[0032] The composition containing useful microorganisms of the present invention can be applied to compositions containing useful microorganisms in food and beverage compositions and pharmaceutical compositions, and can be advantageously applied as a composition containing useful microorganisms in the field of food and beverages. In the present invention, the polycationic polymer selected from chitosan, chitosan oligosaccharide, chitin, polylysine, and protamine to be blended in the composition containing useful microorganisms can be used alone or in combination, and the type, combination, dosage, etc. of the polycationic polymer can be selected depending on the composition containing the useful microorganisms to be used. For example, when the composition containing useful microorganisms is applied as a composition for food and beverages, it can be appropriately adjusted according to the type of food and beverage to be used and the composition of the desired composition. For example, when it is a thickening composition for food and beverages, the thickening property (viscosity) of the thickening polysaccharide can be used as it is.

[0033] The composition containing the useful microorganism of the present invention can be advantageously applied as a composition containing the useful microorganism in the field of food and drink.The food and drink in the composition containing the useful microorganism can be, for example, drinking water, milk, milk drink, lactic acid bacteria drink, soft drink containing fruit juice, carbonated drink, fruit juice drink, vegetable juice drink, tea drink, sports drink, functional drink, vitamin supplement drink, nutrition supplement balanced drink, various soups such as consommé soup, potage soup, cream soup, Chinese soup, miso soup, clear soup, liquid nutrients usable for gastrostomy, etc., as well as solid foods such as bread, confectionery, and noodles.

[0034] When preparing a composition containing the useful microorganism of the present invention, a known method for promoting dissolution and mixing can be used in combination in order to promote dissolution, mixing, etc. of the composition when producing the composition containing the useful microorganism, within a range that does not impair the effects of the present invention, for example, within a range that does not adversely affect the survival of the useful microorganism in artificial gastric juice or gastric juice.

[0035] The method for producing the composition containing the useful microorganism can be produced by a method used in a method for producing a composition containing a useful microorganism, as long as the useful microorganism and the polycationic polymer selected from chitosan, chitosan oligosaccharide, chitin, polylysine, and protamine used in the present invention are mixed and the useful microorganism and the polycationic polymer are allowed to coexist in a contact state. The method for allowing the useful microorganism and the polycationic polymer to coexist in a contact state is not particularly limited, but a method of mixing the useful microorganism powder with the polycationic polymer can be adopted, and a method of mixing the useful microorganism and the polycationic polymer and further granulating the mixture can be adopted. In the method for granulating the useful microorganism and the polycationic polymer as described above, it is preferable to mix the useful microorganism and the polycationic polymer in advance and then granulate. Furthermore, the means for mixing the powder of useful microorganisms and the powder of the polycationic polymer may be any known method or device that can sufficiently mix the powder of useful microorganisms and the powder of polycationic polymer uniformly.

[0036] As a method for granulating the composition containing the useful microorganisms of the present invention, a known method can be used, but spray granulation, vacuum freeze granulation, fluidized bed granulation, rolling granulation, stirring granulation, etc. are preferably used. When using these methods, in addition to the above-mentioned raw materials, water or water containing a polycationic polymer can be used as a binder. The granulation conditions may be appropriately selected and set according to each granulation method, but conditions that do not involve excessive heating or heat generation are preferable so as not to adversely affect the survival of the useful microorganisms, and it is preferable to use a method of granulation at room temperature or below in order to avoid inactivation of the useful microorganisms due to heat. In the granulation, a method of granulating by adding a starch hydrolyzate containing a metal salt that can be added to food, such as a sodium salt, a potassium salt, a calcium salt, or a magnesium salt, can be adopted in order to suppress the formation of lumps (JP Patent Publication No. 2016-26507).

[0037] In the composition of the present invention containing the useful microorganism, the amount of the useful microorganism to be contained in the composition may be appropriately determined depending on the type of the useful microorganism and the expected activity. However, the viable cell count in 1 g of the composition of the present invention is usually 1×10 6 Contains more than 1×10 CFU, preferably 1×10 7 CFU or more, and more preferably 1×10 8 It is preferable that the content is more than CFU.

[0038] In the composition containing the useful microorganism of the present invention, the amount of the polycationic polymer used in the present invention to be coexisted with the useful microorganism may be appropriately set depending on the purpose of use, but is usually 0.2 parts by weight or more, particularly preferably 1 part by weight or more, and most preferably 10 parts by weight or more per part by weight of the dry cell weight of the useful microorganism. There is no upper limit, but the upper limit is 2500 parts by weight or less, more preferably 2000 parts by weight or less per part by weight of the dry cell weight of the probiotics.

[0039] In the composition containing the useful microorganisms of the present invention, in addition to the polycationic polymer used in the present invention, excipients, sugars, proteins, vitamins, minerals, coloring agents, fragrances, etc. that are generally used in compositions containing useful microorganisms can be used as necessary, as long as they do not adversely affect the survival of the useful microorganisms.

[0040] The composition containing the useful microorganisms of the present invention can be provided in an appropriate product form depending on the target, and for example, when the target is food and drink, it can be provided as a food and drink itself containing the useful microorganisms, or as a composition for preparing a food and drink. The composition for preparing a food and drink can be consumed in the same way as normal food and drink, but in special cases, such as food and drink for people with swallowing difficulties, administration methods such as feeding via a gastrostomy tube can also be mentioned.

[0041] Ingested foods and beverages are delivered to the intestines after passing through a stomach environment that is harsh for the survival of useful microorganisms, and it is known that the pH in the stomach is usually 1 to 2, but becomes 4 to 5 immediately after eating and then drops again 2 to 3 hours later. The residence time of food in the stomach varies depending on the type of food, but if the residence time of food and beverages is about 2 hours, as shown in the examples, by improving the survival rate of useful microorganisms in a composition containing the useful microorganisms of the present invention for at least about 1 hour in an artificial gastric juice that simulates the stomach environment, it is possible to achieve the effective exertion of the effects of the useful microorganisms.

[0042] As described above, in the composition containing the useful microorganisms of the present invention, (A) dried cells or dried cell preparation of the useful microorganisms and (B) polycationic polymer selected from chitosan, chitosan oligosaccharide, chitin, polylysine, and protamine are mixed so that the useful microorganisms and the polycationic polymer coexist in a contact state to prepare a powder mixture, and the powder mixture is added to the composition containing the useful microorganisms, thereby making it possible to provide a useful microorganism composition that maintains the storage stability of the useful microorganisms and resistance to gastric juice degradation. In addition, the method of the present invention, which is characterized by including a step of coexisting the useful microorganisms and the polycationic polymer in a contact state, can be used as a method for imparting stability such as storage stability not only in a low pH environment such as gastric juice, but also in environments that are harsh for the survival of the microorganisms, such as a high pH environment, a high temperature environment, a low temperature environment, a high salt concentration environment, and a dry environment.

[0043] The present invention will be described below with reference to examples, but the present invention is not limited to these examples. EXAMPLES

[0044] <Experimental Condition 1: Preparation and Testing of Artificial Gastric Juice> The artificial gastric juice used in the present invention is an aqueous solution containing 0.03% by weight of yeast extract, 0.06% by weight of peptone, 0.01% by weight of lactose, 0.01% by weight of polysorbate 80 (also called Tween 80), 0.001% by weight of cysteine ​​hydrochloride, and 0.03% by weight of sodium chloride, and has a pH of 2.7 to 4.0, preferably 2.8 to 3.5, and more preferably 2.9 to 3.2. The retention time in the artificial gastric juice was 1 to 3 hours.

[0045] <Experimental condition 2: Quantification of viable probiotic bacteria> In the present invention, the viable cell count of probiotics was determined by the following method: a liquid in which a composition containing probiotics was dissolved or suspended in a diluent was added to the diluent and artificial gastric fluid, respectively, and mixed; after a predetermined time had elapsed, the probiotics were appropriately diluted with the diluent, and then the probiotics were mixed into a detectable medium (e.g., MRS medium) and cultured for 2 to 3 days at a temperature (e.g., 37°C) and under conditions (such as anaerobic conditions as necessary) in which the probiotics can grow.

[0046] <Experimental Condition 3: Amount of Probiotic Composition Used> The amount of the probiotic composition of the present invention to be used depends on the type of food or drink, the degree of viscosity to be imparted to the food or drink, etc., but when the amount of probiotics to be ingested is used as an indicator, it is usually at least 1 × 10 7 CFU, preferably 1 x 10 8 CFU, more preferably 3 x 10 8 Use a dose that provides sufficient CFU of probiotics.

[0047] [Test example] (1) Testing with E. faecalis and various polycationic polymers

[0048] <Test Method> [sample] Faecalis bacteria preparation: Faecalis powder 120, 5% dried bacteria + 95% potato starch (manufactured by Biofermin Pharmaceutical Co., Ltd.) Chitosan (low molecular weight): "Koyo Chitosan FL-80" molecular weight 50,000 to 100,000, 100% chitosan (manufactured by Koyo Chemical Co., Ltd.) Chitosan (medium molecular weight): "Koyo Chitosan FM-80" molecular weight 100,000 to 1,000,000; 100% chitosan (manufactured by Koyo Chemical Co., Ltd.) Chitosan (polymer): "Koyo Chitosan FH-80" molecular weight over 1 million, 100% chitosan (manufactured by Koyo Chemical Co., Ltd.) Chitin: "TG-L" manufactured by Koyo Chemical Co., Ltd. Chitosan oligosaccharide: "Koyo Oligoglucosamine WG" manufactured by Koyo Chemical Co., Ltd. Polylysine: 50% polylysine powder, 50% polylysine + 50% cassava-derived dextrin (manufactured by JNC Corporation) Protamine: "Shirako Protein Preparation MC-70" 16.4% Shirako Protein (containing 13.2-14.6% protamine) + 83.6% cassava-derived dextrin (manufactured by Ueno Food Techno Co., Ltd.)

[0049] [Culture medium] 4% salted MRS agar medium (selective medium for Lactobacillus faecalis)

[0050] [Table 1]

[0051] [Preparation of sample solution and artificial gastric juice test conditions] A 0.1g sample was weighed from each sample and transferred to a centrifuge tube. 40ml of diluent was added and mixed with a vortex for 1 minute to prepare the sample stock solution. After dissolving, the sample stock solution was left to stand at 37℃ for 30 minutes. The sample stock solution was diluted to 120,000 cells / ml with diluent, then diluted to 12,000 cells / ml with artificial gastric fluid, adjusted to pH 3.2 with 0.1N or 1.0N hydrochloric acid, and further diluted with diluent as appropriate. The sample was inoculated immediately and designated as 0-minute artificial gastric fluid treatment. The sample was diluted to 12,000 cells / ml with artificial gastric fluid, adjusted to pH 3.2 with 0.1N or 1.0N hydrochloric acid, and left to stand in a 37℃ incubator for 60, 120, and 180 minutes, designated as 60-minute, 120-minute, and 180-minute artificial gastric fluid treatment.

[0052] [Artificial gastric juice / pH conditions] Based on prior verification, the pH of the artificial gastric juice was set to 3.2.

[0053] [Inoculation and culture conditions] 20 ml of medium was added to 1 ml of each sample solution and allowed to solidify in a petri dish (n=6). Culture was carried out at 37°C under aerobic conditions for 48 hours.

[0054] <Test Results> The number of viable bacteria (cells / ml) detected under each condition is shown in Table 2. As shown in Table 2, it was confirmed that the polycationic polymer has the effect of significantly increasing the number of viable bacteria in artificial gastric fluid. Among them, protamine maintained a viable bacteria survival rate of 60% or more even after 180 minutes of treatment with artificial gastric fluid, and was extremely effective. It was also confirmed that the larger the molecular weight of chitosan, the greater the effect tends to be.

[0055] [Table 2]

[0056] (2) Tests using Lactobacillus acidophilus combined with various polycationic polymers The target bacteria was changed to L. acidophilus, and an acid resistance test using artificial gastric juice was carried out in the same manner as in (1) above.

[0057] <Test Method> [sample] Acidophilus preparation: Acidophilus powder 120, 5% dried bacteria + 95% potato starch (Biofermin Pharmaceutical Co., Ltd.) Chitosan (low molecular weight): "Koyo Chitosan FL-80" molecular weight 50,000 to 100,000, 100% chitosan (manufactured by Koyo Chemical Co., Ltd.) Chitosan (medium molecular weight): "Koyo Chitosan FM-80" molecular weight 100,000 to 1,000,000; 100% chitosan (manufactured by Koyo Chemical Co., Ltd.) Chitosan (polymer): "Koyo Chitosan FH-80" molecular weight over 1 million, 100% chitosan (manufactured by Koyo Chemical Co., Ltd.) Polylysine: 50% polylysine powder, 50% polylysine + 50% cassava-derived dextrin (manufactured by JNC Corporation) Protamine: "Shirako Protein Preparation MC-70" Shirako protein (containing 13.2-14.6% protamine) 16.4% + cassava-derived dextrin 83.6% (manufactured by Ueno Food Techno Co., Ltd.)

[0058] [Culture medium] 4% salted MRS agar medium (selective medium for Lactobacillus faecalis) Salt-free MRS agar medium (selective medium for Lactobacillus acidophilus and Lactobacillus faecalis)

[0059] [Table 3]

[0060] [Preparation of sample solution and artificial gastric juice test conditions] A 0.1g sample was weighed out from each sample and transferred to a centrifuge tube. 40ml of diluent was added and mixed with a vortex for 1 minute to prepare the sample stock solution. After dissolving, the sample stock solution was left to stand at 37℃ for 30 minutes. The sample stock solution was diluted to 120,000 cells / ml with diluent, then diluted to 12,000 cells / ml with artificial gastric fluid, adjusted to pH 2.9 with 0.1N or 1.0N hydrochloric acid, and then further diluted with diluent as appropriate. The sample was inoculated immediately and designated as 0-minute artificial gastric fluid treatment. The sample was diluted to 12,000 cells / ml with artificial gastric fluid, adjusted to pH 2.9 with 0.1N or 1.0N hydrochloric acid, and left to stand in a 37℃ incubator for 60, 120, and 180 minutes, designated as 60-minute, 120-minute, and 180-minute artificial gastric fluid treatment.

[0061] [Artificial gastric juice / pH conditions] Through prior verification, the pH of the artificial gastric juice was set to 2.9.

[0062] [Inoculation and culture conditions] 20 ml of medium was added to 1 ml of each sample solution and allowed to solidify in a petri dish (n=6). Both Lactobacillus faecalis selective medium and Lactobacillus acidophilus-Lactobacillus faecalis selective medium were used. After 48 hours of cultivation at 27°C under aerobic conditions, the number of colonies in each medium was counted, and the number of colonies in salt-added MRS medium (number of Lactobacillus acidophilus + number of Lactobacillus faecalis) was subtracted from the number of colonies in salt-free MRS medium (number of Lactobacillus acidophilus + number of Lactobacillus faecalis) to calculate the number of Lactobacillus acidophilus.

[0063] <Test Results> The results are shown in Table 4. As shown in Table 4, even when the target bacteria was L. acidophilus, the same effect was confirmed as when L. faecalis was targeted. In particular, protamine was extremely effective, with over 30% viable bacteria confirmed even after 180 minutes of treatment with artificial gastric fluid. Furthermore, the smaller the molecular weight of chitosan, the greater its effect, which was a different trend from the results of a similar test targeting L. faecalis.

[0064] [Table 4]

[0065] (4) Test to change the ratio of E. faecalis + chitosan

[0066] <Test Method> [sample] Faecalis bacteria preparation: Faecalis powder 120, 5% dried bacteria + 95% potato starch (manufactured by Biofermin Pharmaceutical Co., Ltd.) Chitosan (polymer): Koyo Chitosan FH-80 molecular weight over 1 million, 100% chitosan (manufactured by Koyo Chemical Co., Ltd.)

[0067] [Culture medium] 4% salted MRS agar medium (selective medium for Lactobacillus faecalis)

[0068] [Table 5]

[0069] [Preparation of sample solution and artificial gastric juice test conditions] A 0.1g sample was weighed from each sample and transferred to a centrifuge tube. 40ml of diluent was added and mixed with a vortex for 1 minute to prepare the sample stock solution. After dissolving, the sample stock solution was left to stand at 37℃ for 30 minutes. The sample stock solution was diluted to 120,000 cells / ml with diluent, then diluted to 12,000 cells / ml with artificial gastric fluid, adjusted to pH 3.2 with 0.1N or 1.0N hydrochloric acid, and further diluted appropriately with diluent. The sample was inoculated immediately and designated as 0-minute artificial gastric fluid treatment. The sample was diluted to 12,000 cells / ml with artificial gastric fluid, adjusted to pH 3.2 with 0.1N or 1.0N hydrochloric acid, and left to stand in a 37℃ incubator for 60, 120, and 180 minutes, designated as 60-minute, 120-minute, and 180-minute artificial gastric fluid treatment.

[0070] [Artificial gastric juice / pH conditions] Based on prior verification, the pH of the artificial gastric juice was set to 3.2.

[0071] [Inoculation and culture conditions] 20 ml of medium was added to 1 ml of each sample solution and allowed to solidify in a petri dish (n=6). Culture was carried out at 37°C under aerobic conditions for 48 hours.

[0072] <Test Results> The results are shown in Table 6. As shown in Table 6, the effect was confirmed even in the test plots where the chitosan blending ratio was lower than that of test plot 1, which was the conventional blending ratio.

[0073] [Table 6] [Industrial Applicability]

[0074] The present invention is applied to compositions containing various useful microorganisms to provide compositions containing useful microorganisms that are imparted with gastric fluid decomposition stability when ingested from the oral cavity, and in particular, to compositions containing useful microorganisms provided in the form of foods and beverages or compositions for preparing foods and beverages, the present invention provides compositions containing useful microorganisms that are imparted and maintained with storage stability during distribution and storage, and stability against decomposition by gastric fluid when the composition containing the useful microorganisms is ingested, i.e., storage stability and gastric fluid decomposition stability. In the present invention, the powder mixture or granulated product thereof used to impart storage stability and gastric fluid decomposition stability to the composition containing useful microorganisms can be prepared as a mixture of (A) dried cells or dried cell preparation of useful microorganisms and (B) a polycationic polymer, so that there are no restrictions on the materials used or the product form when applied to products containing various useful microorganisms such as foods and beverages, and the present invention can be applied to the preparation of a wide range of products containing useful microorganisms to provide products containing useful microorganisms that have the functions of useful microorganisms, such as effective storage stability and gastric fluid decomposition stability. The present invention is expected to be useful not only for protecting useful microorganisms under acidic conditions as described above, but also for imparting stability to useful microorganisms in environments that are harsh for the survival of useful microorganisms, such as high pH environments, high temperature environments, low temperature environments, high salt concentration environments, and dry environments.

Claims

1. A composition for imparting stability to gastric juice degradation to a useful microorganism, comprising (A) a dried cell or a dried cell preparation of the useful microorganism, and (B) a powder of a polycationic polymer selected from chitosan, chitosan oligosaccharide, chitin, polylysine, and protamine, which are coexisted in a contact state as a powder mixture.

2. 2. The composition according to claim 1, wherein the composition containing useful microorganisms is a food or drink, or a composition for preparing a food or drink.

3. The composition according to claim 1 or 2, characterized in that the composition contains useful microorganisms, and the polycationic polymer (B) is blended in an amount ranging from 0.2 parts by weight to 2500 parts by weight per 1 part by weight of the dried cells of the useful microorganisms (A) or the dried cells of the useful microorganisms in the dried cell preparation.

4. The composition according to any one of claims 1 to 3, wherein the chitosan blended as the polycationic polymer (B) is a low molecular weight chitosan polymer having a molecular weight of 50,000 to 100,000, a medium molecular weight chitosan polymer having a molecular weight of 100,000 to 1,000,000, or a high molecular weight chitosan polymer having a molecular weight of 1,000,000 or more.

5. The composition according to any one of claims 1 to 4, which is prepared by mixing and processing dried useful microbial cells or a dried microbial cell preparation with a powder of a polycationic polymer to prepare a powder mixture, and then granulating the mixture.

6. A method for producing a composition for imparting gastric fluid degradation stability to a useful microorganism, comprising: (1) a step of mixing (A) dried cells or a dried cell preparation of a useful microorganism with (B) a powder of a polycationic polymer selected from chitosan, chitosan oligosaccharide, chitin, polylysine, and protamine in an amount of 0.2 to 2500 parts by weight of the powder of the polycationic polymer (B) per 1 part by weight of the dry cells in the dried cells or dried cell preparation of the useful microorganism (A), thereby preparing a powder mixture in which the useful microorganism and the polycationic polymer coexist in a contact state; and (2) a step of blending the powder mixture with a raw material composition containing the useful microorganism.

7. 7. A method for producing a composition for imparting gastric fluid decomposition stability to the useful microorganism described in claim 6, characterized in that the powder mixture obtained in step (1) is further subjected to a granulation step.

8. The method for producing a composition for imparting gastric fluid decomposition stability to useful microorganisms is a method for producing a food or beverage comprising blending a powder mixture or a granulated product thereof prepared by mixing and processing useful microorganisms with a polycationic polymer into raw materials for producing a food or beverage, or a method for producing a composition for preparing a food or beverage comprising blending the powder mixture or a granulated product thereof into raw materials for the composition for preparing a food or beverage.

9. A method for imparting resistance to gastric juice decomposition stability to useful microorganisms in a composition containing useful microorganisms, comprising: mixing (A) dried cells or a dried cell preparation of useful microorganisms with (B) a powder of a polycationic polymer selected from chitosan, chitosan oligosaccharide, chitin, polylysine, and protamine so that the useful microorganisms and the powder of the polycationic polymer coexist in a contact state to prepare a powder mixture; or further granulating the powder mixture.

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