Immunoregulator

Abstract

To provide an immunoregulator having excellent immunoregulation effect.SOLUTION: The present invention provides an immunoregulator and an immunoregulatory food composition that contains a water dispersion of particles including yeast residue of the genus Candida yeast and has a β-glucan content of the water dispersion of 34 wt.% or less. Furthermore, the invention includes a first cleaning step that suspends the yeast residue of the genus Candida yeast in an aqueous solvent and dilutes it more than ten times to separate the solid components, a second cleaning step that preferably suspends the solids separated in the first cleaning step in water and dilutes them more than fifty times to obtain a treatment liquid containing solids, and a dispersion step that disperses the treatment liquid obtained from the first or second cleaning step to obtain a water dispersion of particles containing yeast residue of the genus Candida yeast.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to an immunomodulator.

Background Art

[0002] Patent Document 1 describes that an immunomodulator containing at least one oxidized product and / or an extract thereof selected from the group consisting of yeast belonging to the genus Candida, yeast culture, and yeast residue can regulate the secretion amount of cytokines in immune cells and can treat, prevent, and relieve symptoms of autoimmune diseases.

[0003] Patent Document 2 describes that a composition excellent in anti-inflammatory effect can be obtained by using a yeast protein degradation product obtained from yeast cells after yeast extract extraction or yeast cells without yeast extract extraction.

[0004] Patent Document 3 describes that an immunomodulator containing cells of lactic acid bacteria, β1,3-1,4 glucan derived from barley, and β1,3-1,6 glucan derived from black yeast, a health supplement feed comprising the immunomodulator, and a feed containing the immunomodulator enhance immunity.

[0005] Patent Document 4 describes that a biological fiber composition containing β-1,4 glucan derived from Gluconacetobacter has an anti-inflammatory effect on the skin.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Summary of the Invention

Problems to be Solved by the Invention

[0007] However, in the inventions described in Patent Documents 1 to 4, the immunomodulatory effect is insufficient. Further, the production of the agent described in Patent Document 1 requires drying treatment of residues, oxidation treatment, alcohol extraction, etc., and the production of the agent described in Patent Document 2 requires treatment with cell wall-lysing enzymes, resulting in a complicated production process and low production efficiency.

[0008] The present invention has been made in view of the above, and an object thereof is to provide an immunomodulatory agent having a good immunomodulatory effect.

Means for Solving the Problems

[0009] The present invention provides the following [1] to [9]. [1] An immunomodulatory agent containing an aqueous dispersion of particles containing yeast residues of Candida yeast, wherein the β-glucan content of the aqueous dispersion is 34% by weight or less. [2] The agent according to [1], wherein the volume cumulative 50% particle diameter of the particles is 2.5 μm or more and 5.5 μm or less. [3] The agent according to [1] or [2], wherein the volume cumulative 10% particle diameter of the particles is less than 2.5 μm. [4] The agent according to any one of [1] to [3], wherein the volume cumulative 90% particle diameter of the particles exceeds 4.5 μm. [5] The agent according to any one of [1] to [4], wherein the solid content concentration of the aqueous dispersion is 0.01 to 10%. [6] The agent according to any one of [1] to [5], wherein the Candida yeast includes Candida utilis. [7] An immunomodulatory food composition containing an aqueous dispersion of particles containing yeast residues of Candida yeast, wherein the β-glucan content of the aqueous dispersion is 34% by weight or less. 〔8〕The method for producing the agent according to 〔1〕 or 〔2〕, comprising: a first washing step of suspending yeast residues of Candida yeast in an aqueous solvent, diluting the suspension by 10 times or more, and separating the solid content; and a dispersion step of dispersing the solid content obtained in the washing step in a dispersion medium to obtain an aqueous dispersion of particles containing yeast residues of Candida yeast. 〔9〕The production method according to 〔8〕, further comprising, after the first washing step and before the dispersion step, a second washing step of suspending the solid content separated in the first washing step in water, diluting the suspension by 50 times or more, and separating the solid content.

Advantages of the Invention

[0010] According to the present invention, it is possible to increase the production amount of cytokines in immune cells without performing treatments such as drying treatment, oxidation treatment, and alcohol extraction of the residue, and to provide an immunomodulatory agent having a good immunomodulatory effect. Further, the aqueous dispersion composition of the present invention is useful as an immunomodulatory food composition.

Brief Description of the Drawings

[0011]

Figure 1

Embodiments for Carrying Out the Invention

[0012] [1. Aqueous Dispersion] The aqueous dispersion contained in the immunomodulatory agent is an aqueous dispersion of particles containing yeast residues of Candida yeast.

[0013] [Candida Yeast] The yeast residue is a yeast residue of yeast belonging to the genus Candida. The Candida yeast is not particularly limited, and examples include Candida utilis, Cyberlindnera jadinii (Torula yeast), Candida kefyr, Candida etchellsiii, Candida stellata, Candida versatilis, etc., and preferably includes Candida utilis.

[0014] [Yeast residue] In the present specification, the yeast residue is obtained by removing at least a part of the yeast cells from the yeast culture, and preferably is the residue after extracting the yeast extract from the yeast culture. The yeast residue usually contains components derived from the cell wall. Examples of the method for extracting the yeast extract include autolysis method, hot water extraction method, enzyme extraction method, acid-alkali extraction method, etc. The hot water extraction method and the enzyme extraction method are preferred, and the enzyme extraction method (for example, extraction using a protease) is more preferred. The yeast residue may be one kind, or a combination of two or more kinds of yeast residues having different yeast species and extraction methods.

[0015] [Dispersion medium] In the aqueous dispersion, the particles containing the above yeast residue are dispersed in an aqueous solvent which is the dispersion medium. The aqueous solvent is preferably water such as pure water, ion-exchanged water, distilled water, tap water, etc., but may further contain components such as a dispersion aid and a pH adjuster used as necessary during production.

[0016] [Particles] The aqueous dispersion contains particles containing the above yeast residue. By adjusting the particle diameter of the particles as described below, aggregation of the particles can be suppressed and the immunomodulatory effect can be more efficiently exerted.

[0017] -Volume cumulative 10% particle diameter- The volume cumulative 10% particle size (D10) of the particles only needs to be smaller than the following volume cumulative 50% particle size, and is not particularly limited, but is preferably less than 2.5 μm, more preferably less than 2.4 μm. The lower limit is preferably 0.5 μm or more, more preferably 1.0 μm or more, and still more preferably 2.0 μm or more. Therefore, D10 is preferably less than 2.5 μm, more preferably 0.5 μm or more and less than 2.5 μm, still more preferably 1.0 μm or more and less than 2.4 μm, and even more preferably 2.0 μm or more and less than 2.4 μm.

[0018] -Volume cumulative 50% particle size- The volume cumulative 50% particle size (D50, average particle size) is preferably 2.5 μm or more, more preferably 2.6 μm or more, and still more preferably 2.7 μm or more. The upper limit is preferably 5.0 μm or less, more preferably 4.5 μm or less, and still more preferably 4.0 μm or less. Therefore, D50 is preferably 2.5 μm or more and 5.0 μm or less, more preferably 2.6 μm or more and 4.5 μm or less, and still more preferably 2.7 μm or more and 4.0 μm or less.

[0019] -Volume cumulative 90% particle size- The volume cumulative 90% particle size (D90, average particle size) is preferably more than 4.5 μm, more preferably 5.0 μm or more, and still more preferably 5.5 μm or more. The upper limit is preferably 10.0 μm or less, more preferably 9.5 μm or less, and still more preferably 9.0 μm or less. Therefore, D90 is preferably more than 4.5 μm and 10.0 μm or less, more preferably 5.0 μm or more and 9.5 μm or less, and still more preferably 5.5 μm or more and 9.0 μm or less.

[0020] D10, D50, and D90 can be measured by a laser diffraction particle size distribution measuring device as the maximum particle sizes below which 10%, 50%, and 90% of the sample volume are respectively.

[0021] [Glucans] The aqueous dispersion contains β-glucan. As used herein, β-glucan means a polysaccharide in which glucose is linked by β-bonds. For example, it includes linear β-glucans composed of a backbone linked by linear β-1,3 bonds and / or β-1,4 bonds, and branched β-glucans in which side chains (glucose linked by β-1,6 bonds) are branched from a backbone in which glucose is linked by linear β-1,3 bonds. The length of the side chain is usually about 10 to 35 residues. The content of β-glucan in the aqueous dispersion is usually 5% by weight or more, preferably 7% by weight or more, more preferably 8% by weight or more, and still more preferably 9% by weight or more. The upper limit is preferably 34% by weight or less, more preferably 32% by weight or less, and still more preferably 30% by weight or less. Therefore, the content of β-glucan is preferably 34% by weight or less, more preferably 5 to 34% by weight, still more preferably 7 to 32% by weight, and even more preferably 9 to 30% by weight. Thereby, the immunomodulatory effect can be preferably exerted.

[0022] The aqueous dispersion may contain glucans other than β-glucan. Examples of glucans other than β-glucan include α-glucans such as amylose, dextran, glycogen, amylopectin, and pullulan.

[0023] The content of total glucan in the aqueous dispersion is preferably 6% by weight or more, more preferably 10% by weight or more. The upper limit is preferably 60% by weight or less, more preferably 55% by weight or less, and still more preferably 50% by weight or less. Therefore, the total glucan content is preferably 6 to 60% by weight, more preferably 6 to 55% by weight, and still more preferably 10 to 50% by weight.

[0024] In the aqueous dispersion, glucans such as β-glucan may be contained in the particles of yeast residue or in the dispersion medium.

[0025] The β-glucan content and the total glucan content can be measured by the method described in the examples.

[0026] [Total protein amount] The aqueous dispersion contains proteins. Examples of the proteins include yeast cells and proteins derived from culture broth. The total protein content in the aqueous dispersion is preferably 15% by weight or more, more preferably 20% by weight or more. The upper limit is preferably 80% by weight or less, more preferably 70% by weight or less.

[0027] The total protein content can be measured by the method described in the examples.

[0028] [Other Components] The aqueous dispersion may contain components other than the above components. For example, glycoproteins such as chitosan, sugars other than glucan such as mannan, lipids, ash, and nucleotides can be mentioned.

[0029] [Solid Content Concentration] The solid content concentration of the aqueous dispersion is preferably 0.01% by weight or more, more preferably 0.03% by weight or more. The upper limit is preferably 10% by weight or less, more preferably 7% by weight or less, and even more preferably 5% by weight or less. Therefore, the solid content concentration of the aqueous dispersion is preferably 0.01 to 10% by weight, more preferably 0.03 to 7% by weight, and even more preferably 0.03 to 5% by weight.

[0030] [2. Immunomodulator] An aqueous dispersion of particles containing yeast residues of Candida yeast has an immunomodulatory effect and is therefore useful as an active ingredient of an immunomodulator.

[0031] [Immunomodulatory Action] In this specification, immunomodulation means at least one selected from improvement of immunity in vivo, suppression of excessive immune responses, control to an appropriate immune state, and suppression of inflammatory symptoms. The immunomodulatory action may be confirmed by an increase in the production amount of cytokines such as interleukin-10 (IL-10) in bone marrow dendritic cells administered with the above aqueous dispersion as compared with the case without addition. Since the immunomodulator has an immunomodulatory action, it can be used for the treatment, prevention, symptom relief of diseases such as autoimmune diseases, cancer, viral diseases, and allergic diseases such as pollen, food, and house dust, and improvement of immunity reduced by stress.

[0032] Even if the β-glucan content in the aqueous dispersion is relatively small, it can efficiently exert an immunomodulatory effect. The immunomodulatory effect is mainly considered to be brought about by β-glucan. However, since β-glucan is contained in the dispersion medium and particles, it is presumed that the immunomodulatory effect of β-glucan is efficiently exerted as compared with dry yeast residue.

[0033] [Optional Component] The immunomodulator may contain the above aqueous dispersion, and may consist only of the aqueous dispersion, but may also contain other components (optional components) as necessary. The optional components may be any pharmacologically acceptable components, and examples include: seasonings; acidulants (such as citric acid and succinic acid); preservatives (such as ascorbic acid, acetate, and ε-polylysine); pH adjusters; emulsifiers (such as sucrose fatty acid esters, glycerin fatty acid esters, organic acid monoglycerides, and lecithin); fragrances; pigments; thickeners (such as carrageenan and xanthan gum); swelling agents; proteins (such as soy protein and milk protein); saccharides (such as starch, sucrose, fructose, reduced starch saccharides, erythritol, and xylitol); sweeteners (such as sucralose and thaumatin); vitamins (such as vitamin A, vitamin C, vitamin E, and vitamin K); and minerals (such as iron and calcium).

[0034] [Dosage Form] Since the immunomodulator uses the aqueous dispersion as an active ingredient, it is usually liquid or syrup-like. Capsules containing the aqueous dispersion may also be used.

[0035] [Administration Form] There is no particular limitation on the administration form of the immunomodulator. For example, oral administration, parenteral administration (such as pulmonary administration, subcutaneous administration, intravenous administration, intrathecal administration, nasal administration, transdermal administration, and rectal administration), sublingual administration, inhalation administration, etc. may be mentioned. Usually, oral administration, nasal administration, transdermal administration, and rectal administration are used.

[0036] [Food, Feed] The immunomodulator can be used as a medicine, quasi-drug, food, or feed, and among them, use as food or feed is preferred. When used as food, it can be used as a food composition or feed composition for immunomodulation, for example, as a health food (functional food, dietary supplement, food for specified health use); a food for a specific subject (medical food, food for patients, food for infants, food for nursing care, food for the elderly), or as an additive for these foods. The type of food is not particularly limited, and examples include beverages (soft drinks, carbonated drinks, nutritional drinks, powdered drinks, fruit drinks, milk drinks, jelly drinks, beer, Japanese sake, Western liquor, Chinese liquor, herbal liquor, etc.); rice foods (cooked rice, porridge, etc.); breads; seasonings (sauces, miso, soy sauce, mayonnaise, shortening, dressings, sauces, spices, etc.); soy foods (natto, tofu, deep-fried bean curd, etc.); processed fishery products (kamaboko, fish cakes, chikuwa, processed products, etc.); processed meat products (ham, sausage, wiener, etc.); processed agricultural products (vegetables, fruits, etc.); pickles; noodles (udon, soba, spaghetti, etc.); soups (powdered soups, liquid soups, etc.). As feed, it can be used for animals other than humans, for example, livestock such as horses, cows, sheep, pigs, and goats; poultry such as chickens, turkeys, and quails; experimental animals such as mice, rats, guinea pigs, hamsters, and rabbits; and pets such as dogs, cats, budgerigars, and parrots. [Administration Conditions] In the case of medicines and health foods, administration conditions such as the administration time, dose, and administration interval can be appropriately selected according to appropriate conditions and are not particularly limited.

[0037] [3. Manufacturing Method] The above-mentioned aqueous dispersion can be produced by a method including a first washing step, a second washing step performed as necessary, and a dispersion step. Each step will be described below.

[0038] [First Washing Step] In the first washing step, the yeast residue of Candida yeast is suspended in an aqueous solvent and diluted more than 10 times to separate the solid content. The yeast residue is usually a heat-dried product with particles aggregated. By performing this step, the aggregation can be dispersed. Also, impurities such as soluble components contained in the yeast residue can be preliminarily removed. The yeast residue of Candida yeast and the aqueous solvent are as described in the previous section.

[0039] - Suspension treatment - First, when suspending the yeast residue in the aqueous solvent, the solid content concentration of the prepared suspension is preferably 0.5 wt% or more, more preferably 3 wt% or more. The upper limit is preferably 10 wt% or less, more preferably 7 wt% or less.

[0040] Also, a dispersion aid may be added to the above-mentioned suspension. By adding the dispersion aid, pH adjustment and dispersion degree adjustment in the system can be performed, and the dispersion efficiency can be improved. Examples of the dispersion aid include hydroxides such as sodium hydroxide, calcium hydroxide, potassium hydroxide, magnesium hydroxide, ammonium hydroxide; carbonates such as sodium carbonate, potassium carbonate, sodium bicarbonate, ammonium carbonate, guanidine carbonate; borates such as potassium borate, sodium borate; silicates such as potassium silicate, sodium silicate No. 1, sodium silicate No. 2, sodium silicate No. 3, sodium orthosilicate, sodium metasilicate; sulfites such as sodium sulfite; pyrophosphates such as disodium hydrogen phosphate, sodium pyrophosphate, potassium pyrophosphate. Hydroxides are preferred, and sodium hydroxide is more preferred. When the dispersion aid is sodium hydroxide, it can usually be added as a 0.01 - 2N, preferably 0.08 - 1.5N aqueous solution. Note that the dispersion aid preferably does not contain an oxidizing agent (such as sodium hypochlorite).

[0041] As a method of suspending yeast residue in an aqueous solvent, for example, a method of adding an aqueous solvent to the yeast residue and stirring it as necessary can be mentioned. Stirring is carried out while dispersing using a stirrer (for example, 300 rpm or less, 250 rpm or less, 200 rpm or less), usually for 1 hour or more, preferably 5 hours or more, more preferably 10 hours or more, still more preferably 13 hours or more. The upper limit is not particularly limited, but usually it is within 30 hours. The temperature conditions can be set near room temperature (for example, 15 to 35 °C, preferably 20 to 30 °C).

[0042] -Dilution and solid separation treatment- The dilution ratio (dilution ratio with respect to solids) when diluting the suspension of yeast residue is usually 10 times or more, preferably 15 times or more, more preferably 20 times or more, still more preferably 25 times or more. The upper limit is not particularly limited, but usually it is 50 times or less.

[0043] Separation of the solids after dilution can be carried out by centrifugation. Dilution and separation of the solids can be carried out one or more times, and preferably two or more times.

[0044] [Second washing step] In the second washing step, it is a step of suspending and diluting the solids separated in the first washing step in water and separating the solids. By performing this step between the first washing step and the dispersion step, the dispersion of the aggregated state of the yeast residue and the effect of removing impurities can be enhanced more. When a dispersion aid is added in the first washing step, it is preferable to perform the second step. Thereby, the added dispersion aid can be neutralized and the influence on the physical properties of the dispersion can be reduced.

[0045] -Suspension treatment- When suspending the solid content obtained in the first washing step in an aqueous solvent, the solid content concentration of the prepared suspension is preferably 0.5% by weight or more, more preferably 1% by weight or more. The upper limit is preferably 10% by weight or less, more preferably 5% by weight or less. It is preferably lower than the concentration of the suspension adjusted in the first washing step. The dispersing aid that can be added as necessary and the method of suspending the solid content in the aqueous solvent are the same as the suspension methods and conditions described in the first washing step.

[0046] -Dilution and solid content separation treatment- When diluting the suspension of the solid content, the dilution ratio (dilution ratio with respect to the solid content) is usually more than 50 times, preferably 60 times or more, more preferably 70 times or more, and still more preferably 80 times or more. The upper limit is not particularly limited, but is usually 200 times or less. It is preferably lower than the dilution ratio in the second washing step.

[0047] When diluting the suspension to obtain a treatment liquid, the dilution concentration is preferably 50 times or more, more preferably 80 times or more with respect to the solid content of the yeast residue. The upper limit is not particularly limited, but it is preferably diluted 150 times or less, more preferably 120 times or less. Therefore, the dilution concentration of the suspension is preferably 50 times or more, more preferably diluted 50 to 150 times, and still more preferably diluted 80 to 120 times.

[0048] -Neutralization treatment- In the second washing step, a neutralization treatment may be performed. Thereby, the change in pH due to the influence of the dispersing aid added in the first washing step can be adjusted to near neutral. The neutralization treatment may be performed so that the pH after treatment is usually 6 to 8, preferably 6.5 to 7.5. The neutralization treatment can be performed using a pH adjuster (for example, an acid such as hydrochloric acid or acetic acid).

[0049] After dilution, the preparation of the treatment liquid containing the solid content is preferably by centrifugation. The centrifugation can be performed one or more times, and preferably two or more times.

[0050] The first washing step and the second washing step that is performed as necessary can each be performed one or more times. For example, the first step may be performed one or more times, or the second washing step may be performed one or more times after the first washing step has been performed one or more times, or after the first washing step and the second washing step have each been performed one or more times, the first washing step and the second washing step may each be performed one or more times again. The conditions and number of times of each washing step can be appropriately adjusted using, as an index, the β-glucan content in the resulting treatment liquid and the like.

[0051] [Dispersion step] In the dispersion step, the solid content obtained in the washing step is dispersed in a dispersion medium to obtain an aqueous dispersion of particles containing yeast residues of Candida yeast.

[0052] The dispersion treatment can be performed using equipment such as a homogenizer after adding the solid content obtained in the first or second washing step to the dispersion medium. The dispersion medium is usually water, and the solid content concentration after addition is usually 0.5% by weight or more, preferably 1% by weight or more, more preferably 1.5% by weight or more. The upper limit is usually 5% by weight or less, preferably 3% by weight or less. The conditions of the dispersion treatment are not particularly limited, but an example is as follows. When using a homogenizer, the rotation speed can usually be 5,000 to 20,000 rpm. The treatment time is usually 1 to 5 hours.

[0053] By the above method, the above-mentioned aqueous dispersion can be efficiently obtained. The reason why an aqueous dispersion having an immunomodulatory action can be efficiently obtained by the above method is not clear, but by performing a predetermined washing and dispersion step, β-glucan (generally present as a glucan layer constituting the cell wall and located below the mannan layer) derived from yeast residues can be exposed on the surface of the particles, and aggregation of the particles can be suppressed. Therefore, it is presumed that this improves the contact efficiency between β-glucan and immune cells, and an immunomodulatory action can be exhibited even with a small β-glucan content.

Example

[0054] Hereinafter, the present invention will be described by way of examples. The following examples do not limit the present invention. In the following description, “%” means “% by weight” unless otherwise specified.

[0055] Example 1 (KR yeast, water dispersion treatment) [Washing treatment 1] KR yeast, which is yeast residue of Candida utilis (KR yeast, manufactured by Mitsubishi Corporation Life Sciences, hot water extract product), was added to water so as to be 5% of the total amount (25 g of yeast residue, 475 g of water, total weight 500 g), and treated at 28°C for 15 hours while stirring (150 rpm) using a stirrer to prepare a suspension. Thereafter, the treatment of diluting to about 32 times the solid content and centrifuging to separate the solid content was repeated 3 times.

[0056] [Washing treatment 2] The solid content separated in washing treatment 1 was added to water so as to be 2%, hydrochloric acid was added to adjust the pH to 6.8, and then the mixture was treated at 28°C for 15 hours while stirring using a stirrer to prepare a suspension. Thereafter, the treatment of diluting to about 107 times the solid content and centrifuging to separate the solid content was repeated 3 times.

[0057] [Dispersion treatment] After adjusting the treatment liquid obtained in washing treatment 2 to a solid content concentration of 1%, a homogenizer treatment (10,000 rpm, 2 hours) was performed to obtain Sample 1 (Table 1).

[0058] Example 2 (KR yeast, NaOH treatment) In addition to KR yeast and water, 1N NaOH was added so as to be 1 / 10 of the total amount in the system (25 g of yeast residue, 425 g of water, 50 g of 1N NaOH, total weight 500 g). Otherwise, the same procedure was carried out, and then washing treatment 2 and dispersion treatment were also carried out in the same manner as in Example 1 to obtain Sample 2 (Table 1).

[0059] Example 3 (GL yeast, water dispersion treatment) Except that GL yeast, which is another yeast residue of Candida utilis (manufactured by Mitsubishi Corporation Life Sciences), was used instead of KR yeast, the same procedure as in Example 1 was carried out to obtain Sample 3 (Table 1).

[0060] Example 4 (GL yeast, NaOH treatment) Except for using the same GL yeast as that used in Example 3 instead of KR yeast, the procedure was the same as in Example 2 to obtain Sample 4 (Table 1).

[0061] Comparative Example 1 (KR yeast, NaClO treatment) Except for adding 1N NaClO so that the chlorine concentration was 0.5% with respect to the liquid in addition to KR yeast and water (50 g of KR yeast, total amount of water and 1N NaClO: 450 g, total amount: 500 g), the procedure was the same. Thereafter, Washing Treatment 2 and Dispersion Treatment were also carried out in the same manner as in Example 1 to obtain Sample 5 (Table 1).

[0062] Comparative Example 2 (GL yeast, NaClO treatment) Except for using the same GL yeast as that used in Example 3 instead of KR yeast, the procedure was the same as in Comparative Example 1 to obtain Sample 6 (Table 1).

[0063] Comparative Example 3 (black yeast) Black yeast β-glucan (a yeast belonging to the genus Aureobasidium, Itochu Foodstuff Co., Ltd., water-soluble β-glucan derived from black yeast, 90% or more, dry product) was suspended in water so that the solid content was 1% and used as Sample 7 (Table 1).

[0064] Comparative Example 4 (bread yeast residue) The residue of baker's yeast (manufactured by Oriental Yeast Co., Ltd., trade name: Zymosan, solid content 1%), which is a yeast belonging to the genus Saccharomyces, was used as Sample 8 (Table 1).

[0065] When the viable cell count in each sample was measured by 1,000-fold dilution and 10,000-fold dilution, Samples 1 to 4 were undetectable at any dilution ratio, Sample 5 was 1 at 1,000-fold dilution and 0 at 10,000-fold dilution, Samples 6 and 7 were 0 at any dilution ratio, and Sample 8 was 10 or less. Thus, it was confirmed that each sample hardly contained viable cells.

[0066]

Table 1

[0067] [Measurement method] -Particle size- The particle size distribution was measured using a Mastersizer 3000 manufactured by Malvern, and the particle sizes (D10, 50, and 90) at cumulative volume of 10%, 50%, or 90% were calculated.

[0068] -Final yield- The final yield was calculated as the weight of the finally recovered yeast residue / the weight of the yeast at the time of charging × 100%.

[0069] -β-glucan content- It was measured using the method described in the β-glucan (β-1,3:1,6 yeast type) assay kit manufactured by Nippon Biotest Laboratories Co., Ltd.

[0070] -Total glucan content- It was measured using the method described in the β-glucan (β-1,3:1,6 yeast type) assay kit manufactured by Nippon Biotest Laboratories Co., Ltd.

[0071] -Total protein amount- The nitrogen content was measured by the Kjeldahl method, and the total protein amount was calculated by multiplying the measured value by a coefficient of 6.25.

[0072] [Evaluation of immunomodulatory effect] The immunomodulatory effect of each sample was evaluated by the IL-10 production induction test in human peripheral blood-derived dendritic cells (PBMC-derived dendritic cells) as follows.

[0073] (1) Preparation of dendritic cells Human PBMC (HemaCare) was washed with Lymphocyte Medium (Zen-bio, LYMPH-1), and CD14-positive cells were separated using anti-CD14-MicroBeads (Miltenyi Biotec). The cell density was 1.0×10 6Suspended in a standard dendritic cell differentiation medium (cDC differentiation medium) containing human recombinant GM-CSF and human recombinant IL-4 at cells / mL and seeded on a 12-well plate. These cells (conventional myeloid cells; cDC) were cultured in a CO 2 incubator at 37 °C for 5 days and then used for co-culture. The purity of cDC was evaluated by flow cytometry.

[0074] (2) Culture of cDC and samples (1) After collection in (1), the cDC were washed with cell culture medium and resuspended in the same medium again to prepare a cell suspension of 2.0×10 6 cells / mL. 100 μL of this was added to each well on a 96-well plate (2.0x10 5 cells / well). Furthermore, 10 μL each of Samples 1 to 8 and control (LPS; lipopolysaccharide 200 ng / mL) were added to make the total volume 200 μL (final concentration of LPS: 10 ng / mL, final solid content concentration of each sample: 30 μg / mL). Two wells of culture were prepared for each sample or control. This was cultured in a CO 2 incubator at 37 °C for 24 hours. After the culture was completed, the plate was centrifuged at 1,500 rpm for 5 minutes to precipitate the cells, and the culture supernatant (about 150 μL) was collected. This was stored at -80 °C and used for ELISA at a later date.

[0075] (3) Measurement of IL-10 IL-10 in the culture supernatant was measured by ELISA. A commercially available kit (IL-10, Human, ELISA Kit, Quantikine, 2 nd Generation, D1000B: R&D systems) was used, and the measurement method followed the attached protocol. The culture supernatant used for ELISA was diluted 10-fold immediately before measurement.

[0076] Also, the concentrations of the standard substances used for making the calibration curve were as follows: IL-10 standard substances: 1000, 500, 250, 125, 62.5, 31.25, 15.6, 7.8, 3.9 pg / mL

[0077] The measurement was performed using a microplate reader (Chromate, Awareness Technology Inc.), and the absorbance at 450 nm / 570 nm was measured. Then, the IL-10 concentration in each sample was calculated based on the calibration curve (Figure 1, Table 2).

[0078]

Table 2

[0079] The IL-10 production amounts of Samples 5 to 8 of Comparative Examples 1 to 4 were less than 400, whereas those of Samples 1 to 4 of Examples 1 to 4, which had a smaller β-glucan content than the comparative examples, exceeded 3,000 (Figure 1, Table 2). This result indicates that the immunomodulator of the present invention can efficiently exhibit a good immunomodulatory effect.

Claims

1. It contains an aqueous dispersion of particles containing yeast residue of Candida yeast, wherein the β-glucan content of the aqueous dispersion is 34% by weight or less, an immunomodulator.

2. The agent according to claim 1, wherein the cumulative volume 50% particle size of the particles is 2.5 μm or more and 5.5 μm or less.

3. The agent according to claim 1 or 2, wherein the cumulative volume 10% particle size of the particles is less than 2.5 μm.

4. The agent according to claim 1 or 2, wherein the cumulative volume 90% particle size of the particles exceeds 4.5 μm.

5. The agent according to claim 1 or 2, wherein the solid content concentration of the aqueous dispersion is 0.01 to 10% by weight.

6. The agent according to claim 1 or 2, wherein the Candida yeast includes Candida utilis.

7. An immunomodulatory food composition containing an aqueous dispersion of particles containing yeast residue of Candida yeast, wherein the β-glucan content of the aqueous dispersion is 34% by weight or less.

8. A first washing step of suspending the yeast residue of Candida yeast in an aqueous solvent, diluting it by 10 times or more, and separating the solid content, and a dispersion step of dispersing the solid content obtained in the washing step in a dispersion medium to obtain an aqueous dispersion of particles containing yeast residue of Candida yeast, a method for producing the agent according to claim 1 or 2.

9. The production method according to claim 8, wherein after the first washing step and before the dispersion step, a second washing step of suspending the solid content separated in the first washing step in water, diluting it by 50 times or more, and separating the solid content is further performed.

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