Composition for supplying alkaline phosphatase, method for producing composition for supplying alkaline phosphatase, and method for designing composition for supplying alkaline phosphatase

Abstract

To provide a composition comprising an active ingredient derived from lactic acid bacteria that allows efficient intake of alkaline phosphatase (AP).SOLUTION: The present invention provides a composition for supplying alkaline phosphatase comprising, as an active ingredient, one or more selected from the group consisting of the following (a) to (c): (a) a lactic acid bacterial body, wherein the bacterial body, after culturing under the condition below and recovery, exhibits within 8 hours alkaline phosphatase activity of 0.8 U or more per 1 mg of dry weight of the bacterial body, wherein the culturing condition being that 3 vol.% of a preculture having an OD600 value of 0.8 to 1.0 is added to 3 mL of MRS medium supplemented with 0.5 mg / mL cysteine and statically cultured at 37°C for 20 hours; (b) a culture resulting from (a); and (c) a processed material of (b).SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a composition for supplying alkaline phosphatase, a method for producing a composition for supplying alkaline phosphatase, and a method for designing a composition for supplying alkaline phosphatase. [Background technology]

[0002] Animals, including humans, need to ingest and digest (break down) food to maintain their lives. Absorbing nutrients through digestion of food is crucial for the survival of animals. However, if digestion is impaired due to disease or other factors, bodily function declines in the short or medium term, and in the long term, life becomes impossible to maintain. Digestive enzyme replacement therapy, in which digestive enzymes are administered to animals to compensate for the decline in the secretory function of digestive enzymes required for food digestion, has long been known. For example, it is known that the enzyme takadiastase has become widely used as a digestive enzyme preparation to compensate for the decline in the digestive capacity of humans with a reduced stomach (Non-Patent Document 1).

[0003] Furthermore, recent animal experiments have demonstrated that oral intake of alkaline phosphatase (AP), which decreases in the intestine with age, can compensate for its ability (to detoxify endotoxins), thereby extending lifespan and improving intestinal function. Alkaline phosphatase is an enzyme that hydrolyzes phosphate ester compounds under alkaline conditions and is widely distributed throughout the body, including the liver, osteoblasts, placenta, and small intestine. Its detoxifying effect has been reported to be exerted by AP dephosphorylating the phosphate group attached to lipid A, a component of lipopolysaccharide (LPS), an endotoxin, thereby reducing its ability to bind to receptors (Non-Patent Document 2).

[0004] For example, Non-Patent Document 2 describes that the activity of intestinal alkaline phosphatase (IAP) in humans decreases with age, and also describes that oral supplementation of alkaline phosphatase derived from bovine intestine significantly reduced age-related intestinal permeability and intestinal-derived systemic inflammation in mice, alleviated frailty, and extended lifespan. Furthermore, Non-Patent Document 3 describes that IAP dephosphorylates lipid A, which constitutes lipopolysaccharide (LPS), to detoxify LPS. Non-Patent Document 4 describes that IAP promotes the growth of intestinal bacteria by reducing the ATP concentration in the intestine. It also describes that IAP promotes the growth of aerobic and anaerobic bacteria in stool, and exerts a growth-promoting effect on various resident bacteria by inactivating (dephosphorylating) ATP in the intestine.

[0005] Incidentally, Patent Document 1 describes a culture of a strain of Lactobacillus salivarius selected from strains UCC1 (NCIMB40830) and UCC118 (NCIMB40829). Patent Document 1 describes that all the tested strains had alkaline phosphatase activity that was weaker than their acid phosphatase activity (average 5.0). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent No. 4743925 [Non-patent literature]

[0007] [Non-Patent Document 1] Hong, Shigeru. Characteristics of various digestive enzyme preparations available in Japan and their appropriate use. Pancreas, 2017;32:125-139. [Non-patent document 2] Florian Kuhn, et al., Intestinal alkaline phosphatase targets the gut barrier to prevent aging, JCI Insight. 2020;5(6):e134049. [Non-patent document 3] Gloria Komazin, et al., Substrate structure-activity relationship reveals a limited lipopolysaccharide chemotype range for intestinal alkaline phosphatase, J Biol Chem. 2019;Dec 13;294(50):19405-19423. [Non-patent document 4] Madhu S. Malo, et al., Intestinal alkaline phosphatase promotes gut bacterial growth by reducing the concentration of luminal nucleotide triphosphates, Am J Physiol Gastrointest Liver Physiol. 2014;306: G826 -G838. Summary of the Invention [Problem to be solved by the invention]

[0008] As mentioned above, alkaline phosphatase is known to have various beneficial effects. Therefore, ingesting alkaline phosphatase is expected to contribute to maintaining human health. However, there is no information on how to provide alkaline phosphatase safely and inexpensively. For example, alkaline phosphatase derived from bovine intestine, as described in Non-Patent Document 2, was difficult to mass-produce industrially. For these reasons, no studies have been conducted to date on products intended for ingesting alkaline phosphatase.

[0009] Therefore, an object of the present invention is to provide a composition for supplying alkaline phosphatase.

[0010] Another objective of the present invention is to provide a composition for reducing the toxicity of LPS, dephosphorylating ATP, suppressing intestinal inflammation, ameliorating chronic inflammation, improving frailty, increasing the intestinal flora, improving the balance of intestinal bacteria, and extending lifespan. Another object of the present invention is to provide a method for producing and designing a composition for supplying alkaline phosphatase. [Means for solving the problem]

[0011] After extensive research and efforts, the present inventors discovered that certain lactic acid bacteria can be cultured to have alkaline phosphatase activity above a certain level, and came to the realization that these lactic acid bacteria could be used for the supply of alkaline phosphatase, a purpose that had not been explored before, and thus completed the present invention.

[0012] [1] A composition for supplying alkaline phosphatase, comprising, as an active ingredient, one or more selected from the group consisting of the following (a) to (c): (a) Lactic acid bacteria that exhibit alkaline phosphatase activity of 0.8 U or more per 1 mg of dry weight of cells within 8 hours after culturing under the following conditions: [Conditions] 3% by volume of pre-cultured bacterial solution with an OD600 value of 0.8-1.0 was added to 3 mL of MRS medium supplemented with 0.5 mg / mL cysteine, and the mixture was left to stand at 37°C for 20 hours. (b) Culture of (a) (c)(b) processed material

[0013] [2] The alkaline phosphatase-supplying composition according to [1], which has an alkaline phosphatase activity of 0.2 U or more per 1 mg of dry weight of the composition.

[0014] [3] Contains one or more active ingredients selected from the group consisting of the following (a) to (c): A composition for supplying alkaline phosphatase, having an alkaline phosphatase activity of 0.2 U or more per 1 mg of dry weight of the composition. (a) Lactic acid bacteria cells exhibiting alkaline phosphatase activity (b) Culture of (a) (c)(b) processed material

[0015] [4] The alkaline phosphatase supplying composition according to any one of [1] to [3], wherein the lactic acid bacterium is one or more selected from the group consisting of Lacticaseibacillus paracasei, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus acidophilus, Lactobacillus helveticus, Streptococcus thermophilus, Lactobacillus amylovorus, Lactobacillus gallinarum, and Lactobacillus crispatus.

[0016] [5] The lactic acid bacteria may be Lacticaseibacillus paracasei MCC1375 (Accession Number: FERM BP-11313), Lacticaseibacillus paracasei ATCC25302, Lacticaseibacillus paracasei MCC1849 (Accession Number: NITE BP-01633), Lactobacillus delbrueckii subsp. bulgaricus ATCC11842, Lactobacillus acidophilus MCC1847 (Accession Number: NITE BP-01695), Lactobacillus helveticus, or the like. The alkaline phosphatase supply composition according to any one of [1] to [4], wherein the alkaline phosphatase supply composition is one or more selected from the group consisting of Lactobacillus helveticus MCC1844 (Accession Number: NITE BP-02185), Lactobacillus helveticus MCC1848 (Accession Number: NITE BP-01671), Streptococcus thermophilus ATCC19258, Lactobacillus amylovorus JCM1126, Lactobacillus acidophilus ATCC4356, Lactobacillus gallinarum JCM2011, and Lactobacillus crispatus JCM1185.

[0017] [6] The alkaline phosphatase-supplying composition according to any one of [1] to [5], which is a composition for reducing LPS toxicity.

[0018] [7] The alkaline phosphatase supplying composition according to any one of [1] to [6], which is one or more selected from the group consisting of a composition for suppressing intestinal inflammation, a composition for improving chronic inflammation, and a composition for improving weakness caused by inflammation.

[0019] [8] A composition for supplying alkaline phosphatase according to any one of [1] to [7], which is a composition for extending lifespan.

[0020] [9] A composition for supplying alkaline phosphatase according to any one of [1] to [8], which is a composition for dephosphorylating ATP in the intestine.

[0021]

[10] The alkaline phosphatase supplying composition according to any one of [1] to [9], which is at least one selected from the group consisting of compositions for increasing the intestinal flora and compositions for improving the balance of intestinal bacteria.

[0022]

[11] The alkaline phosphatase supplying composition according to any one of [1] to

[10] , which is a food, drink or pharmaceutical product.

[0023]

[12] The alkaline phosphatase supplying composition according to any one of [1] to

[11] , wherein the active ingredient is administered so that the alkaline phosphatase activity per day is 80 U or more.

[0024]

[13] The alkaline phosphatase supplying composition according to any one of [1] to

[12] , which is administered in an amount of 200 mg or more per day.

[0025]

[14] A method for producing a composition for supplying alkaline phosphatase according to any one of [1] to

[13] above, A method for producing a composition for supplying alkaline phosphatase, comprising the step of adding one or more active ingredients selected from the group consisting of the following (a) to (c) to other raw materials: (a) Lactic acid bacteria that exhibit alkaline phosphatase activity of 0.8 U or more per 1 mg of dry weight of cells within 8 hours after culturing under the following conditions: [Conditions] 3% by volume of pre-cultured bacterial solution with an OD600 value of 0.8-1.0 was added to 3 mL of MRS medium supplemented with 0.5 mg / mL cysteine, and the mixture was left to stand at 37°C for 20 hours. (b) Culture of (a) (c)(b) processed material

[0026]

[15] A method for producing a composition for supplying alkaline phosphatase according to any one of [3] to

[13] above, A method for producing a composition for supplying alkaline phosphatase, comprising the step of adding one or more active ingredients selected from the group consisting of the following (a) to (c) to other raw materials: (a) Lactic acid bacteria cells exhibiting alkaline phosphatase activity (b) Culture of (a) (c)(b) processed material

[0027]

[16] The method for producing a composition for supplying alkaline phosphatase according to

[14] or

[15] , wherein the adding step comprises adding the active ingredient so that the alkaline phosphatase activity per 1 mg of dry weight of the composition is 0.2 U or more.

[0028]

[17] A method for designing a composition for supplying alkaline phosphatase, comprising: a selection step of selecting lactic acid bacteria that exhibit alkaline phosphatase activity; A method for designing a composition for supplying alkaline phosphatase, comprising a design step of designing the content of lactic acid bacteria cells based on the alkaline phosphatase activity of the lactic acid bacteria so that the alkaline phosphatase activity of the composition is at a predetermined concentration or higher.

[0029]

[18] The method for designing a composition for supplying alkaline phosphatase according to

[17] , wherein the selection step selects lactic acid bacteria that exhibit alkaline phosphatase activity of 0.8 U or more per mg of dry weight of bacterial cells within 8 hours after culturing under the following conditions and recovering the cells: [Conditions] 3% by volume of pre-cultured bacterial solution with an OD600 value of 0.8-1.0 was added to 3 mL of MRS medium supplemented with 0.5 mg / mL cysteine, and the mixture was left to stand at 37°C for 20 hours. [Effects of the Invention]

[0030] According to the present invention, it is possible to provide a composition for supplying alkaline phosphatase that is suitable for ingesting alkaline phosphatase. In particular, since the active ingredient contained in the composition of the present invention is a lactic acid bacterium that has been widely consumed as a food and there is a wealth of knowledge about its culture and quality maintenance, it is possible to provide a product containing a predetermined amount of alkaline phosphatase. Furthermore, the present invention can provide novel compositions for reducing LPS toxicity, dephosphorylating ATP, suppressing intestinal inflammation, ameliorating chronic inflammation, improving frailty, increasing intestinal flora, improving the balance of intestinal bacteria, and extending lifespan. [Brief explanation of the drawings]

[0031] [Figure 1] FIG. 1 shows the results of measuring the alkaline phosphatase activity of lactic acid bacteria, which are active ingredients in the alkaline phosphatase-supplying composition of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0032] Preferred embodiments of the present invention will be described below. However, the present invention is not limited to the following preferred embodiments and can be freely modified within the scope of the present invention. In this specification, percentages are expressed in mass % unless otherwise specified.

[0033] <Active ingredient> The alkaline phosphatase-supplying composition of the present invention contains, as an active ingredient, one or more selected from the group consisting of the following (a) to (c): (a) Lactic acid bacteria cells exhibiting alkaline phosphatase (also referred to as "AP" in this specification) activity (b) Culture of lactic acid bacteria showing AP activity (c) Processed culture of lactic acid bacteria exhibiting AP activity In this specification, for the sake of convenience, when there is no need to distinguish between the above (a) to (c), they may be collectively referred to as "active ingredients derived from lactic acid bacteria." The active ingredients derived from lactic acid bacteria will be explained below.

[0034] (a) Lactic acid bacteria cells exhibiting AP activity AP activity refers to the activity of hydrolyzing phosphate ester compounds under alkaline conditions, and in the present invention, this activity is defined as the activity of dephosphorylating disodium p-nitrophenyl phosphate to produce p-nitrophenol. Specifically, when quantitatively expressing the activity of the subject to be measured, 1 unit is defined as the enzyme activity that produces 1 μmol of p-nitrophenol per minute, and is defined as the activity per mg of dry weight of the subject to be measured (specific activity, unit: U / mg). For example, when quantitatively expressing the AP activity of bacterial cells, it is expressed as the activity per mg of dry weight of bacterial cells, and when quantitatively expressing the AP activity of a composition, it is expressed as the activity per mg of dry weight of the composition. The method for measuring the AP activity of lactic acid bacteria for this definition will be explained in more detail in the Examples.

[0035] The lactic acid bacteria cells used in the present invention may be commercially available products or may be obtained by appropriate production. Furthermore, the lactic acid bacteria cells used in the present invention can be obtained, for example, by culturing lactic acid bacteria and separating the resulting culture into medium components and bacteria. The culture method is not particularly limited as long as the lactic acid bacteria can grow, and conventional conditions can be adopted depending on the type of lactic acid bacteria. For example, a medium containing 0.5 mg / mL of cysteine ​​can be used, and the culture temperature can be 121° C. or higher, and the culture time can be 15 minutes or longer.

[0036] In the present invention, the term "microbial cells" may include both live microbial cells and sterilized microbial cells. Furthermore, the microbial cells may be in the form of a microbial cell concentrate, freeze-dried microbial cells, spray-dried microbial cells, sterilized microbial cells, heat-sterilized microbial cells, or crushed microbial cells. These forms of microbial cells can be obtained by subjecting the cultured microbial cells to appropriate treatments such as dilution, concentration, freeze-drying, spray-drying, or sterilization. Sterilization methods include retort sterilization, UHT (Ultra High Temperature) sterilization, pressure sterilization, high-pressure steam sterilization, dry heat sterilization, circulation steam disinfection, electromagnetic wave sterilization, electron beam sterilization, high frequency sterilization, radiation sterilization, ultraviolet sterilization, ethylene oxide gas sterilization, hydrogen peroxide gas plasma sterilization, and chemical sterilization (alcohol sterilization, formalin fixation, electrolyzed water treatment).

[0037] Here, the lactic acid bacteria are preferably cultured under the following conditions, and within 8 hours after harvesting, the cells exhibit an AP activity of 0.8 U or more per mg of dry mass of the cells. [Conditions] 3% by volume of pre-cultured bacterial solution with an OD600 value of 0.8-1.0 was added to 3 mL of MRS medium supplemented with 0.5 mg / mL cysteine, and the mixture was left to stand at 37°C for 20 hours. The lactic acid bacteria may be cultured under the above-mentioned culture conditions to prepare a desired amount. Specific examples of the culture procedure can be found in the Examples.

[0038] In addition, the lactic acid bacteria are preferably classified into the genus Lactobacillus, Lacticaseibacillus, Ligilactobacillus, Limosilactobacillus, Lactiplantibacillus, and Streptococcus. Here, lactic acid bacteria previously classified into the genus Lactobacillus were reclassified in the International Journal of Systematic and Evolutionary Microbiology (IJSEM) in 2020 in accordance with the rules (ICNP) of the International Committee on Prokaryotic Nomenclature (ICSP). Therefore, in the description of the present invention, the names of the lactic acid bacteria will be described in accordance with the reclassification.

[0039] Furthermore, the lactic acid bacteria preferably exhibit AP activity that is at least 1.1 times, more preferably at least 1.3 times, more preferably at least 1.5 times, more preferably at least 1.6 times, more preferably at least 1.7 times, more preferably at least 1.8 times, more preferably at least 1.9 times, more preferably at least 2 times, more preferably at least 2.1 times, more preferably at least 2.5 times, more preferably at least 2.8 times, or more preferably at least 3 times that of Ligilactobacillus salivarius JCM1231 strain or Lacticaseibacillus casei ATCC393 strain, which are bacteria known to exhibit AP activity from Patent Document 1 (Japanese Patent No. 4743925).

[0040] Examples of lactic acid bacteria include Lacticaseibacillus paracasei, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus acidophilus, Lactobacillus helveticus, Streptococcus thermophilus, Lactobacillus amylovorus, Lactobacillus gallinarum, Lactobacillus crispatus, and Ligilactobacillus salivarius. salivarius, Lacticaseibacillus casei, Lactobacillus gasseri, Limosilactobacillus reuteri, Lactobacillus delbrueckii subsp. lactis, Lactobacillus johnsoni, Lactiplantibacillus plantarum subsp. plantarum, and Limosilactobacillus fermentum.

[0041] The strains disclosed in this specification and assigned accession numbers are available from the respective depositories listed below. "NPMD" is the abbreviation for the National Institute of Technology and Evaluation (NITE) Patent Microorganisms Depositary, located at Room 120 or 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan. In this specification, strains assigned accession numbers beginning with "NITE" are those deposited at NPMD. "IPOD" is the abbreviation for the National Institute of Advanced Industrial Science and Technology (AIST) Patent Organism Depositary. In 2012, the status of international deposit was transferred from the AIST to the National Institute of Technology and Evaluation (NITE), and patent microorganism deposit operations were centralized at NPMD. In this specification, strains assigned accession numbers beginning with "FERM" were deposited at IPOD, and their management was subsequently transferred to NPMD. "ATCC" is an abbreviation for American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110, United States of America. As used herein, strains assigned accession numbers beginning with "ATCC" have been deposited with the ATCC. "JCM" is an abbreviation for Japan Collection of Microorganisms, Microbial Materials Development Division, RIKEN BioResource Research Center, National Research and Development Agency, located at 3-1-1 Takanodai, Tsukuba, Ibaraki Prefecture, 305-0074. In this specification, strains assigned accession numbers beginning with "JCM" have been deposited at JCM.

[0042] The preferred strain of Bacillus paracasei is strain MCC1375, which was deposited under the Budapest Treaty on November 5, 2010, in Room 120 of the National Museum of Natural Sciences (NMPD) under the accession number FERM BP-11313. The preferred strain of Bacillus paracasei is strain MCC1849, which was deposited under the Budapest Treaty on June 6, 2013, with Room 122 of the National Museum of Natural History (NPMD) under the accession number NITE BP-01633. Furthermore, the ATCC25302 strain can be preferably used as the Lacticase Bacillus paracasei strain.

[0043] As the strain of Lactobacillus delbrueckii subsp. bulgaricus, the ATCC11842 strain can be preferably used.

[0044] The Lactobacillus acidophilus strain that can be used is preferably strain MCC1847. Strain MCC1847 was internationally deposited under the Budapest Treaty on August 23, 2013, in Room 122 of the National Pesticide and Mold Research Institute (NPMD) under the accession number NITE BP-01695. Furthermore, the Lactobacillus acidophilus strain that can be used is preferably the ATCC4356 strain.

[0045] The Lactobacillus helveticus strain that can be used is preferably strain MCC1844, which was internationally deposited under the Budapest Treaty on December 25, 2015, in Room 122 of the National Pesticide and Mold Research Institute (NPMD) under the accession number NITE BP-02185. The Lactobacillus helveticus strain that can be used is preferably strain MCC1848. Strain MCC1848 has been internationally deposited in Room 122 of the National Pesticide and Medical Devices Agency (NPMD) under the accession number NITE BP-01671 in accordance with the Budapest Treaty.

[0046] As the strain of Streptococcus thermophilus, the ATCC19258 strain can be preferably used. As the strain of Lactobacillus amylovorus, the JCM1126 strain can be preferably used. As the strain of Lactobacillus gallinarum, the JCM2011 strain can be preferably used. As the strain of Lactobacillus crispatus, the JCM1185 strain can be preferably used. If these strains are commercially available, they can also be used.

[0047] In the present invention, the above-mentioned lactic acid bacteria exhibiting AP activity may be used alone, or two or more types of lactic acid bacteria may be used in combination.

[0048] The lactic acid bacteria contained in the composition of the present invention are not limited to the above strains, and may be strains having substantially equivalent genes to the above strains. The term "strains having substantially equivalent genes" refers to strains belonging to the genus Lactobacillus, Lacticaseibacillus, Rizilactobacillus, Rimosilactobacillus, Lactipranchibacillus, or Streptococcus, and having AP activity comparable to that of the deposited strain. Furthermore, a strain having substantially equivalent genes has a base sequence of its 16S rRNA gene that is 98% or more, preferably 99% or more, and more preferably 100% homologous to the base sequence of the 16S rRNA gene of the deposited strain, and preferably has the same mycological properties as the deposited strain. Furthermore, the lactic acid bacteria of the present invention may be strains bred from deposited strains or substantially equivalent strains by mutation treatment, genetic recombination, selection of natural mutants, etc., as long as the effects of the present invention are not impaired.

[0049] (b) Culture of lactic acid bacteria showing AP activity In the present invention, a culture of lactic acid bacteria cells exhibiting AP activity is a culture obtained by culturing the cells of (a) and contains the cells of (a). The culture may be a commercially available product or may be prepared as needed. The culture conditions typically used are as described above. The culture obtained can be used as is or after drying. In this case, heating may be performed under conditions that will kill the cells of (a).

[0050] (c) Processed culture of lactic acid bacteria exhibiting AP activity In the present invention, the processed culture product of lactic acid bacteria cells exhibiting AP activity is obtained by subjecting the culture product of (b) above to some treatment. The treatment method is not particularly limited as long as it does not significantly inactivate AP, and examples include centrifugation, concentration, dilution, freeze-drying, spray-drying, sterilization, crushing, etc. More specific examples include immobilizing the bacterial cells with acrylamide, carrageenan, etc., partially or completely crushing the cell walls and membranes of the bacterial cells, collecting the supernatant (water-soluble fraction) after centrifugation, partially purifying the supernatant with ammonium sulfate treatment, etc., or concentrating the supernatant. Furthermore, from the viewpoint of efficient recovery of AP, the treated material preferably contains bacterial cells and / or components contained within the cells of the bacterial cells.

[0051] <Alkaline phosphatase (AP) supplying composition> The composition of the present invention preferably has an AP activity per 1 mg of dry weight of the composition of 0.2 U or more, more preferably 0.5 U or more, more preferably 0.6 U or more, and more preferably 0.8 U or more. When the composition of the present invention is in liquid form, "AP activity per 1 mg of dry weight of the composition" can be replaced with "AP activity per mL of the composition." The AP activity can be calculated based on the AP activity per dry weight of the bacterial cells of the active ingredient contained in the composition and the mass of the active ingredient contained in the composition. That is, the composition of the present invention preferably contains the active ingredient derived from the lactic acid bacteria described above so that the AP activity of the composition is equal to or greater than the above-mentioned value.

[0052] The AP activity of a composition can be measured by measuring its activity to dephosphorylate disodium p-nitrophenyl phosphate to produce p-nitrophenol. The method for measuring the activity can be found in the Examples.

[0053] For example, the content of (a) the bacterial cells among the active ingredients derived from lactic acid bacteria may be, as a guideline, 1 to 99% by mass, 10 to 90% by mass, 20 to 80% by mass, 30 to 70% by mass, or 40 to 60% by mass, in terms of the dry weight of the bacterial cells, relative to the total mass of the composition. In the case of live bacteria, preferably 1 x 10 6 ~1×10 12 cfu / g or 1 x 10 6 ~1×10 12 cfu / mL, more preferably in the range of 1 x 10 7 ~1×10 11 cfu / g or 1 x 10 7 ~1×10 11 cfu / mL. "cfu" stands for "Colony Forming Unit." (b) When a culture is included, the content may be, as a guideline, 1 to 99 mass%, 10 to 90 mass%, 20 to 80 mass%, 30 to 70 mass%, or 40 to 60 mass% of the total composition, as the dry weight of the culture. (c) When a processed material is included, the content may be, as a guideline, 1 to 99 mass %, 10 to 90 mass %, 20 to 80 mass %, 30 to 70 mass %, or 40 to 60 mass % of the total composition, as the dry weight of the processed material.

[0054] The compositions of the present invention are used to deliver AP to a subject. The composition of the present invention can be preferably administered or ingested by a healthy subject to provide AP to the body of the subject, more preferably to the intestine of the subject.

[0055] It is known that the activity of intestinal alkaline phosphatase (IAP) in humans decreases with aging (Non-Patent Document 2). Therefore, the composition of the present invention can be used to supply AP to a subject whose AP activity has decreased due to aging, such as an elderly person, in the body.

[0056] <Composition for reducing LPS toxicity> The present invention is a composition for reducing LPS toxicity, which contains an active ingredient derived from lactic acid bacteria exhibiting AP activity. LPS is a complex compound composed of lipids and sugars present in the outer membrane surrounding the peptidoglycan of the cell walls of Gram-negative bacteria such as Escherichia coli, Salmonella, and Bordetella pertussis. LPS is an endotoxin, and it is known that LPS binds to immune system cells and activates them, increasing inflammatory cytokines. As will be described later, various pathological conditions develop due to the induction of an excessive inflammatory reaction by this LPS. That is, when LPS is present in excess, it exerts toxicity on the living body. Here, AP is known to have the action of dephosphorylating the above LPS and reducing its activity. It is also known that dephosphorylating LPS inhibits the binding of LPS to receptors of immune system cells (Non-Patent Document 3). Based on the above findings, the present inventors have found that the composition for supplying AP containing the active ingredient derived from lactic acid bacteria of the present invention can reduce the toxicity of LPS. Therefore, the composition for supplying AP of the present invention can suppress the inflammatory reaction induced by LPS and can be used for reducing the toxicity of LPS.

[0057] <Composition for suppressing intestinal inflammation, improving chronic inflammation, and improving debilitation due to inflammation> The present invention is a composition for suppressing intestinal inflammation, improving chronic inflammation, and improving debilitation due to inflammation, which contains an active ingredient derived from lactic acid bacteria exhibiting AP activity. As described above, various pathological conditions develop due to the induction of an excessive inflammatory reaction by LPS. Specifically, it induces intestinal inflammation by increasing inflammatory cytokines in the intestine. It also reduces the ability of intestinal epithelial cells to form tight junctions, allowing inflammatory substances to enter the bloodstream and causing systemic chronic inflammation. Furthermore, the induction of the intestinal inflammation and systemic chronic inflammation leads to inflammatory weakness (Non-Patent Document 2). As described above, the AP-supplying composition of the present invention has the effect of dephosphorylating LPS, and thus can be used to suppress LPS-induced intestinal inflammation, improve chronic inflammation, and alleviate weakness caused by inflammation. Here, the chronic inflammation is preferably systemic inflammation, more preferably systemic inflammation caused by the permeation of inflammatory substances derived from the intestine.

[0058] It is also known that the addition of LPS to the intestine induces the production of inflammatory cytokines (Non-Patent Document 4). The AP-supplying composition of the present invention dephosphorylates LPS and suppresses the production of inflammatory substances in the intestine, and therefore can be used to suppress the production of inflammatory cytokines in the intestine. It is also known that the addition of LPS to the intestine reduces the intestinal barrier function (Non-Patent Document 4). Therefore, the AP supplying composition of the present invention can be used to suppress a decrease in intestinal barrier function caused by inflammation.

[0059] Furthermore, the AP supplying composition of the present invention can be used to improve or prevent symptoms caused by the aforementioned inflammation and pathologies resulting from chronic impairment of intestinal barrier function, such as leaky gut, depression, anxiety, cognitive impairment, neurodegenerative diseases, cardiovascular diseases, liver injury, necrotizing enterocolitis (NEC), kidney injury, autoimmune diseases, asthma, and chronic obstructive pulmonary disease.

[0060] <Composition for life extension> The present invention is a composition for extending lifespan, which comprises an active ingredient derived from lactic acid bacteria that exhibits AP activity. As described above, the AP-supplying composition of the present invention dephosphorylates LPS, thereby suppressing inflammatory responses caused by LPS and the onset of various pathologies. Furthermore, it is known that ingestion of IAP increases the lifespan of mice (Non-Patent Document 2). Therefore, the AP supply composition of the present invention can be used to extend life. Here, "extending lifespan" is a concept that includes extending the median lifespan (the period until 50% of the population dies) and extending healthy lifespan, which is the period during which one can live an independent and healthy life both physically and mentally. Furthermore, the subject for which lifespan extension is to be performed is not particularly limited, and is preferably a human, but can also be a non-human animal (for example, a dog, cat, horse, mouse, or rabbit).

[0061] <Composition for dephosphorylating ATP in the intestine> The present invention relates to a composition for dephosphorylating ATP in the intestine, which comprises an active ingredient derived from lactic acid bacteria that exhibits AP activity. ATP is known to suppress the growth of bacteria in the intestine. Furthermore, as mentioned above, AP is a dephosphorylating enzyme and is known to be able to dephosphorylate the intestinal ATP and decompose it into ADP or AMP (Non-Patent Document 4). Therefore, the AP-supplying composition of the present invention can be used to dephosphorylate ATP in the intestine.

[0062] <Composition for increasing intestinal flora, composition for improving the balance of intestinal bacteria> The present invention relates to a composition for increasing the intestinal flora and a composition for improving the balance of intestinal bacteria, which contains an active ingredient derived from lactic acid bacteria that exhibits AP activity. As mentioned above, ATP is known to suppress the growth of intestinal bacteria, and its dephosphorylated forms, AMP and ADP, are known to serve as food for bacteria. Therefore, it is known that dephosphorylating ATP in the intestine and increasing the amounts of AMP and ADP increases the intestinal flora, and furthermore, it is known that increasing the intestinal flora improves the balance of intestinal bacteria (Non-Patent Document 4). Here, the AP supplying composition of the present invention is capable of dephosphorylating ATP in the intestine. Therefore, the AP supplying composition of the present invention can be used to increase the intestinal flora and improve the balance of intestinal bacteria. Here, the balance of intestinal bacteria means improving the balance between gram-positive and gram-negative bacteria in the intestine, and preferably means improving the proportion of gram-positive bacteria in the intestine.

[0063] <Dosage form> The composition of the present invention is preferably in the form of a food or drink or a pharmaceutical product. The composition of the present invention may be in the form of an oral composition or a parenteral composition, but is preferably an oral composition. Examples of parenteral compositions include suppositories, ointments, injections, etc. Examples of the form of the food, drink, or pharmaceutical product include solid preparations such as granules, powders, tablets, and capsules. Preferably, tablets and capsules are used. When the composition of the present invention is made into a food, drink, or pharmaceutical product, it may contain optional ingredients such as excipients as long as the effects of the present invention are not impaired.

[0064] The composition of the present invention can preferably be in the form of a food or drink or a pharmaceutical product for the purpose of promoting health. When the composition of the present invention is in the form of a food or drink, it can be made into a functional food, a food for specified health uses, or a food with nutrient functions. Furthermore, the composition of the present invention can be provided and sold as a food, drink, or pharmaceutical product labeled with a specific use (especially a health use) or function. Examples of uses and functions include extending lifespan, extending healthy lifespan, improving chronic inflammation, improving weakness caused by inflammation, improving the intestinal environment, a composition for increasing intestinal flora, and improving the balance of intestinal bacteria.

[0065] The term "indication" as used above refers to all actions aimed at informing consumers of the above-mentioned uses, and any indication that can recall or infer the above-mentioned uses falls under the category of "indication" in this invention, regardless of the purpose of the indication, the content of the indication, the object or medium on which it is displayed, etc. However, it is preferable to display the use in an expression that allows consumers to directly recognize the above-mentioned uses. Specifically, examples include the act of describing the above-mentioned uses on the food and beverage composition or food and beverage product of the present invention or on the product packaging; the act of transferring, delivering, displaying for transfer or delivery, or importing the product or product packaging on which the above-mentioned uses are described; the act of describing the above-mentioned uses in advertisements, price lists, or transaction documents for the product and displaying or distributing them; or the act of describing the above-mentioned uses in information containing these contents and providing them by electromagnetic means (such as the Internet), and particularly displaying them on promotional materials at the point of sale such as packaging, containers, catalogs, pamphlets, POP, and other documents.

[0066] Furthermore, the labeling is preferably one approved by the government or the like (e.g., labeling approved under various government systems and made in a manner based on such approval). Examples include labeling as a health food, more specifically, a health food, a functional food, an enteral nutritional food, a food for special dietary uses, a food with nutrient functions, a quasi-drug, etc. Other examples include labeling approved by the Consumer Affairs Agency, such as a food for specified health uses, a food with nutrient functions, a functional food, or labeling approved under similar systems. Examples of the latter include labeling as a food for specified health uses, a labeling as a conditional food for specified health uses, a labeling claiming to affect the structure or function of the body, a labeling claiming to reduce disease risk, and a labeling claiming functionality based on scientific evidence. More specifically, examples include labeling as a food for specified health uses (especially a labeling claiming health uses) as defined in the Cabinet Office Ordinance on the Permission for Labeling for Special Dietary Uses Provided in the Health Promotion Act (Cabinet Office Ordinance No. 57 of August 31, 2009), and similar labeling. Examples of such claims include "for extending life expectancy," "for extending healthy life expectancy," "for those who want to live longer," "for staying vibrant forever," "for improving chronic inflammation," "for improving weakness caused by inflammation," "for improving the intestinal environment," "for a composition that increases intestinal flora," "for improving the balance of intestinal bacteria," and "regulates the stomach."

[0067] <Usage, dosage> The composition of the present invention can be ingested or administered so that the alkaline phosphatase activity derived from the active ingredient per dose is preferably 1.2 U / kg body weight or more, more preferably 1.5 U / kg body weight or more, more preferably 1.6 U / kg body weight or more, more preferably 2 U / kg body weight or more, more preferably 2.5 U / kg body weight or more, more preferably 3 U / kg body weight or more, and particularly preferably 4 U / kg body weight or more. In addition, it can usually be ingested or administered so that the dose per administration is preferably 7 U / kg body weight or less, more preferably 6 U / kg body weight or less, and more preferably 5 U / kg body weight or less.

[0068] The composition of the present invention can be ingested or administered so that the alkaline phosphatase activity derived from the active ingredient per dose is preferably 20 U or more, more preferably 25 U or more, more preferably 30 U or more, more preferably 35 U or more, more preferably 40 U or more, more preferably 50 U or more, more preferably 60 U or more, and more preferably 80 U or more. In addition, it can usually be ingested or administered so that the amount per dose is preferably 300 U or less, more preferably 280 U or less, and more preferably 240 U or less.

[0069] The composition of the present invention can be ingested or administered so that the alkaline phosphatase activity derived from the active ingredient is preferably 80 U or more, more preferably 100 U or more, even more preferably 120 U or more, more preferably 160 U or more, more preferably 200 U or more, even more preferably 200 U or more, and particularly preferably 240 U or more per day. In addition, it can usually be ingested or administered so that the daily dose is preferably 500 U or less, more preferably 400 U or less, more preferably 350 U or less, and more preferably 300 U or less.

[0070] The composition of the present invention can be ingested or administered so that the daily dose is preferably 200 mg or more, more preferably 300 mg or more, more preferably 400 mg or more, more preferably 500 mg or more, and more preferably 600 mg or more. In addition, it can usually be ingested or administered so that the daily dose is preferably 1000 mg or less, more preferably 800 mg or less, and more preferably 700 mg or less.

[0071] The composition of the present invention can be ingested or administered multiple times per day, preferably at least once, more preferably at least twice, and even more preferably at least three times. The timing of ingestion or administration is not particularly limited, and may be, for example, before meals, after meals, between meals, or before bedtime.

[0072] When the composition of the present invention is in the form of a food or pharmaceutical product, the content of the composition in the food or pharmaceutical product can be determined based on the daily intake or dosage (U or mg) of the composition. Furthermore, when the composition is formulated into a food or pharmaceutical product, the amount of the composition contained in a single dose of the food or pharmaceutical product can be adjusted depending on the dosage form and the number of times it is taken or administered.

[0073] Furthermore, when the composition of the present invention is in the form of a food or pharmaceutical product, the content of lactic acid bacteria contained in the daily intake or daily administration of the food or pharmaceutical product can be determined based on the intake or administration amount (U or mg) of the composition. For example, when the lactic acid bacteria are live bacteria, the content of bacterial cells is usually 1×10 6 ~1×10 12 cfu / g or 1 x 10 6 ~1×10 12cfu / mL, and more preferably in the range of 1×10 7 ~1×10 11 cfu / g or 1 x 10 7 ~1×10 11 cfu / mL range.

[0074] In addition, when the lactic acid bacteria are killed or sterilized, the content of the bacteria is 1.0 × 10 per day of the daily intake or daily administration amount of the food, drink, or medicine. 10 Individual cells ~1.0×10 11 The percentage is 0.001 to 90% by weight in terms of individual cells or dry weight. In this specification, when the lactic acid bacteria are killed or sterilized, "cfu" can be replaced with "cells."

[0075] When the composition of the present invention is in the form of tablets or capsules, the number of tablets to be ingested or administered at one time can be determined depending on the dosage form so that the total daily intake or daily dosage falls within the above-mentioned numerical range. The weight per tablet can be determined depending on the number of tablets to be taken or administered at one time. For example, when the composition of the present invention is made into tablets, each tablet preferably contains 50 mg or more, more preferably 100 mg or more, more preferably 150 mg or more, and more preferably 200 mg or more. In addition, the AP activity per tablet can be determined depending on the number of tablets taken at one time. For example, the amount per tablet can be preferably 10 U or more, more preferably 20 U or more, more preferably 30 U or more, more preferably 35 U or more, more preferably 40 U or more, more preferably 60 U or more, and more preferably 80 U or more.

[0076] The subject to which the composition of the present invention is administered is not particularly limited, and the subject can be humans, but the subject can also be animals other than humans (for example, dogs, cats, horses, mice, rabbits, etc.). When the subject to which the composition of the present invention is administered is humans, the composition can be administered to minors under 20 years of age, adults, or elderly people aged 65 years or older.

[0077] The present invention can also be used for non-therapeutic purposes. Non-therapeutic purposes are a concept that does not include medical procedures, i.e., treatment of the human body through therapy. For example, health promotion is one example. In this specification, improvement includes the concepts of improving a disease, symptom or condition; preventing or delaying the worsening of a disease, symptom or condition; reversing, preventing or delaying the progression of a disease or symptom, and prevention.

[0078] <Method for producing a composition for supplying alkaline phosphatase> The present invention also relates to a method for producing an AP delivery composition. The production method of the present invention includes an addition step of adding one or more active ingredients selected from the group consisting of the above-mentioned (a) to (c) to other raw materials. The preferred forms of each active ingredient are as described above.

[0079] In the production method of the present invention, the adding step preferably includes a step of incorporating the active ingredient so that the AP activity of the composition is 0.2 U or more per 1 mg of dry weight of the composition.

[0080] <Design method> The present invention also relates to a method for designing a composition based on the AP activity of a bacterium. The design method of the present invention first involves a selection step of selecting bacteria that exhibit AP activity. The selection step may include a measurement step of measuring the AP activity of the target lactic acid bacteria. The method for measuring the AP activity of the target lactic acid bacteria can be found in the description of the composition.

[0081] Furthermore, in the selection step, for example, when the test bacterium is cultured and measured under the following measurement conditions, the test bacterium can be selected if the alkaline phosphatase activity of the bacterial cells is 0.8 U or more per 1 mg of dry weight of the bacterial cells. [Conditions] 3% by volume of pre-cultured bacterial solution with an OD600 value of 0.8-1.0 was added to 3 mL of MRS medium supplemented with 0.5 mg / mL cysteine, and the mixture was left to stand at 37°C for 20 hours. In another preferred embodiment, for example, the test bacterium can be selected when the alkaline phosphatase activity measured is 1.1 times or more higher than when JCM1231 cells are added under the same conditions after culturing the test bacterium.

[0082] Then, a design step is carried out in which the content of the lactic acid bacteria cells is designed based on the alkaline phosphatase activity of the lactic acid bacteria so that the alkaline phosphatase activity of the composition is at a predetermined concentration or higher. Preferably, the content of the bacteria in the entire composition is designed so that the total amount per 1 mg of dry weight of the composition is 0.2 U or more. In the design step, the content of the bacteria may be designed using multiple types of bacteria. [Example]

[0083] The lactic acid bacteria (type strains and deposited strains) used in the test are shown in Table 1.

[0084] [Table 1]

[0085] <Sample preparation> In the test, MRS medium was prepared by dissolving 0.1% cysteine ​​(Kanto Chemical Co., Ltd.) in MRS Broth (Solabia Biokar Diagnostics) and sterilizing it in an autoclave (121°C, 15 minutes). Various lactic acid bacteria were inoculated into the MRS medium and cultured and passaged anaerobically at 37°C using square jars and Anaeropacks (Sugiyamagen Co., Ltd.).

[0086] All lactic acid bacteria used in the test had been passaged at least five times and were used in a stable state. Test tubes (manufactured by Thermo Fisher Scientific) containing 3 mL of MRS medium were prepared for each strain, and each strain was added with 3% of the bacterial solution, followed by static culture for 20 hours. After the cultivation, the test tube containing the cultured bacterial solution was centrifuged (3000 rpm, 10 minutes), and the supernatant was removed by suction using an aspirator. To the remaining precipitate, 2 mL of ultrapure water (Fujifilm Wako Pure Chemical Industries, Ltd.) was added, and the mixture was stirred and shaken, and then centrifuged again. This process of suctioning off the supernatant and centrifugation was repeated twice, and finally 0.5 mL of ultrapure water was added to the precipitate, and the suspension was frozen to prepare a freeze-dried sample. The freeze-dried sample was stored dry at room temperature and, immediately before use, was weighed and prepared into a 6 mg / mL suspension in ultrapure water.

[0087] <Activity measurement> Alkaline phosphatase activity was measured as the activity to produce p-nitrophenol from disodium p-nitrophenyl phosphate using "Labo Assay™ ALP" (Fujifilm Wako Pure Chemical Industries, Ltd.). 20 μL / well of each sample suspension was added to a 96-well plate (Thermo Fisher Scientific), and 100 μL / well of a substrate solution prepared by dissolving 6.7 mM p-nitrophenyl phosphate disodium in 0.1 M carbonate buffer (pH 9.8, 2 mM magnesium chloride) was added and stirred to initiate the enzyme reaction. Immediately after the start of the reaction, the absorbance (405 nm) of each well was measured over time (every 2 minutes) for 20 minutes using a plate reader. The absorbance value of each well was calculated by subtracting the absorbance value at the start of the reaction, and the activity was calculated using the value 10 minutes after the start of the reaction, within the period when the increase in the value showed linearity. The amount of p-nitrophenol produced was calculated by preparing a calibration curve using a standard solution. The activity was calculated as 1 unit, which is the enzyme activity required to produce 1 μmol of p-nitrophenol per minute, and the activity per mg of dry weight of the bacterial cells of each sample (specific activity, U / mg).

[0088] The results of measuring alkaline phosphatase activity of each strain are shown in FIG. As shown in Figure 1, the AP activity of all lactic acid bacteria contained as active ingredients in the composition of the present invention was 0.8 U or more per 1 mg of dry mass of bacterial cells. Ligilactobacillus salivarius JCM1231 and Lacticaseibacillus casei ATCC393 are known to have AP activity, as described in Japanese Patent No. 4743925 (Patent Document 1). The lactic acid bacteria strain contained as an active ingredient in the composition of the present invention was shown to have significantly higher AP activity than these known bacteria.

[0089] [Manufacturing example] Examples of producing pharmaceutical compositions and food compositions using the compositions of the present technology are shown below.

[0090] [Production Example 1] Bacillus paracasei MCC1849 (NITE BP-01633) was added to 3 mL of MRS liquid medium and cultured anaerobically at 37°C for 16 hours. The culture was then concentrated and lyophilized to obtain a lyophilized bacterial powder. The bacterial powder was homogeneously mixed with whey protein concentrate (WPC) and prebiotics (lactulose, raffinose, and galactooligosaccharides) to obtain a composition. 20 g of this composition was dissolved in 200 g of water to obtain a composition for alkaline phosphatase. This composition can be used to reduce LPS toxicity, dephosphorylate ATP, inhibit intestinal inflammation, improve chronic inflammation, improve frailty, increase intestinal flora, improve intestinal bacterial balance, and extend lifespan.

[0091] [Production Example 2] Bacillus paracasei MCC1849 (NITE BP-01633) was added to 3 mL of MRS liquid medium and cultured anaerobically at 37°C for 16 hours. The culture was then concentrated and lyophilized to obtain a lyophilized bacterial powder. The bacterial powder was homogeneously mixed with a dried milk protein concentrate powder (MPC480, Fonterra, protein content 80% by weight, casein protein:whey protein ratio approximately 8:2) and prebiotics (lactulose, raffinose, and galactooligosaccharides). 20 g of the resulting composition was dissolved in 200 g of water to obtain a composition for alkaline phosphatase. This composition can be used to reduce LPS toxicity, dephosphorylate ATP, inhibit intestinal inflammation, improve chronic inflammation, improve frailty, increase intestinal flora, improve intestinal bacterial balance, and extend lifespan.

[0092] [Production Example 3] Bacillus paracasei MCC1849 (NITE BP-01633) was added to 3 mL of MRS liquid medium and cultured anaerobically at 37°C for 16 hours. The culture was then concentrated and lyophilized to obtain a lyophilized bacterial powder. Prebiotics (lactulose, raffinose, and galactooligosaccharides) and crystalline cellulose were then added to a mixing granulator and mixed. Purified water was then added to the mixture, which was then dried to obtain a granulated product (pharmaceutical composition) containing bacterial extracts, prebiotics, and excipients. This granulated product is a composition for supplying alkaline phosphatase and can be used for reducing LPS toxicity, dephosphorylating ATP, inhibiting intestinal inflammation, improving chronic inflammation, improving frailty, increasing intestinal flora, improving intestinal bacterial balance, and extending lifespan.

[0093] [Production Example 4] The method for producing fermented milk with the addition of Lacticase Bacillus paracasei MCC1849 (NITE BP-01633) is shown below.

[0094] First, the raw milk ingredients, and optionally water and other ingredients, are mixed, preferably homogenized, and then heat sterilized. Homogenization and heat sterilization can be carried out by conventional methods. After heat sterilization, a lactic acid bacteria starter is added (inoculated) to the sterilized milk preparation that has been cooled to a certain temperature, and then mixed. After filling into a container, the mixture is maintained at a predetermined fermentation temperature and fermented. Curd is formed by fermentation. Lactic acid bacteria starters that can be used include those commonly used in yogurt production, such as Lactobacillus bulgaricus, Lactococcus lactis, and Streptococcus thermophilus. In the present technology, these bacteria may be used alone or in any combination of two or more. Once the pH reaches the target value, the curd formed is broken down by stirring and cooled to below 10°C to obtain the fermented product. Cooling to below 10°C reduces the activity of lactic acid bacteria and inhibits the production of lactic acid. The fermented product obtained in the fermentation step is then heat-treated to a degree that does not kill the lactic acid bacteria, thereby obtaining a heated fermented product (heat-treated fermented product). By heating the fermented product appropriately, it is possible to suppress the production of lactic acid by the lactic acid bacteria in the heated fermented product. It is also possible to suppress a decrease in pH during the subsequent production steps and / or storage of the concentrated fermented milk.

[0095] Next, Lacticase Bacillus paracasei MCC1849 (NITE BP-01633) and prebiotics (lactulose, raffinose, and galactooligosaccharides) are added to the heated fermented product obtained in the heat treatment step. The amount of Lacticase Bacillus paracasei MCC1849 (NITE BP-01633) added is 1 × 10 7 ~1×10 11 cfu / ml is preferred, 1 x 10 8 ~1×10 10 cfu / ml is more preferred. Subsequently, Lacticase Bacillus paracasei MCC1849 (NITE BP-01633) and prebiotics are added to the heated fermented product, followed by concentration. The concentration step can be carried out using any conventionally known concentration method. For example, centrifugation, membrane separation, etc. can be used. In the centrifugation method, whey is removed from the concentrated product (heated fermented product to which lactic acid bacteria and prebiotics have been added), yielding concentrated fermented milk containing lactic acid bacteria and prebiotics and having an increased solids concentration. The fermented milk obtained as described above is a composition for supplying alkaline phosphatase, and can be used as a composition for reducing the toxicity of LPS, dephosphorylating ATP, suppressing intestinal inflammation, improving chronic inflammation, improving frailty, increasing the intestinal flora, improving the balance of intestinal bacteria, and extending lifespan.

[0096] [Production Example 5] The manufacturing method for infant formula containing Lacticase Bacillus paracasei MCC1849 (NITE BP-01633) is shown below.

[0097] 10 kg of desalted milk whey protein powder (Mirai Co., Ltd.), 6 kg of milk casein powder (Fonterra), 48 kg of lactose (Mirai Co., Ltd.), 920 g of mineral mixture (Tomita Pharmaceutical Co., Ltd.), 32 g of vitamin mixture (Tanabe Pharmaceutical Co., Ltd.), 500 g of lactulose (Morinaga Milk Industry Co., Ltd.), 500 g of raffinose (Nippon Beet Sugar Co., Ltd.), and 900 g of galactooligosaccharide liquid sugar (Yakult Pharmaceutical Co., Ltd.) were dissolved in 300 kg of warm water and further heated to 90°C for 10 minutes. 28 kg of modified fat (Taiyo Yushi Co., Ltd.) was added and homogenized. The mixture was then sterilized, concentrated, and spray-dried to prepare approximately 95 kg of modified milk powder. To this was added 1.8 x 10 micrograms of Lacticase Bacillus paracasei MCC1849 (NITE BP-01633) cell powder (1.8 x 10 micrograms) dispersed in starch. 11 cfu / g, manufactured by Morinaga Milk Industry Co., Ltd.) to prepare approximately 95 kg of lactic acid bacteria-oligosaccharide-containing infant formula. The resulting infant formula was dissolved in water to prepare a standard infant formula with a total solids concentration of 14% (w / v). The number of lactic acid bacteria in the infant formula was 2.7 x 10 9 cfu / 100 mL can be obtained. The prepared powdered milk obtained as described above is a composition for supplying alkaline phosphatase, and can be used as a composition for reducing the toxicity of LPS, dephosphorylating ATP, suppressing intestinal inflammation, improving chronic inflammation, improving weakness, increasing the intestinal flora, improving the balance of intestinal bacteria, and extending lifespan. [Industrial Applicability]

[0098] The lactic acid bacteria contained as an active ingredient in the composition of the present invention are easy to mass-produce and have a long history of use in food. Therefore, they can be added to supplements or foods and are highly safe. In other words, the present invention provides a composition for supplying alkaline phosphatase that is highly safe and has few side effects even when continuously ingested or administered.

Claims

1. A composition for supplying alkaline phosphatase, comprising one or more active ingredients selected from the group consisting of the following (a) to (c): (a) Lactic acid bacteria that exhibit alkaline phosphatase activity of 0.8 U or more per mg of dry weight of bacterial cells within 8 hours after culturing under the following conditions: [Conditions] 3% by volume of a pre-cultured bacterial solution with an OD600 value of 0.8 to 1.0 was added to 3 mL of MRS medium supplemented with 0.5 mg / mL cysteine, and the mixture was left to stand at 37°C for 20 hours. (b) the culture of (a) (c) The processed product of (b)

2. 2. The alkaline phosphatase-supplying composition according to claim 1, wherein the alkaline phosphatase activity per 1 mg of dry weight of the composition is 0.2 U or more.

3. It contains one or more active ingredients selected from the group consisting of the following (a) to (c): A composition for supplying alkaline phosphatase, having an alkaline phosphatase activity of 0.2 U or more per 1 mg of dry weight of the composition. (a) Lactic acid bacteria cells exhibiting alkaline phosphatase activity (b) the culture of (a) (c) The processed product of (b)

4. The lactic acid bacteria may be Lacticaseibacillus paracasei, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus acidophilus, Lactobacillus helveticus, Streptococcus thermophilus, Lactobacillus amylovorus, or the like.

4. The alkaline phosphatase supplying composition according to claim 1, wherein the alkaline phosphatase supplying agent is one or more selected from the group consisting of Lactobacillus amylovorus, Lactobacillus gallinarum, and Lactobacillus crispatus.

5. The lactic acid bacteria may be Lacticaseibacillus paracasei MCC1375 (Accession Number: FERM BP-11313), Lacticaseibacillus paracasei ATCC25302, Lacticaseibacillus paracasei MCC1849 (Accession Number: NITE BP-01633), Lactobacillus delbrueckii subsp. Lactobacillus bulgaricus ATCC11842, Lactobacillus acidophilus MCC1847 (Accession number: NITE BP-01695), Lactobacillus helveticus MCC1844 (Accession number: NITE BP-02185), Lactobacillus helveticus MCC1848 (Accession number: NITE BP-01671), Streptococcus thermophilus 5. The alkaline phosphatase supplying composition according to claim 4, wherein the alkaline phosphatase supplying bacterium is one or more selected from the group consisting of Lactobacillus thermophilus ATCC19258, Lactobacillus amylovorus JCM1126, Lactobacillus acidophilus ATCC4356, Lactobacillus gallinarum JCM2011, and Lactobacillus crispatus JCM1185.

6. 4. The alkaline phosphatase supplying composition according to claim 1, which is a composition for reducing LPS toxicity.

7. The alkaline phosphatase supplying composition according to claim 6, which is one or more selected from the group consisting of a composition for suppressing intestinal inflammation, a composition for improving chronic inflammation, and a composition for improving weakness caused by inflammation.

8. The alkaline phosphatase supplying composition according to claim 7, which is a lifespan extension composition.

9. 4. The alkaline phosphatase supplying composition according to claim 1, which is a composition for dephosphorylating ATP in the intestine.

10. 10. The alkaline phosphatase supplying composition according to claim 9, which is at least one selected from the group consisting of compositions for increasing intestinal flora and compositions for improving the balance of intestinal bacteria.

11. The alkaline phosphatase supplying composition according to any one of claims 1 to 3, which is in the form of a food, drink or pharmaceutical product.

12. 4. The alkaline phosphatase supplying composition according to claim 1, wherein the active ingredient is administered so that the alkaline phosphatase activity per day is 80 U or more.

13. 4. The alkaline phosphatase supplying composition according to claim 1, which is administered in an amount of 200 mg or more per day.

14. A method for producing the alkaline phosphatase supplying composition according to any one of claims 1 to 3, comprising: A method for producing a composition for supplying alkaline phosphatase, comprising the step of adding one or more active ingredients selected from the group consisting of (a) to (c) below to other raw materials: (a) Lactic acid bacteria that are cultured under the following conditions and, within 8 hours after harvesting, exhibit alkaline phosphatase activity of 0.8 U or more per mg of dry weight of the bacteria. [Conditions] 3% by volume of a pre-cultured bacterial solution with an OD600 value of 0.8 to 1.0 was added to 3 mL of MRS medium supplemented with 0.5 mg / mL cysteine, and the mixture was left to stand at 37°C for 20 hours. (b) the culture of (a) (c) The processed product of (b)

15. 15. The method for producing a composition for supplying alkaline phosphatase according to claim 14, wherein the adding step comprises adding the active ingredient so that the alkaline phosphatase activity per 1 mg of dry weight of the composition is 0.2 U or more.

16. A method for designing a composition for supplying alkaline phosphatase, comprising: a selection step of selecting lactic acid bacteria that exhibit alkaline phosphatase activity; A method for designing a composition for supplying alkaline phosphatase, comprising a design step of designing the content of lactic acid bacteria cells based on the alkaline phosphatase activity of the lactic acid bacteria so that the alkaline phosphatase activity of the composition is at a predetermined concentration or higher.

17. The method for designing a composition for supplying alkaline phosphatase according to claim 16, wherein the selection step selects lactic acid bacteria whose cells exhibit alkaline phosphatase activity of 0.8 U or more per mg of dry weight of the cells within 8 hours after culturing and recovering under the following conditions: [Conditions] 3% by volume of a pre-cultured bacterial solution with an OD600 value of 0.8 to 1.0 was added to 3 mL of MRS medium supplemented with 0.5 mg / mL cysteine, and the mixture was left to stand at 37°C for 20 hours.

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