JP2024049199A - Plasmacytoid composition for activating dendritic cells - Google Patents

Abstract

[Problem] The objective of the present invention is to provide a composition capable of maintaining, improving, or enhancing immune function by activating plasmacytoid dendritic cells (pDC). [Solution] One or more selected from the group consisting of Lactobacillus paracasei MCC1849 strain (NITE BP-01633) cells, a culture of said bacteria, and a processed product of said bacteria are contained as active ingredients in a composition for activating pDC. Here, activation of pDC includes an increase in the number of CD86-positive pDC cells and/or the amount of CD86 expression in pDC. The composition can be ingested in the form of a food or beverage or a pharmaceutical. [Selected Figure] Figure 1

Description

The present invention relates to a composition for activating plasmacytoid dendritic cells that contains a specific lactic acid bacterium as an active ingredient.

Dendritic cells are powerful and important components of the innate immune system. Plasmacytoid dendritic cells (hereafter referred to as pDCs), a type of dendritic cell, are known to act as a command center for immune cells, activating various immune cells such as NK cells, B cells, and T cells. pDCs are also the main producers of type I interferon (IFN), which exhibits growth inhibitory activity against viruses. For this reason, pDCs are considered to be extremely important cells in terms of immune function and biological defense, such as antiviral infection defense.

Conventionally, there has been much interest in enhancing immune function and increasing virus defense activity, and components that produce IFN have been studied.
For example, Patent Document 1 describes that a specific strain of Lactobacillus helveticus increases pDC. Patent Document 2 discloses that a specific strain of Lactococcus lactis can activate pDC and induce IFN production. Non-Patent Document 1 describes that Lactobacillus rhamnosus GG activates pDC.

Incidentally, Lactobacillus paracasei MCC1849 strain (NITE BP-01633) has been reported to promote the production of IL-12 and to have immunostimulatory and protective effects against viral infections (Patent Document 3), and is known as Shield Lactic Acid Bacteria (registered trademark) (Non-Patent Document 2).
However, it is not known that the lactic acid bacteria activate pDC.

Patent No. 6705628 Patent No. 5950827 Patent No. 5646124

S. Toki et al., J. Allergy Clin. Immunol., Vol.119, No.110, 1013 https://morinagamilk-ingredients.com/paraprobiotics/immunogenic/

The objective of the present invention is to provide a composition that can maintain, improve, or enhance immune function by activating pDC.

As a result of intensive research aimed at solving the above problems, the inventors discovered that when pDCs recovered from human peripheral blood are cultured in the presence of a specific strain of Lactobacillus paracasei, expression of the activation marker CD86 on the pDC surface is enhanced, thereby completing the present invention.

That is, a first aspect of the present invention is a composition for activating plasmacytoid dendritic cells (pDC), comprising one or more selected from Lactobacillus paracasei MCC1849 strain (NITE BP-01633) cells, a culture of the bacteria, and a treated product of the bacteria.
In this aspect, the activation is preferably an increase in the number of CD86-positive pDC cells and/or an increase in the amount of CD86 expression in pDC.
A second aspect of the present invention is a composition for promoting CD86 expression in pDC, comprising one or more selected from Lactobacillus paracasei MCC1849 strain (NITE BP-01633) cells, a culture of the bacteria, and a processed product of the bacteria.
The compositions of the first and second aspects are preferably used for maintaining, improving, or strengthening immune function, and are more preferably foods and beverages used for these purposes.
The compositions of the first and second aspects are preferably pharmaceutical agents.
A third aspect of the present invention is a composition for activating pDC, which contains one or more components selected from bacterial cells of the Lactobacillus paracasei species, cultures of the bacteria, and processed products of the bacteria, and has a higher pDC activation effect than a composition containing the same amount of bacterial cells of Lactococcus lactis JCM5805 strain, one or more components selected from cultures of the bacteria, and processed products of the bacteria.

According to the present invention, immune function can be maintained, improved, or strengthened by activating pDC.

1 is a graph showing the expression level of CD86 per pDC 24 hours after addition of the test sample in terms of fluorescence intensity in the in vitro test of Example 1 (n=3 for each group, *: p=0.003, Dunnett SPSS). 1 is a graph showing the CD86 expression level per pDC in subjects 0, 2, 4, and 6 weeks after ingestion of L. paracasei MCC1849 strain in the clinical trial of Example 2, as a fluorescence intensity (n=20, *: p<0.05, paired t-test SPSS). A graph showing the CD86 expression level per pDC in subjects 4 weeks after ingestion of the test sample in the clinical trial of Example 3 as a fluorescence intensity (control group n=49, L. paracasei MCC1849 strain ingestion group n=47), (*: p=0.037, ANCOVA).

Next, the present invention will be described in detail. However, the present invention is not limited to the following embodiments, and can be freely modified within the scope of the present invention.

The composition of the present invention contains, as an active ingredient, one or more selected from Lactobacillus paracasei MCC1849 strain cells, a culture of said bacteria, and a processed product of said bacteria (these may be collectively referred to as Lactobacillus paracasei MCC1849 strain cells).

Lactobacillus paracasei MCC1849 strain is a bacterium that was deposited at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary (NPMD; Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818) under the accession number of NITE P-01633 on June 6, 2013, and transferred to an international deposit based on the Budapest Treaty on December 19, 2013, to which the accession number of NITE BP-01633 has been assigned.
In addition, bacteria previously classified as the Lactobacillus genus were reclassified in 2020 in the International Journal of Systematic and Evolutionary Microbiology (IJSEM) in accordance with the rules (ICNP) of the International Committee on Nomenclature of Prokaryotes (ICSP), with Lactobacillus paracasei being reclassified into the Lacticaseibacillus genus.

In this specification, the Lactobacillus paracasei MCC1849 strain is not limited to the strain deposited or registered in a specified institution under the name of the bacterium (hereinafter, for convenience of explanation, also referred to as the "deposited strain"), but also includes strains substantially equivalent thereto (also referred to as "derived strains" or "derived strains"). In other words, it is not limited to the strain deposited in the depository institution under the above accession number, but also includes strains substantially equivalent thereto. Regarding bacteria, "strain substantially equivalent to the deposited strain" refers to a strain that belongs to the same species as the deposited strain, has a genome sequence similarity (average nucleotide identity value) with the deposited strain of preferably 99.0% or more, more preferably 99.5% or more, and even more preferably 100% identity, and preferably has the same bacteriological properties as the deposited strain. Regarding bacteria, a strain substantially equivalent to the deposited strain may be, for example, a derived strain with the deposited strain as a parent strain. Derivative strains include strains bred from the deposited strain and strains that have arisen naturally from the deposited strain. Breeding methods include modification by genetic engineering techniques and modification by mutation treatment. Mutation treatments include irradiation with X-rays, irradiation with ultraviolet light, and treatment with mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methanesulfonate, and methyl methanesulfonate. Strains that have arisen naturally from the deposited strain include strains that have arisen naturally during use of the deposited strain. Such strains include mutant strains that have arisen naturally by culturing (e.g., subculturing) the deposited strain. Derivative strains may be constructed by one type of modification, or by two or more types of modifications.

The bacteria contained in the composition of the present invention may be commercially available products, may be obtained by appropriate production, or may be obtained by culturing the above-mentioned bacteria.
The culture method is not particularly limited as long as the bacteria can grow. As the culture method, for example, a method generally used for culturing lactic acid bacteria can be used as is or with appropriate modification. The culture temperature may be, for example, 25 to 50°C, and preferably 35 to 42°C. The culture can be preferably carried out under anaerobic conditions, for example, while aerating with anaerobic gas such as carbon dioxide gas. The culture can also be carried out under microaerobic conditions such as liquid static culture. The culture can be carried out, for example, until the bacteria grow to a desired extent.

The medium used for the culture is not particularly limited as long as the bacteria can grow. As the medium, for example, a medium generally used for culturing lactic acid bacteria can be used as it is or after appropriate modification. That is, as the carbon source, for example, sugars such as galactose, glucose, fructose, mannose, cellobiose, maltose, lactose, sucrose, trehalose, starch, starch hydrolysate, blackstrap molasses, etc. can be used depending on the assimilation. As the nitrogen source, for example, ammonium salts such as ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate, and nitrates can be used. In addition, as the inorganic salt, for example, sodium chloride, potassium chloride, potassium phosphate, magnesium sulfate, calcium chloride, calcium nitrate, manganese chloride, ferrous sulfate, etc. can be used. In addition, organic components such as peptone, soybean flour, defatted soybean meal, meat extract, yeast extract, etc. may be used. Specific examples of media commonly used for culturing lactic acid bacteria include Reinforced Clostridial medium, de Man, Rogosa, and Sharpe medium (MRS medium), modified MRS medium (mMRS medium), TOS propionate medium (TOSP medium), TOS propionate mupirocin medium (TOSP Mup medium), Gifu Anaerobic Medium (GAM) medium, and Yeast Extract-casein Hydrolysate Acid (YCFA) medium.

The Lactobacillus paracasei MCC1849 strain contained in the composition of the present invention may be in the form of any of bacteria, a bacterial culture, and a treated bacterial product.
The bacterial cells may be live bacterial cells, killed bacterial cells, or a mixture of live and killed bacterial cells, with killed bacterial cells being preferred.
As the bacterial culture, for example, the culture obtained by culturing may be used as it is, the culture may be diluted or concentrated, or the bacterial cells recovered from the culture may be used. In addition, the culture may be a culture supernatant or a culture fraction. When the culture supernatant is used, for example, the supernatant of the culture solution obtained by culturing in GAM medium at 37° C. for 16 hours can be preferably used.
The processed bacterial products include cells or cultures that have been crushed, heated, or dried, and dilutions, dried products, or fractions thereof.
The Lactobacillus paracasei MCC1849 strain to be contained in the composition of the present invention is particularly preferably in the form of a heat-treated (heat-sterilized) bacterial body. The heat-sterilized body refers to, for example, a bacterium obtained by treating the bacterium at 70 to 100°C for 10 to 40 minutes or at 90 to 150°C for 5 to 30 seconds. Note that pressure may be applied during the heat treatment. Note that the temperature does not necessarily need to be constant during the heat treatment, and it is sufficient that the temperature is within the above range for a predetermined time.
The heat-sterilized bacteria may be used as is after the heat treatment, or may be subjected to a process such as crushing, heat drying, freeze drying, or spray drying before use.

The content of Lactobacillus paracasei MCC1849 strain bacteria in the composition of the present invention is not particularly limited and is appropriately set depending on the form of the composition, but for example, the amount of bacteria is preferably 1×10 ⁴ to 1×10 ¹³ cfu/g or 1×10 ⁴ to 1×10 ¹³ cfu/mL, more preferably 1×10 ⁵ to 1×10 ¹² cfu/g or 1×10 ⁵ to 1×10 ¹² cfu/mL, and even more preferably 1×10 ⁶ to 1×10 ¹¹ cfu/g or 1×10 ⁶ to 1×10 ¹¹ cfu/mL. In this specification, "cfu" stands for colony forming unit. In addition, when dead bacteria are used, cfu/g or cfu/mL may be read as individual cells/g or individual cells/mL.
When a culture supernatant is used as the bacterial culture, the content is preferably 0.1 to 100% by mass, more preferably 1 to 90% by mass, and even more preferably 10 to 80% by mass of the entire composition.
In the composition of this embodiment, the content of one or more selected from Lactobacillus paracasei MCC1849 strain cells, a culture of the bacteria, and a processed product of the bacteria relative to the total composition is preferably 0.001% by mass or more but less than 100% by mass, more preferably 0.005 to 95% by mass, and even more preferably 0.01 to 85% by mass.
These contents may be within the range generally used when distributed as oral compositions.

The composition of the present invention is capable of activating pDC. Activation of pDC refers to enhanced expression of the activation marker CD86 on the surface of pDC.
Specifically, activation of pDC includes the increase in the expression amount of CD86 on the surface of pDC.This means that the amount of CD86 expressed in unit pDC (in other words, per pDC cell) increases after application of the composition of the present invention compared to before application, or when the composition of the present invention is applied compared to when it is not applied.In other words, the composition of the present invention can promote the expression of CD86 in pDC, and the second aspect of the present invention provides a composition for promoting the expression of CD86 in pDC, which contains the bacteria of Lactobacillus paracasei MCC1849 strain.
Specifically, activation of pDC may include an increase in cells expressing CD86 on the surface of pDC (CD86-positive cells). The increase in CD86-positive cells may be
This includes an increase in the absolute number of CD86-positive cells and an increase in the proportion of the number of CD86-positive cells. The proportion refers to the proportion of the number of CD86-positive cells in the entire pDC population in any unit. An increase in the absolute number or proportion refers to an increase in the absolute number or proportion of CD86-positive cells after application of the composition of the present invention compared to before application, or in the case of application of the composition of the present invention compared to when it is not applied.
Here, the expression of CD86 can be confirmed qualitatively or quantitatively by known methods, for example, immunological techniques using a fluorescently labeled antibody.

The composition of the present invention has been confirmed in the examples described below to have a significantly higher pDC activation effect by Lactobacillus paracasei MCC1849 strain cells than one or more selected from the same amount of Lactococcus lactis JCM5805 strain cells, cultures of the bacteria, and processed products of the bacteria.
Therefore, in a third aspect, the present invention provides a composition for activating pDCs, which contains bacterial cells of Lactobacillus paracasei, and has a higher pDC activation effect than a composition containing the same amount of Lactococcus lactis JCM5805 strain bacterial cells as the above-mentioned components, or one or more selected from a culture of the above-mentioned bacteria and a processed product of the above-mentioned bacteria.

Here, the bacteria belonging to Lactobacillus paracasei refer to bacteria belonging to Lacticaseibacillus paracasei as a new classification, and specifically, in addition to Lactobacillus paracasei MCC1849, Lactobacillus paracasei MCC1375, Lactobacillus paracasei KW3110 (Accession Number: FERM BP-08634), Lactobacillus paracasei WON0604 (Accession Number: FERM BP-08634), ...
BP-11468), Lactobacillus paracasei Lpc-37 (Accession Number: DSM32661), etc.

The composition of the present invention activates pDC, and is therefore expected to activate the functions of various immune cells and promote IFN production, thereby maintaining, improving, or strengthening immune function. Maintaining immune function includes maintaining a state in which normal or good immune function is being exerted and preventing the function from declining, and improving immune function includes restoring a declined immune function to a normal state.

The subject to which the composition of the present invention is administered (ingested) is not particularly limited as long as it is an animal, but is usually a mammal, and preferably a human. In addition, the subject is not particularly limited, but is preferably a healthy person. In this case, a healthy person refers to a person who is not suffering from any disease or illness.

Another aspect of the present invention is the use of one or more selected from Lactobacillus paracasei MCC1849 strain cells, a culture of the bacteria, and a treated product of the bacteria in the production of a composition for activating pDC.
Another aspect of the present invention is the use of one or more selected from Lactobacillus paracasei MCC1849 strain cells, a culture of said bacteria, and a treated product of said bacteria in pDC activation.
Another aspect of the present invention is one or more selected from Lactobacillus paracasei MCC1849 strain cells, a culture of the bacterium, and a treated product of the bacterium, which are used for pDC activation.
Another aspect of the present invention is a method for activating pDC, comprising administering to a subject one or more selected from Lactobacillus paracasei MCC1849 strain cells, a culture of the bacteria, and a treated product of the bacteria.

In addition, "administering Lactobacillus paracasei MCC1849 strain cells to a subject" may be synonymous with "allowing Lactobacillus paracasei MCC1849 strain cells to be ingested by a subject". Ingestion may be spontaneous (free intake) or forced (forced intake). That is, the administration step may specifically be, for example, a step of blending Lactobacillus paracasei MCC1849 strain cells with food, drink or feed and supplying the cells to a subject, thereby allowing the subject to freely ingest the cells of Lactobacillus paracasei MCC1849 strain.

The timing and duration of ingestion (administration) of the composition of the present invention are not particularly limited and can be selected appropriately depending on the condition of the subject to be administered.

The composition of the present invention may be in the form of a food or drink, a medicine, etc., or may be in the form of an additive contained in a food or drink, a medicine, etc.
The composition of the present invention may be administered orally or parenterally, but is usually administered orally. Parenteral administration includes transdermal, intravenous, rectal, and inhalation routes.

The intake (administration) amount of the composition of the present invention is appropriately selected depending on the age (months), sex, condition, and other conditions of the subject to be ingested (administered).
The intake (administration) amount of the composition of the present invention, as the intake (administration) amount of Lactobacillus paracasei MCC1849 strain of the present invention, is, for example, preferably 1 x 10 ⁴ to 1 x 10 ¹³ cfu per day for an adult, more preferably 1 x 10 ⁵ to 1 x 10 ¹² cfu, and even more preferably 1 x 10 ⁶ to 1 x 10 ¹¹ cfu.
Regardless of the amount or period of ingestion (administration), the composition of the present invention can be ingested (administered) once a day or in multiple divided doses.

When the composition of the present invention is intended to be orally ingested, it is preferably in the form of a food or drink.
As for the food and beverage products, there are no particular limitations on their form or properties as long as they do not impair the effects of the present invention and can be taken orally, and they can be produced by conventional methods using raw materials normally used for food and beverage products, except that they contain Lactobacillus paracasei MCC1849 strain cells.

Food and drink products include, regardless of their form, liquid, paste, gel-like solid, powder, etc., for example, tablet confectionery; liquid food (nutritional food for tube feeding); wheat flour products such as bread, macaroni, spaghetti, noodles, cake mix, fried chicken flour, breadcrumbs, etc.; instant noodles, cup noodles, retort/prepared foods, canned foods, microwave foods, instant soups/stews, instant miso soup/cleansing liquids, canned soups, freeze-dried foods, other instant foods, etc.; canned agricultural products, canned fruit, jams, etc. Agricultural processed products such as marmalades, pickles, boiled beans, dried agricultural goods, cereals (grain processed products); marine processed products such as canned seafood, fish ham and sausages, seafood paste products, seafood delicacies, and tsukudani (fried fish dishes); livestock processed products such as canned livestock and pastes, livestock ham and sausages; dairy products such as processed milk, milk drinks, yogurt (fermented milk), lactic acid bacteria drinks, cheese, ice cream, cream, and other dairy products; fats and oils such as butter, margarines, and vegetable oils; soy sauce, miso, sauces, Basic seasonings such as processed tomato seasonings, mirin, vinegar, etc.; complex seasonings and foods such as cooking mixes, curry bases, sauces, dressings, noodle soups, spices, and other complex seasonings; frozen foods such as frozen ingredient foods, semi-cooked frozen foods, and cooked frozen foods; sweets such as caramel, candy, chewing gum, chocolate, cookies, biscuits, cakes, pies, snacks, crackers, Japanese sweets, rice sweets, bean sweets, dessert sweets, jellies, and other sweets; These include carbonated drinks, natural fruit juice, fruit juice drinks, soft drinks with fruit juice, fruit pulp drinks, fruit drinks with fruit particles, vegetable drinks, soy milk, soy milk drinks, coffee drinks, tea drinks, powdered drinks, concentrated drinks, sports drinks, nutritional drinks, alcoholic drinks, other beverages, and other commercial foods such as baby food, furikake, and ochazuke nori; nutritional compositions such as supplements and prepared milk (including powdered milk, liquid milk, etc.); enteral nutritional foods; and functional foods (foods for specified health uses, foods with nutritional functions).

When made into a food or drink supplement, it can be formulated into solid preparations such as powders, granules, tablets, capsules, etc., which may be enteric-coated, etc.; liquid preparations such as solutions, syrups, suspensions, emulsions, etc. When formulating such preparations, the ingredients, carriers, and methods for formulating pharmaceuticals described below can be followed.

In addition, one aspect of the food and drink may be feed, such as pet food, livestock feed, and fish feed.
The form of the feed is not particularly limited, and may contain, in addition to the Lactobacillus paracasei MCC1849 strain fungus bodies, for example, grains such as corn, wheat, barley, rye, milo, etc.; vegetable oil cakes such as soybean oil cake, rapeseed oil cake, palm oil cake, linseed oil cake, etc.; bran such as wheat bran, rice bran, defatted rice bran, etc.; manufacturing residues such as corn gluten meal and corn jam meal; animal feeds such as fish meal, skim milk powder, whey, yellow grease, tallow, etc.; yeasts such as torula yeast and brewer's yeast; mineral feeds such as calcium tertiary phosphate and calcium carbonate; oils and fats; simple amino acids; sugars, etc.

When the composition of the present invention is in the form of a food or drink (including feed), it can be provided and sold as a food or drink labeled with a purpose such as maintaining immune function. Furthermore, the Lactobacillus paracasei MCC1849 strain cells according to this specification can be used for the manufacture of such food or drink.

Such "indication" acts include all acts aimed at informing consumers of the above-mentioned uses, and any expression that can recall or infer the above-mentioned uses falls under the category of "indication" acts 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.
Furthermore, it is preferable that the "labeling" be done in an expression that allows consumers to directly recognize the above-mentioned uses.Specific examples include the act of transferring, delivering, displaying for the purpose of transferring or delivering, or importing food and beverage products or product packaging on which the above-mentioned uses are written, displaying or distributing advertisements, price lists, or transaction documents related to the products and writing the above-mentioned uses in information containing the above-mentioned uses and providing them by electromagnetic means (such as the Internet), etc.

On the other hand, it is preferable that the content of the labeling be approved by the government or the like (for example, a labeling approved based on various systems established by the government and made in a manner based on such approval). It is also preferable that such content of the labeling be affixed to promotional materials at the point of sale such as packaging, containers, catalogs, pamphlets, and POP, as well as other documents, etc.

In addition, "labeling" also includes labeling as health food, functional food, enteral nutrition food, special purpose food, health functional food, food for specified health uses, nutrient functional food, functional food, functional labeling food, medical drug, etc. Among these, in particular, labeling approved by the Consumer Affairs Agency, for example, labeling approved under the system related to food for specified health uses, nutrient functional food, or functional labeling food, or a similar system, can be mentioned. Specifically, labeling as food for specified health uses, labeling as food for conditional specified health uses, labeling that affects the structure or function of the body, labeling that reduces the risk of disease, labeling of functionality based on scientific evidence, etc. can be mentioned, and more specifically, labeling as food for specified health uses (especially labeling of health uses) and similar labeling as specified in the Cabinet Office Ordinance on the Permission of Labeling for Special Uses Prescribed in the Health Promotion Act (Cabinet Office Ordinance No. 57 of August 31, 2009) are typical examples.
Such indications include, for example, "acting on pDC (plasmacytoid dendritic cells)" and
Examples of claims that may be made include "acts on pDC immune cells,""activates pDC immune cells,""increases pDC immune cells,""helps maintain immune function in healthy people,""enhances immune function,""supports immune function,""supports maintaining immune function in healthy people,""acts on pDC (plasmacytoid dendritic cells) and helps maintain immune function in healthy people," etc.

The composition of the present invention may also be in the form of a pharmaceutical product.
The route of administration of pharmaceuticals may be either oral or parenteral, with oral being preferred. Parenteral intake (administration) includes transdermal, intravenous, rectal, inhalation, etc.
The pharmaceutical form can be formulated into a desired dosage form depending on the administration method. For example, in the case of oral administration, it can be formulated into solid preparations such as powders, granules, tablets, and capsules; liquid preparations such as solutions, syrups, suspensions, and emulsions. It can also be made into an enteric-coated preparation by enteric coating, etc. In the case of parenteral administration, it can be formulated into suppositories, ointments, injections, etc.
In the formulation, in addition to the bacteria of Lactobacillus paracasei MCC1849 strain, ingredients such as excipients, pH adjusters, colorants, and flavorings that are usually used in formulations can be used. In addition, it is also possible to use other medicinal ingredients, or ingredients having a known or future pDC activating effect, etc. in combination.
In addition, formulation can be carried out by a known method according to the dosage form. When formulating, a carrier usually used in formulation may be appropriately blended. Such carriers include excipients, binders, disintegrants, lubricants, stabilizers, flavorings, etc.

Examples of excipients include sugar derivatives such as lactose, sucrose, glucose, mannitol, and sorbitol; starch derivatives such as corn starch, potato starch, α-starch, dextrin, and carboxymethyl starch; cellulose derivatives such as crystalline cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, carboxymethyl cellulose, and calcium carboxymethyl cellulose; gum arabic; dextran; pullulan; silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, and magnesium aluminometasilicate; phosphate derivatives such as calcium phosphate; carbonate derivatives such as calcium carbonate; and sulfate derivatives such as calcium sulfate.

Examples of binders include the above excipients, gelatin, polyvinylpyrrolidone, macrogol, etc.

Disintegrants include, for example, the above-mentioned excipients as well as chemically modified starch or cellulose derivatives such as croscarmellose sodium, sodium carboxymethyl starch, and cross-linked polyvinylpyrrolidone.

Examples of lubricants include talc; stearic acid; metal stearates such as calcium stearate and magnesium stearate; colloidal silica; waxes such as veegum and galangal; boric acid; glycol; carboxylic acids such as fumaric acid and adipic acid; sodium carboxylates such as sodium benzoate; sulfates such as sodium sulfate; leucine; lauryl sulfates such as sodium lauryl sulfate and magnesium lauryl sulfate; silicic acids such as silicic anhydride and silicic acid hydrate; and starch derivatives.

Examples of stabilizers include paraoxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol, and phenylethyl alcohol; benzalkonium chloride; acetic anhydride; and sorbic acid.

Examples of flavoring agents include sweeteners, acidulants, and fragrances.
In the case of a liquid preparation for oral administration, examples of the carrier used include solvents such as water.

The timing of taking the pharmaceutical of the present invention is not particularly limited, and may be, for example, before or after a meal, between meals, or before going to bed.

The present invention will be explained in more detail below using examples, but the present invention is not limited to these examples.

<Production Example> Preparation of heat-sterilized lactic acid bacteria Lactobacillus paracasei (hereinafter referred to as "L. paracasei") MCC1849 strain (NITE BP-01633) was cultured in MRS (de Man Rogasa Sharpe) medium (manufactured by Difco) at 37 ° C for 16 hours. Lactococcus lactis JCM5805 strain was cultured in M-17 medium supplemented with lactose under aerobic conditions at 30 ° C for 16 hours. After washing the cultured bacteria with PBS, they were suspended in PBS to a concentration of 2 × 10 ⁹ cells / mL, and heat-treated at 90 ° C for 15 minutes to prepare a heat-sterilized body. The obtained heat-sterilized body was used in the examples described below.

Example 1: Confirmation of pDC activation effect in in vitro test Human normal peripheral blood cells (PBMC) were purchased from Veritas, and human DCs were isolated according to the protocol of the EasySep (registered trademark) Human Pan-DC Pre-Enrichment Kit (STEMCELL Technologies). Human DCs were seeded at 2 x ¹⁰⁵ cells/well in a 96-well round-bottom flask for adherent cells, and 2 x ¹⁰⁷ cells of heat-killed L. paracasei MCC1849 strain or L. lactis JCM5805 strain were added, followed by culturing for 24 hours in a _CO2 incubator. After the culture, the cells were collected and centrifuged, and the cell pellet was suspended in PBS (-). In order to analyze this cell population by fluorescence-activated cell sorting (FACS) using a flow cytometer, the cell surface was stained with FITC-labeled anti-Lineage (CD3, CD14, CD19, CD56) antibody (BioLegend), PE-labeled anti-CD86 antibody (BioLegend), PerCP-Cy5.5-labeled anti-HLA-DR antibody (BioLegend), and APC-labeled anti-CD11c antibody (BioLegend). Cell groups that were Lineage negative, CD11c negative, CD123 positive, and HLA-DR positive detected by FACS were counted as plasma cell dendritic cells (pDC), and the activation state of pDC was evaluated by the expression level of CD86 measured by the fluorescence intensity (Geometric Mean) of the PE-labeled anti-CD86 antibody.

The results are shown in Figure 1. The L. paracasei MCC1849 strain-administered group showed higher CD86 expression levels than the control and Lactococcus lactis JCM5805 strain-administered groups. In other words, it can be seen that administration of L. paracasei MCC1849 strain can activate pDCs more effectively. In particular, the L. paracasei MCC1849 strain-administered group showed significantly higher CD86 expression levels than the control group (Dunnett's test, p=0.003). Furthermore, it can be seen that a composition containing L. paracasei MCC1849 strain can activate pDCs more effectively than a composition containing the same amount of Lactococcus lactis JCM5805 strain.

Example 2: Confirmation of pDC activation effect in human clinical trial 1
Twenty healthy adults were given 250 billion heat-killed L. paracasei MCC1849 strains per day for four weeks, and blood was collected at 0, 2, 4, and 6 weeks after ingestion to obtain peripheral blood mononuclear cells (PBMCs). In order to analyze this cell population by fluorescence-activated cell sorting (FACS) using a flow cytometer, the cell surface was stained with FITC-labeled anti-CD304 antibody, PE-labeled anti-HLA-DR antibody, PE-Cy7-labeled anti-CD123 antibody, and APC-labeled anti-CD86 antibody. Cell groups positive for both CD123 and CD304 detected by FACS were counted as plasma cell dendritic cells (pDCs), and the activation state of pDCs was evaluated by the expression level of CD86 measured by the fluorescence intensity (geometric mean) of the APC-labeled anti-CD86 antibody. Comparisons were made using paired t-tests with the baseline being before intake (week 0).

The results are shown in Figure 2. After 4 weeks of ingestion of L. paracasei MCC1849 strain, significantly higher CD86 expression levels were observed compared to 0 weeks. In other words, it can be seen that pDCs were activated in humans by ingestion of L. paracasei MCC1849 strain.

Example 3: Confirmation of pDC activation effect in human clinical trials 2
100 healthy adults were divided into two groups, and 50 billion heat-killed L. paracasei MCC1849 strain cells or a placebo food were consumed per day for 4 weeks. Blood was collected before (week 0) and after 4 weeks of intake, and peripheral blood mononuclear cells (PBMCs) were obtained. The activation state of pDCs in this cell population was evaluated in the same manner as in Example 2. Comparison between groups after 4 weeks of intake was performed by covariance analysis.

The results are shown in Figure 3. Four weeks after ingestion, the L. paracasei MCC1849 strain intake group showed significantly higher CD86 expression levels than the placebo group (control group). In other words, it can be seen that pDCs were activated in humans by ingestion of the L. paracasei MCC1849 strain.

Claims (7)

A composition for activating plasmacytoid dendritic cells (pDCs), comprising one or more selected from the group consisting of Lactobacillus paracasei MCC1849 strain (NITE BP-01633), a culture of said bacteria, and a processed product of said bacteria. The composition according to claim 1, wherein the activation is an increase in the number of CD86-positive pDC cells and/or the amount of CD86 expression in pDC. A composition for promoting CD86 expression in pDCs, comprising one or more selected from Lactobacillus paracasei MCC1849 strain (NITE BP-01633) cells, a culture of said bacteria, and a processed product of said bacteria. The composition according to any one of claims 1 to 3, which is used to maintain, improve, or strengthen immune function. The composition according to claim 4, which is a food or drink. The composition according to any one of claims 1 to 3, which is a pharmaceutical product. A composition for activating pDC, comprising one or more selected from a bacterial cell belonging to Lactobacillus paracasei, a culture of the bacteria, and a processed product of the bacteria,
A composition having a higher pDC activation effect than a composition containing one or more selected from Lactococcus lactis JCM5805 strain cells, a culture of the bacteria, and a processed product of the bacteria in the same amount as the above-mentioned contained components.

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