JP7466166B2 - Lactic acid bacteria-containing agent for preventing viral infection and method for producing same - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Act FOOD STYLE 21, Vol. 21, No. 4, pp. 58-62 (April 1, 2018, Food Chemical Newspaper Co., Ltd.)

The present invention relates to a lactic acid bacteria-containing agent for protecting against viral infections, which contains lactic acid bacteria that have an improved ability to reach Peyer's patches when orally ingested, and a method for producing the same .

The present applicant has already proposed a method for producing a Th1 inducer, which is characterized by adding a dispersant or excipient to lactic acid bacteria cells capable of inducing IL-12 production, grinding and dispersing the cells to a particle size of less than 1 micron by wet dispersion treatment, preventing the cells from re-aggregating with the dispersant or excipient, and powdering the cells in this state (see Patent Document 1).

Japanese Patent No. 4621218

It is known that lactic acid bacteria are taken up by Peyer's patches and passed to immune cells, but it is also known that not all lactic acid bacteria are necessarily taken up.

An object of the present invention is to provide a lactic acid bacteria-containing agent for protecting against viral infections which enhances the ability of orally ingested lactic acid bacteria to reach Peyer's patches, and a method for producing the same .

In order to achieve the above-mentioned object, the present inventors conducted various studies and found that a lactic acid bacteria powder obtained by adding a soluble excipient to a lactic acid bacteria cell culture solution to prepare a desired state, crushing and dispersing the cell culture solution, and further drying and powdering the culture solution, has high reachability to Peyer's patches when orally ingested and has an excellent effect in preventing viral infections , which led to the completion of the present invention.

That is, one aspect of the present invention provides a lactic acid bacteria-containing virus infection protection agent containing, as an active ingredient, a lactic acid bacteria powder having a median particle diameter of 1.0 μm or less as measured by a wet laser diffraction scattering method. According to the virus infection protection agent of the present invention, since the median particle diameter of the lactic acid bacteria is as small as 1.0 μm or less, it can slip through the mesh structure of the mucus covering the mucous membrane surface of the digestive tract and reach Peyer's patches. Then, it is taken up by M cells on Peyer's patches and captured by dendritic cells and macrophages waiting below, and these cells produce cytokines and activate immune responses. As a result, an excellent virus infection protection effect is exerted.

The lactic acid bacteria powder in the lactic acid bacteria-containing agent for protecting against viral infections of the present invention preferably has the ability to reach Peyer's patches when orally ingested.

The lactic acid bacteria-containing agent for preventing viral infection of the present invention is preferably one against norovirus.

The lactic acid bacteria in the lactic acid bacteria powder is preferably one or more species selected from the group consisting of Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus helveticus, Lactobacillus pentosus, Enterococcus faecalis, and Enterococcus faecium.

Another aspect of the present invention provides a method for producing a lactic acid bacteria-containing virus infection protection agent, comprising: preparing a lactic acid bacteria cell culture solution so that the concentration is 0.01% by mass or more and less than 88% by mass in terms of dry cell content; adding a soluble excipient to the cell culture solution so that the cell culture solution has a final dry cell concentration of 12% by mass or more; pulverizing and dispersing the cell culture solution to which the soluble excipient has been added, and then drying and powdering the cell culture solution to obtain a lactic acid bacteria powder having a cell particle median diameter of 1.0 μm or less as measured by a wet laser diffraction scattering method; and incorporating the lactic acid bacteria powder as an active ingredient.
In the present invention, the grinding and dispersion are preferably carried out using a mixer or a homogenizer.

According to the lactic acid bacteria-containing virus infection protection agent of the present invention, the median diameter of the lactic acid bacteria cell particles is as small as 1.0 μm or less, so that they can slip through the mesh structure of the mucus covering the mucous membrane surface of the digestive tract and reach Peyer's patches. They are then taken up by M cells on Peyer's patches and captured by dendritic cells and macrophages waiting below, which causes these cells to produce cytokines and activate immune responses. As a result, excellent virus infection protection effects are exhibited.

1 is a graph showing the results of measuring the median diameter of lactic acid bacteria cell particles at various amounts of dextrin added. FIG. 1 is a graph showing the particle size distribution of lactic acid bacteria cell particles (vertical axis: relative particle amount (%), horizontal axis: particle diameter (μm)) for each amount of dextrin added ((A) 0 mass%, (B) 10 mass%, (C) 15 mass%). 1 is a graph showing the particle size distribution of lactic acid bacteria cell particles at various amounts of dextrin added ((D) 20% by mass, (E) 25% by mass, (F) 50% by mass). This is a diagram showing (A) dispersed lactobacilli passing through the mucus mesh structure to reach Peyer's patches, (B) dispersed lactobacilli being taken up by M cells on Peyer's patches, and (C) aggregated lactobacilli unable to pass through the mucus mesh structure and not reaching Peyer's patches. 1 is a graph showing the time course of viral load in feces of immunocompetent MNV mice. 1 is a graph showing the time course of viral load in feces of immunocompromised MNV mice. 1 is a graph showing neutralizing antibody titers in immunocompetent MNV mice and immunocompromised MNV mice.

Lactic acid bacteria as a raw material for the lactic acid bacteria powder used in the lactic acid bacteria-containing viral infection protection agent of the present invention are not particularly limited, and include microorganisms belonging to the genus Lactobacillus, such as Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus helveticus, and Lactobacillus pentosus; microorganisms belonging to the genus Enterococcus, such as Bifidobacterium bifidum, Enterococcus faecalis, and Enterococcus faecium; Lactobacillus gasseri, Streptococcus thermophilus, and the like; Examples of suitable microorganisms include those belonging to the genus Streptococcus, such as Streptomyces thermophilus, and those belonging to the genus Lactococcus, such as Lactococcus lactis.

Among the above lactic acid bacteria, it is preferable to use one or more species selected from the group consisting of Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus helveticus, Lactobacillus pentosus, Enterococcus faecalis, and Enterococcus faecium.

The lactic acid bacteria powder used in the lactic acid bacteria-containing virus infection protection agent of the present invention is prepared by first preparing a cell culture solution of the lactic acid bacteria so that the cell concentration is 0.01% by mass or more and less than 88% by mass, preferably 0.1% by mass or more and less than 80% by mass, and more preferably 1% by mass or more and less than 50% by mass, calculated as dry cell content.

A soluble excipient is added to the bacterial culture solution thus prepared so that the final dry concentration is 12% by mass or more, preferably 13% by mass or more, and more preferably 15% by mass or more. By adding a soluble excipient at 12% by mass or more, it is possible to prevent reagglomeration of the lactic acid bacteria powder during grinding and dispersion, as described below.

The soluble excipients used are not particularly limited, and examples thereof include dextrin; maltodextrin; xanthan gum; sugar alcohols such as lactitol, maltitol, mannitol, sorbitol, and xylitol; sugars such as dextrose, fructose, glucose, lactose, and sucrose; and organic acids such as adipic acid, citric acid, fumaric acid, glutaric acid, malic acid, succinic acid, and tartaric acid.

Next, the bacterial culture solution to which the soluble excipient has been added is pulverized and dispersed. Methods for pulverization and dispersion include known methods using stirring, a mixer, a homogenizer, a ball mill, a bead mill, a jet mill, a generator, etc., but it is preferable to use a mixer or a homogenizer.

After grinding and dispersion, the bacterial culture liquid is dried into a powder. Methods for drying into a powder include known techniques such as freeze drying and reduced pressure spray drying.

The median diameter of the bacterial particles of the thus obtained lactic acid bacteria powder measured by a wet laser diffraction method is 1.0 μm or less, preferably 0.9 μm or less, and more preferably 0.8 μm or less. Since the median diameter of the bacterial particles is as small as 1.0 μm or less, they can slip through the mesh structure of the mucus covering the mucous membrane surface of the digestive tract and reach Peyer's patches. They are then taken up by M cells on Peyer's patches and captured by dendritic cells and macrophages waiting below, which causes these cells to produce cytokines and activate immune responses.

In this specification, "the median diameter of the bacterial particles is 1.0 μm or less" means that the particle size of the bacterial particles is 1.0 μm or less, which corresponds to 50% of the cumulative volume from the small side.

The median size of bacterial particles is measured by a wet laser diffraction scattering method under the conditions described below. Specifically, it is obtained by measuring using a particle size distribution analyzer SALD-3100 manufactured by Shimadzu Corporation in a standard manner, specifically, using a flow cell, water as the measurement solvent, a refractive index of 1.55-0.00i, a maximum value of 0.2 in the measurement absorbance range, and a minimum value of 0.02.

The lactic acid bacteria obtained by the method of the present invention can be used to protect against viral infections, including, but not limited to, rotavirus, poliovirus, influenza virus, AIDS virus, hepatitis A virus, hepatitis B virus, norovirus, adenovirus, sapovirus, astrovirus, Aichi virus, parechovirus, etc.

Furthermore, the lactic acid bacteria obtained by the method of the present invention can be used to enhance immune activity. Since the lactic acid bacteria obtained by the method of the present invention have improved accessibility to Peyer's patches when orally ingested, ingestion of the bacteria activates dendritic cells and macrophages, enhancing immune activity, and enhancing the body's defense power, making it possible to prevent various diseases.

The lactic acid bacteria obtained by the method of the present invention can be used as a product as it is, but various ingredients can be added or blended to improve the flavor or to give it a required shape, and flavors can also be added to make a final product.

Ingredients that can be added or mixed with lactic acid bacteria include various carbohydrates, emulsifiers, sweeteners, acidulants, fruit juice, etc. More specifically, these include sugars such as glucose, sucrose, fructose, honey, etc., sugar alcohols such as sorbitol, xylitol, erythritol, lactitol, palatinit, etc., and emulsifiers such as sucrose fatty acid esters, glycerin sugar fatty acid esters, and lecithin. Other examples include various vitamins such as vitamin A, vitamin B, vitamin C, and vitamin E, herbal extracts, grain ingredients, vegetable ingredients, milk ingredients, etc.

Flavors include yogurt, berry, orange, quince, shiso, citrus, apple, mint, grape, pear, custard cream, peach, melon, banana, tropical, herb, black tea, and coffee, and these can be used alone or in combination of two or more.

The lactic acid bacteria described above can be made into products in any form, such as solid or liquid.

The lactic acid bacteria obtained by the method of the present invention can be used in various forms such as beverages, granules, tablets, capsules, etc., together with pharma- ceutically acceptable salts, excipients, preservatives, colorants, flavorings, etc., by methods known in the pharmaceutical or food manufacturing field.

The above lactic acid bacteria can also be used in health foods. Health foods refer to foods that are intended to promote health, maintenance, and improvement in a more positive sense than regular foods, and include, for example, liquid, semi-solid, and solid products, such as cookies, rice crackers, jellies, yokan, yogurt, manju, and other confectioneries, soft drinks, nutritional drinks, and soups.

The dosage of the lactic acid bacteria obtained by the method of the present invention varies depending on the quality of the lactic acid bacteria powder, the age and symptoms of the subject, etc. For example, when used to prevent diseases such as viral infections and to improve immunity, the dosage for an adult is about 0.001 to 10 g in solid content per dose, and it is preferable to take it daily. When used as a health food, it is appropriate to use an amount that does not adversely affect the taste or appearance of the food, for example, about 0.01 to 100 g in solid content per 1 kg of the target food.

Furthermore, the lactic acid bacteria obtained by the method of the present invention can also be used in various product forms for external skin preparations, such as lotions, cosmetic creams, milky lotions, packs, skin milks, gels, powders, lip balms, lipsticks, under-makeup, foundations, sun care, bath products, body shampoos, body rinses, soaps, cleansing foams, ointments, patches, jellies, and aerosols.

Furthermore, the lactic acid bacteria obtained by the present invention can be appropriately blended with various ingredients and additives commonly used in cosmetics, quasi-drugs, and pharmaceuticals, as described below, if necessary.

That is, moisturizing agents such as glycerin, petrolatum, urea, hyaluronic acid, and heparin; PABA derivatives (para-aminobenzoic acid, Escarol 507, etc.), cinnamic acid derivatives (Neo Heliopan, Parsol MCX, Sunguard B, etc.), salicylic acid derivatives (octyl salicylate, etc.), benzophenone derivatives (ASL-24, ASL-24S, etc.), dibenzoylmethane derivatives (Parsol A, Parsol DAM, etc.), heterocyclic derivatives (Tinuvin series, etc.), and ultraviolet absorbing and scattering agents such as titanium oxide; disodium edetate, trisodium edetate, citric acid, sodium citrate, Sequestering agents such as tartaric acid, sodium tartrate, lactic acid, malic acid, sodium polyphosphate, sodium metaphosphate, gluconic acid, etc.; sebum suppressants such as salicylic acid, sulfur, caffeine, tannin, etc.; bactericides and disinfectants such as benzalkonium chloride, benzethonium chloride, chlorhexidine gluconate, etc.; anti-inflammatory agents such as diphenhydramine hydrochloride, tranexamic acid, guaiazulene, azulene, allantoin, hinokitiol, glycyrrhizic acid and its salts, glycyrrhizic acid derivatives, glycyrrhetinic acid, etc.; vitamin A, B vitamins (B1, B2, B6, B12, B15), folic acid, etc. vitamins such as nicotinic acid, pantothenic acid, biotin, vitamin C, vitamin D group (D2, D3), vitamin E, ubiquinones, and vitamin K (K1, K2, K3, K4); amino acids and derivatives thereof such as aspartic acid, glutamic acid, alanine, lysine, glycine, glutamine, serine, cysteine, cystine, tyrosine, proline, arginine, and pyrrolidone carboxylic acid; skin whitening agents such as retinol, tocopherol acetate, magnesium ascorbyl phosphate, ascorbyl glucoside, arbutin, kojic acid, ellagic acid, and placenta extract; Antioxidants such as butyl hydroxytoluene, butyl hydroxyanisole, and propyl gallate; astringents such as zinc chloride, zinc sulfate, zinc phenolate, zinc oxide, and potassium aluminum sulfate; sugars such as glucose, fructose, maltose, sucrose, trehalose, erythritol, mannitol, xylitol, and lactitol; various plant extracts such as licorice, chamomile, horse chestnut, saxifrage, peony, quince, Scutellaria root, Phellodendron bark, Coptis japonica, Jew's herb, and ginkgo leaf, as well as oily ingredients, surfactants, thickeners, alcohols, powdered ingredients, and pigments can be appropriately blended.

The present invention will be specifically explained below with reference to examples, but these examples are not intended to limit the scope of the present invention.

<I. Preparation of lactic acid bacteria powder>
1. Flocculated lactic acid bacteria powder The lactic acid bacteria Enterococcus faecalis strain was cultured by a known method. The culture solution (dry lactic acid bacteria concentration: 5 to 10% by mass) was freeze-dried (hereinafter referred to as flocculated lactic acid bacteria powder).

2. Dispersed lactic acid bacteria powder The lactic acid bacteria Enterococcus faecalis strain was cultured by a known method. Dextrin was added to the culture solution (5-10% by mass in terms of dry bacteria) to give final concentrations of 10, 15, 20, 25, and 50% by mass (final dry concentration), homogenized to thoroughly disperse the bacteria, and then freeze-dried (hereinafter referred to as "dispersed lactic acid bacteria").

<II. Measurement of particle size distribution>
The lactic acid bacteria aggregate type and the lactic acid bacteria dispersion type obtained above were suspended in purified water to a concentration of 10 mg/mL in terms of dry bacteria. The particle size distribution of the lactic acid bacteria powder in the suspension was measured using a laser diffraction particle size distribution analyzer SALD-3100 (manufactured by Shimadzu Corporation). Specifically, a flow cell was used, water was used as the measurement solvent, the refractive index was 1.50-0.00i, the number of measurements was 2, the average number was 64, the maximum value of the measurement absorbance range was 0.2, and the minimum value was 0.02, and the sample solution was added until the measurement range was reached. The measurement results of the median size are shown in Table 1 and Figure 1, and the particle size distribution is shown in Figures 2 and 3.

As shown in Table 1, the median diameter of the lactic acid bacteria aggregate type was 29.556 μm. The median diameter of the lactic acid bacteria dispersion type in which 10% by mass of dextrin was added to disperse the bacteria was 1.233 μm. On the other hand, the median diameter of the lactic acid bacteria dispersion type in which 15% by mass or more of dextrin was added to disperse the bacteria was 0.796 μm or less, which was significantly smaller than that of the lactic acid bacteria aggregate type.

<III. Observation of the digestive tract>
1. Method Mice were given Cy3 fluorescently stained lactic acid bacteria aggregates (prepared in I above) and lactic acid bacteria dispersions (prepared in I above, dextrin 50% by mass), and the lactic acid bacteria present near the small intestinal Peyer's patches were observed using a fluorescent microscope, and the inside of the Peyer's patches was observed using a confocal laser microscope. Photographing was carried out with the cooperation of Icom Co., Ltd.

2. Results and Discussion It was found that the lactobacillus dispersion type bacteria are individually dispersed and have a small particle size of 1.0 μm or less, so they pass through the mucosal surface of the digestive tract and reach the Peyer's patches (arrow in Figure 4 (A)). They are then taken up by M cells on the Peyer's patches (arrow in Figure 4 (B)), and captured by dendritic cells and macrophages waiting below, where these cells produce cytokines and activate the immune response.

On the other hand, it was found that the aggregated type of lactobacilli, which consists of large bacterial masses, cannot pass through the mucus mesh structure (arrow in Figure 4 (C)) and simply passes through the intestinal tract without reaching the Peyer's patches, which is thought to reduce the original effect of lactobacilli.

<IV. Murine Norovirus (MNV) Infection Experiment>
1. Materials The following virus strains and host cells were used.
・Virus: Mouse norovirus S7-PP3 strain ・Host cells: RAW264.7 cells (mouse macrophage-like cells)
(Growth medium = Dulbecco's MEM medium with 10% fetal bovine serum)

2. Methods (1) BALB/c mice (6 weeks old, female) were divided into two groups: an immunologically normal group (non-treated 5-fluorouracil (5-FU) group) and an immunologically impaired group (treated with 5-FU (0.25 mg/day/mouse)). The mice were subcutaneously injected with the virus every other day from 7 days before to 21 days after inoculation (n = 3 in both groups) (Table 2).

(2) MNV (1 x 106 PFU/0.2 ml/mouse) was orally inoculated.

(3) Lactic acid bacteria aggregate type (prepared as described in I above) or lactobacillus dispersion type (prepared as described in I above, 50% by mass dextrin) was orally administered (twice a day, 9:00 and 18:00) from 7 days before to 21 days after virus inoculation.

(4) After 8 hours, 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, and 21 days, feces were collected from each mouse in a 5 mL tube (the mouse was placed in an empty cage for 15 to 30 minutes).

(5) The amount of virus in feces was measured by the following method (plaque assay method). 10 μL of PBS was added per 1 mg of feces, and the feces was uniformly dispersed by ultrasonic treatment. After centrifugation, the supernatant was diluted 10, 100, and 1000 times with PBS. Each of the above dilutions was added to RAW 264.7 cells cultured in a 24-well plate to infect them. Then, 1.5% SeaPlaque agarose-added DMEM medium was overlaid and cultured at 37°C. After 2 days, the cells were stained by overlaying neutral red solution, and the number of plaques was counted under a microscope.

(6) The neutralizing antibody titer in the serum was measured by the following method. 21 days after infection, whole blood was collected, centrifuged to separate the serum, and then diluted 10, 50, 200, 1000, and 5000 times with PBS. 100 μL of the virus was mixed with 100 μL of the above dilution (PBS was added instead of serum as a control), and added to RAW 264.7 cells cultured in a monolayer on a 24-well plate for infection at room temperature. DMEM medium containing 1.5% SeaPlaque agarose was overlaid and cultured at 37°C. After 2 days, the cells were stained by overlaying neutral red solution, and the number of plaques was measured under a microscope. The percentage of the number of plaques for each dilution was calculated when the number of plaques for the control was set to 100%, and the serum dilution factor that inhibited plaque formation by 50% was determined on a graph and used as the neutralizing antibody titer.

3. Results and Discussion During the 21-day observation period, some of the mice treated with 5-FU had mild loose stools, but no weight loss was observed and all mice survived.

(1) Viral load in feces (Figs. 5 and 6)
Immunocompetent mice (untreated with 5-FU) (Fig. 5): In the control group, the virus load was at its maximum 1 day after infection, then gradually decreased, and was undetectable 18 days after infection. In the group administered with dispersed lactobacillus type, the virus load 1 day after infection decreased to 40% of that in the control group, and in the group administered with aggregate lactobacillus type, it decreased to 50% and remained consistently low thereafter. The virus load up to 12 days after infection was lower in the group administered with dispersed lactobacillus type than in the group administered with aggregate lactobacillus type, and the virus disappeared from feces 16 days after infection in both groups.

Mice with compromised immune function (treated with 5-FU) (Figure 6): 5-FU treatment increased the amount of virus excreted and prolonged the duration of virus excretion. In particular, in the control group, virus was still detected after 21 days. Administration of lactic acid bacteria reduced the amount of virus over a 21-day period. In the group administered with dispersed lactic acid bacteria, the amount of virus in the feces was lower for up to 14 days after infection compared to the group administered with aggregated lactic acid bacteria. In both groups, virus was no longer detectable after 18 days.

Therefore, administration of lactic acid bacteria suppressed viral proliferation in the intestinal tract and stopped viral excretion early. This effect is thought to be greater with dispersed lactic acid bacteria.

(2) Neutralizing Antibody Titer Three weeks after infection, the amount of MNV neutralizing antibody was measured (Table 3, FIG. 7).

Mice with normal immune function (untreated with 5-FU): Antibody titers were significantly higher (p<0.05) in the groups administered with dispersed and aggregated lactobacilli compared to the control group. The dispersed type had higher titers than the aggregated type.

Mice with compromised immune function (treated with 5-FU): Antibody titers were lower in all treatment groups compared to the corresponding 5-FU untreated groups. However, both lactobacillus-treated groups maintained high antibody titers equal to or higher than those of the untreated control group. Both the lactobacillus-dispersed group and the lactobacillus-aggregated group showed significantly (p<0.05) higher antibody titers than the control group, and the dispersed group tended to have higher antibody titers than the aggregated group.

These results suggest that the increase in antibody levels due to administration of lactic acid bacteria inhibits viral proliferation in the intestinal tract, and that dispersed lactic acid bacteria have a greater immunostimulatory effect than aggregated lactic acid bacteria.

Claims (5)

A lactic acid bacteria-containing viral infection protection agent for oral ingestion, comprising as an active ingredient a powder of lactic acid bacteria having a median particle diameter of 1.0 μm or less as measured by a wet laser diffraction scattering method and capable of reaching Peyer's patches when orally ingested . A virus infection prevention agent containing the lactic acid bacteria according to claim 1 against norovirus. 3. The lactic acid bacteria-containing agent for protection against viral infection according to claim 1 or 2 , wherein the lactic acid bacteria in the lactic acid bacteria powder is Enterococcus faecalis . A method for producing an orally ingested lactic acid bacteria-containing virus infection protection agent, comprising: preparing a lactic acid bacteria cell culture solution so that the cell concentration is 0.01% by mass or more and less than 88% by mass in terms of dry cell; adding a soluble excipient to the cell culture solution so that the cell culture solution has a final dry concentration of 12% by mass or more; pulverizing and dispersing the cell culture solution to which the soluble excipient has been added, and then drying and powdering the cell culture solution to obtain a lactic acid bacteria powder having a cell particle median diameter of 1.0 μm or less as measured by a wet laser diffraction scattering method and capable of reaching Peyer 's patches when orally ingested; and containing the lactic acid bacteria powder as an active ingredient. The method for producing a lactic acid bacteria-containing agent for protecting against viral infection according to claim 4 , wherein the grinding and dispersion are carried out using a mixer or a homogenizer.

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