Immunostimulator and glucan composition production method

Abstract

Provided is an immunostimulator that contains β-glucan derived from yeast cell walls and that has a high immunostimulation effect. This immunostimulator contains β-glucan derived from yeast cell walls, and the percentage content of said β-glucan is 25-45 mass%. The immunostimulator may further contain mannan, and the percentage content of said mannan may be 5-20 mass%.

Description

Method for producing immunostimulant and glucan composition

[0001] The present invention relates to a method for producing an immunostimulant and a glucan composition.

[0002] In recent years, there has been concern about the global spread of drug-resistant bacteria. The World Health Organization (WHO) has pointed to the overuse of antibiotics in livestock as one of the causes, and the livestock industry is seeking alternatives to antibiotics, i.e., solutions to infectious diseases. Improving the body's natural immune function is an effective measure against infectious diseases, and beta-glucan, which is well known for its immunostimulating properties, is expected to help.

[0003] It is generally believed that the higher the degree of purification of yeast-derived glucan materials used as immunostimulants, the greater the immunostimulatory effect (see, for example, Jung-Nam Lee et al., Biosci. Biotechnol. Biochem., 65(4), 837-841, 2001). For example, JP-A 2010-533479 proposes a method for preparing a glucan preparation that combines an enzyme treatment and an acid-alkali hydrolysis treatment, which is said to have a high glucan content and be suitable for industrial use.

[0004] Materials containing yeast-derived β-glucan are required to have a stronger immunostimulating effect. One aspect of the present invention aims to provide an immunostimulating agent that contains β-glucan derived from yeast cell walls and has a strong immunostimulating effect.

[0005] A first aspect is an immunostimulant comprising β-glucan derived from yeast cell walls, the β-glucan content being 25% by mass or more and 45% by mass or less. In one embodiment, the immunostimulant further comprises mannan, and the mannan content in the immunostimulant may be 5% by mass or more and 20% by mass or less, and the ratio of the mannan content to the β-glucan content may be 0.1 or more and less than 1. In one embodiment, the immunostimulant further comprises α-glucan, and the α-glucan content in the immunostimulant may be 7% by mass or more and 12% by mass or less, and the ratio of the α-glucan content to the β-glucan content may be 0.15 or more and 0.55 or less. In one embodiment, the α-glucan comprises glycogen, and the glycogen content in the immunostimulant may be 4% by mass or more and 10% by mass or less, and the ratio of the glycogen content to the β-glucan content may be 0.16 or more and 0.5 or less.

[0006] A second aspect is an interleukin-8 production promoter comprising β-glucan derived from yeast cell walls, the β-glucan content being 25% by mass or more and 45% by mass or less. In one embodiment, the interleukin-8 production promoter further comprises mannan, and the mannan content in the interleukin-8 production promoter may be 5% by mass or more and 20% by mass or less, and the ratio of the mannan content to the β-glucan content may be 0.1 or more and less than 1. In one embodiment, the interleukin-8 production promoter further comprises α-glucan, and the α-glucan content in the interleukin-8 production promoter may be 7% by mass or more and 12% by mass or less, and the ratio of the α-glucan content to the β-glucan content may be 0.15 or more and 0.55 or less. In one embodiment, the α-glucan comprises glycogen, and the glycogen content in the interleukin-8 production promoter may be 4% by mass or more and 10% by mass or less, and the ratio of the glycogen content to the β-glucan content may be 0.16 or more and 0.5 or less.

[0007] A third aspect is a method for producing a glucan composition, comprising washing yeast cell walls under alkaline conditions to obtain a first processed product, heat-treating the first processed product under alkaline conditions to obtain a second processed product, and recovering a third processed product as a solid from the second processed product, wherein the third processed product has a β-glucan content of 25% by mass to 45% by mass. In one embodiment, the heat treatment under alkaline conditions may be carried out under conditions of pH 8 to 14 and 70°C to 110°C. In one embodiment, the yeast cell walls may comprise cell walls derived from brewer's yeast or baker's yeast. In one embodiment, the washing under alkaline conditions may include adjusting the pH of a washing solution containing the yeast cell walls to pH 8 to 14 and performing solid-liquid separation in a centrifuge while adding water. In one embodiment, the third processed product may be recovered by solid-liquid separation of the second processed product by centrifugation. In one embodiment, the produced glucan composition may be an immunostimulant or an interleukin-8 production promoter.

[0008] According to one aspect of the present invention, an immunostimulant containing β-glucan derived from yeast cell walls and having a high immunostimulating effect can be provided.

[0009] 1 is a graph showing the interleukin-8 production promoting effect of the glucan compositions according to Example 1 and Comparative Example 1. FIG. 2 is a graph showing the interleukin-8 production promoting effect of the glucan compositions according to Example 1, Comparative Example 2, and Comparative Example 3.

[0010] As used herein, the term "step" refers not only to an independent step, but also to a step that cannot be clearly distinguished from other steps, as long as the intended purpose of the step is achieved. Furthermore, the content of each component in a composition refers to the total amount of the multiple substances present in the composition, unless otherwise specified, when multiple substances corresponding to each component are present in the composition. Furthermore, the upper and lower limits of the numerical ranges described herein can be arbitrarily selected and combined from the numerical values ​​exemplified as numerical ranges. Hereinafter, embodiments of the present invention will be described in detail. However, the embodiments described below are intended to exemplify methods for producing immunostimulants and glucan compositions in order to embody the technical concept of the present invention, and the present invention is not limited to the methods for producing immunostimulants and glucan compositions shown below.

[0011] Immunostimulant The immunostimulant is a glucan composition containing β-glucan derived from yeast cell walls, and the β-glucan content in the immunostimulant is, for example, 25% by mass or more and 45% by mass or less. The immunostimulant according to this embodiment has an excellent immunostimulating effect due to the inclusion of β-glucan derived from yeast cell walls at a predetermined content. This is expected to not only suppress the onset and progression of infectious diseases in livestock, humans, farmed fish, etc., but also provide a means of reducing the threat of the emergence of drug-resistant bacteria.

[0012] The immunostimulatory effect of an immunostimulator can be evaluated, for example, by the effect of promoting the production of immune-related cytokines such as interleukins. That is, the immunostimulator may be an interleukin production promoter, preferably an interleukin-8 production promoter.

[0013] The β-glucan constituting the immunostimulant is a polymer in which D-glucose units are linked by glycosidic bonds, and has a linear main chain backbone with β-1,3 bonds and side chains branched by β-1,6 bonds. The β-glucan constituting the immunostimulant is preferably derived from, for example, yeast cell walls and obtained by hydrolyzing the yeast cell walls, and may be contained in a glucan composition produced by the method for producing a β-glucan composition described below.

[0014] The β-glucan content in the immunostimulant may preferably be 28% by mass or more, 30% by mass or more, 34% by mass or more, or 35% by mass or more. The β-glucan content in the immunostimulant may preferably be 42% by mass or less, 40% by mass or less, 38% by mass or less, or 35% by mass or less. When the β-glucan content is within the above range, a higher immunostimulatory effect tends to be obtained. Note that the β-glucan content in this specification is a value calculated on a dry matter basis, and the contents of mannan, α-glucan, glycogen, etc. below are also similarly calculated on a dry matter basis.

[0015] The immunostimulant may further contain mannan in addition to β-glucan. When the immunostimulant contains mannan, the mannan content in the immunostimulant may be, for example, 5% by mass or more and 20% by mass or less. The mannan content in the immunostimulant may preferably be 7% by mass or more, 9% by mass or more, or 10% by mass or more, and may preferably be 18% by mass or less, 16% by mass or less, 14% by mass or less, or 12% by mass or less. When the mannan content is within the above range, a higher immunostimulatory effect tends to be obtained.

[0016] From the viewpoint of the effect of promoting interleukin production, the content ratio of mannan to β-glucan in the immunostimulant may be, for example, 0.1 or more and less than 1. The content ratio of mannan to β-glucan in the immunostimulant may be preferably 0.2 or more, 0.25 or more, 0.28 or more, or 0.3 or more, and may also be preferably 0.8 or less, 0.6 or less, 0.4 or less, or 0.35 or less.

[0017] The immunostimulant may further contain α-glucan in addition to β-glucan. When the immunostimulant contains α-glucan, the α-glucan content in the immunostimulant may be, for example, 7% by mass or more and 12% by mass or less. The α-glucan content in the immunostimulant may preferably be 8% by mass or more, 9% by mass or more, or 9.5% by mass or more, and may preferably be 18% by mass or less, 11% by mass or less, 10.5% by mass or less, or 10% by mass or less. When the α-glucan content is within the above range, a higher immunostimulatory effect tends to be obtained.

[0018] From the viewpoint of the effect of promoting interleukin production, the content ratio of α-glucan to β-glucan in the immunostimulant may be, for example, 0.15 or more and 0.55 or less. The content ratio of α-glucan to β-glucan in the immunostimulant may be preferably 0.18 or more, 0.2 or more, 0.24 or more, or 0.26 or more, and may be preferably 0.5 or less, 0.4 or less, 0.3 or less, or 0.28 or less.

[0019] The immunostimulant may contain glycogen as part of the α-glucan. When the immunostimulant contains glycogen, the glycogen content in the immunostimulant may be, for example, 4% by mass or more and 10% by mass or less. The glycogen content in the immunostimulant may preferably be 5% by mass or more, 6% by mass or more, or 6.4% by mass or more, and may preferably be 9% by mass or less, 8% by mass or less, 7% by mass or less, or 6.8% by mass or less. When the glycogen content is within the above range, a higher immunostimulatory effect tends to be obtained.

[0020] From the viewpoint of the effect of promoting interleukin production, the glycogen to β-glucan content ratio in the immunostimulant may be, for example, 0.16 or more and 0.3 or less. The glycogen to β-glucan content ratio in the immunostimulant may be preferably 0.17 or more, 0.18 or more, or 0.182 or more, and may be preferably 0.25 or less, 0.22 or less, 0.2 or less, or 0.19 or less.

[0021] The total content of β-glucan and α-glucan in the immunostimulant may be, for example, 35% by mass or more and 52% by mass or less. The total content of β-glucan and α-glucan in the immunostimulant may be preferably 38% by mass or more, 40% by mass or more, 42% by mass or more, or 44% by mass or more, and may be preferably 50% by mass or less, 48% by mass or less, or 46% by mass or less. When the total content of β-glucan and α-glucan is within the above range, a higher immunostimulatory effect tends to be obtained.

[0022] From the viewpoint of the effect of promoting interleukin production, the ratio of the β-glucan content to the total content of β-glucan and α-glucan (hereinafter also referred to as the total glucan content) in the immunostimulant may be, for example, 0.65 or more and 0.85 or less. The ratio of the β-glucan content to the total glucan content in the immunostimulant may be preferably 0.68 or more, 0.7 or more, 0.72 or more, 0.74 or more, 0.76 or more, or 0.78 or more, and may also be preferably 0.82 or less, 0.8 or less, or 0.79 or less.

[0023] From the viewpoint of the effect of promoting interleukin production, the ratio of the α-glucan content to the total glucan content in the immunostimulant may be, for example, 0.14 or more and 0.35 or less. The ratio of the α-glucan content to the total glucan content in the immunostimulant may be preferably 0.16 or more, 0.18 or more, or 0.2 or more, and may also be preferably 0.3 or less, 0.28 or less, 0.26 or less, 0.24 or less, or 0.22 or less.

[0024] From the viewpoint of the effect of promoting interleukin production, the ratio of the mannan content to the total glucan content in the immunostimulant may be, for example, 0.1 or more and 0.6 or less. The ratio of the mannan content to the total glucan content in the immunostimulant may be preferably 0.14 or more, 0.18 or more, 0.2 or more, or 0.22 or more, and may also be preferably 0.5 or less, 0.4 or less, 0.3 or less, 0.28 or less, or 0.26 or less.

[0025] From the viewpoint of the effect of promoting interleukin production, the ratio of the glycogen content to the total glucan content in the immunostimulant may be, for example, 0.11 or more and 0.3 or less. The ratio of the glycogen content to the total glucan content in the immunostimulant may be preferably 0.11 or more, 0.12 or more, 0.13 or more, or 0.14 or more, and may also be preferably 0.2 or less, 0.18% or less, 0.16 or less, or 0.15 or less.

[0026] The immunostimulant can exhibit high immunostimulating activity despite the relatively low degree of purification of β-glucan. This is thought to be because, for example, by undergoing only a low-strength extraction treatment under weakly alkaline conditions, the active ingredient is effectively concentrated without excessively destroying the three-dimensional structure of the yeast cell wall, and the morphology necessary and sufficient for immune response is maintained. Alternatively, this can be thought to be because the immunostimulant contains a glucan composition produced by the method for producing a glucan composition described below.

[0027] The immunostimulant may have a total carbohydrate content of, for example, 60% by mass or more and 70% by mass or less, and preferably 62% by mass or more and 66% by mass or less. Carbohydrates include the above-mentioned β-glucan, α-glucan, mannan, etc. From the viewpoint of the interleukin production-promoting effect, the β-glucan content relative to the total carbohydrate content may be, for example, 35% by mass or more and 70% by mass or less, preferably 45% by mass or more or 50% by mass or more, and preferably 65% ​​by mass or less or 60% by mass or less.

[0028] The immunostimulant may contain a protein. The protein content in the immunostimulant may be, for example, 15% by mass or more and 25% by mass or less, and preferably 18% by mass or more and 22% by mass or less. From the viewpoint of the effect of promoting interleukin production, the ratio of the protein content to the β-glucan content in the immunostimulant may be, for example, 0.38 to 0.1 or less, preferably 0.45 or more or 0.5 or more, and preferably 0.8 or less or 0.6 or less.

[0029] The immunostimulant may contain a lipid. The lipid content in the immunostimulant may be, for example, 10% by mass or more and 18% by mass or less, and preferably 12% by mass or more and 16% by mass or less. From the viewpoint of the effect of promoting interleukin production, the ratio of the lipid content to the β-glucan content in the immunostimulant may be, for example, 0.38 to 0.4 or less, preferably 0.39 or more, and preferably 0.4 or less.

[0030] The immunostimulant may contain ash. The ash content in the immunostimulant may be, for example, 1.8% by mass or more and 3% by mass or less, preferably 1.9% by mass or more and 2.2% by mass or less. From the viewpoint of the effect of promoting interleukin production, the ratio of the ash content to the β-glucan content in the immunostimulant may be, for example, 0.04 or more and 0.065 or less, preferably 0.05 or more and preferably 0.06 or less.

[0031] The immunostimulant may contain a water-insoluble component, and the water-insoluble component may include an active ingredient. The immunostimulant may be substantially composed of a water-insoluble component. Here, "substantially" means that water-soluble components that are inevitably mixed in are not excluded, and the content of the water-soluble component is, for example, less than 5% by mass or less than 1% by mass. The immunostimulant may also contain a water-insoluble component and a water-soluble component. When the immunostimulant contains a water-soluble component, the content thereof may be, for example, 1% by mass or more but less than 50% by mass, preferably 40% by mass or less or 20% by mass or less, and preferably 5% by mass or more. In this specification, "water-insoluble" means that the solubility in 100 g of water at 25°C is less than 1 g, and "water-soluble" means that the solubility in 100 g of water at 25°C is 1 g or more. The immunostimulant may, for example, contain a third processed product obtained by the method for producing a glucan composition described below, or may consist of the third processed product.

[0032] The immunostimulant may further contain, in addition to the main component β-glucan, known components having immunomodulatory effects, such as sweet tea extract, fucoidan, arabinoxylan, lactoferrin, catechin, chitosan, chitosan oligosaccharide, chitin oligosaccharide, L-ascorbic acid, and coenzyme Q10 (including reduced form).

[0033] The immunostimulant may be blended with any component known to be normally used in pharmaceuticals, foods, feeds, etc., as needed, such as water, oils and fats, sugars, vitamins, sweeteners, seasonings, acidulants, preservatives, fragrances, colorants, excipients, bulking agents, binders, thickeners, stabilizers, emulsifiers, pH adjusters, etc.

[0034] The immunostimulant may be in any form, such as a solid (e.g., powder or granules), a liquid, or a paste. The dosage form of the immunostimulant can be appropriately selected depending on the administration method, the recipient, and the like. Examples of dosage forms include oral administration preparations such as tablets, capsules, granules, lozenges, powders, and liquids. These preparations can be produced according to known methods using additives such as excipients, lubricants, binders, disintegrants, stabilizers, flavorings, and diluents. Furthermore, the immunostimulant can also be incorporated into food compositions, such as health foods and health supplements, beverage compositions, feeds, and the like.

[0035] When administered to a target animal, such as a vertebrate or invertebrate, an immunostimulatory effect is exerted in the target. This can suppress the onset of an infectious disease in the target. That is, the immunostimulator can be used in a method for preventing an infectious disease in a target. Examples of vertebrates to which the immunostimulatory agent can be administered include mammals, birds, fish, and the like. Mammals may include humans, as well as non-human mammals. Non-human mammals include pigs, cows, horses, sheep, monkeys, and pet animals such as dogs and cats. Examples of birds include chickens, laying hens, turkeys, ducks, pigeons, and the like. Examples of fish include salmonids such as salmon, trout, yamame trout, char, and huchen, as well as carp, crucian carp, tilapia, catfish, sea bass, yellowtail, amberjack, yellowtail, flounder, sea bream, tuna, and eel. Examples of invertebrates to which the present invention can be administered include arthropods such as crabs and shrimp, echinoderms such as sea urchins and sea cucumbers, mollusks such as octopus, squid and shellfish, and protochordates such as sea squirts.

[0036] When the immunostimulant is administered as a pharmaceutical product or food or drink, it can be orally administered, for example, in an amount of 1 mg to 500 mg (dry weight) per kg body weight once to several times per day. When used as a food or drink, feed, or the like, it can be added in an amount of 0.001 g to 1 g per 100 g of the food or drink, feed, or the like.

[0037] When the immunostimulant is administered as feed, for example, to mammals, birds, and fish, it can be mixed in an amount of 0.001% by mass to 1% by mass of the feed to be administered, and administered once to several times a day.

[0038] 1. Method for Producing Glucan Composition The method for producing a glucan composition includes a washing step of washing yeast cell walls under alkaline conditions to obtain a first processed product, a heat treatment step of heat treating the first processed product under alkaline conditions to obtain a second processed product, and a recovery step of recovering a third processed product as a solid from the second processed product. The resulting third processed product has a β-glucan content of 25% by mass or more and 45% by mass or less and has an excellent immunostimulatory effect. The third processed product also has an excellent interleukin production-promoting effect, particularly an excellent effect of promoting interleukin-8 production.

[0039] Washing yeast cell walls under alkaline conditions followed by hydrolysis under alkaline conditions allows for efficient production of glucan compositions with excellent immunostimulatory effects, which can be attributed to the efficient concentration of components that contribute to the immunostimulatory effects during the washing process.

[0040] The yeast cell walls used in the method for producing a glucan composition may be commercially available or may be obtained by pretreating the yeast as a raw material. The pretreatment step for pretreating the yeast as a raw material may include, for example, a pre-decomposition step, such as an autolysis step in which the yeast is autolyzed to obtain an autolysate, a hot water extraction step in which the yeast is extracted with hot water to obtain a hot water extract, or an enzyme treatment step in which the yeast is enzymatically treated to obtain an enzyme-treated product, alone or in combination, and a separation step in which the pre-decomposition product obtained by the pre-decomposition step is centrifuged to obtain the yeast cell walls.

[0041] Examples of yeasts that can be used as raw materials include yeasts of the genus Saccharomyces, Schizosaccharomyces, Kluyveromyces, Candida, Pichia, and Torulopsis. It is preferable to include at least one species selected from the group consisting of these, and more preferably to include at least a yeast of the genus Saccharomyces. Examples of yeasts that can be used as raw materials include food yeasts such as beer yeast and baker's yeast. Examples of food yeasts include brewer's yeast, beer yeast, miscellaneous alcoholic beverage yeast, sake yeast, wine yeast, whiskey yeast, and other brewer's yeasts, baker's yeast, and Torula yeast.

[0042] In the autolysis step, yeast proteins and the like are decomposed by proteases and the like contained in the yeast itself to obtain an autolysate. The autolysis step is carried out, for example, by adjusting the pH to 2 to 7 and the temperature to 40°C to 65°C for 1 hour to 48 hours. In the separation step, the autolysate is centrifuged to obtain a fraction containing yeast cell walls as a heavy liquid. For example, in industrial production, a nozzle-type centrifuge, an intermittent discharge centrifuge, a Sharpless centrifuge, or the like can be used for centrifugation, and the centrifugation conditions can be appropriately adjusted as long as the insoluble components in the autolysate can be recovered.

[0043] Washing Step In the washing step, the yeast cell walls are washed under alkaline conditions to obtain a first treated product. The washing method may be any method capable of removing at least a portion of the proteins, amino acids, etc. remaining on the yeast cell walls. The washing method may, for example, include mixing the yeast cell walls with a liquid medium to prepare a washing solution containing the yeast cell walls, and removing at least a portion of the liquid medium from the washing solution. The liquid medium may contain at least water and an alkaline substance. The method for removing the liquid medium from the washing solution may be appropriately selected from commonly used solid-liquid separation methods. Examples of solid-liquid separation methods include centrifugation using a yeast separator, etc., and methods using a filter, ultrafiltration membrane, etc.

[0044] Washing of yeast cell walls may include adjusting the pH of a washing solution containing yeast cell walls to between pH 8 and pH 14, preferably between pH 9.5 and pH 12, and performing solid-liquid separation using a centrifuge (e.g., a yeast separator) while adding water. The washing solution may contain at least water as a liquid medium. The washing solution containing yeast cell walls can be adjusted to a desired pH by adding an alkaline substance to the washing solution. Examples of alkaline substances include alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, alkaline earth metal hydroxides such as calcium hydroxide, and alkali metal carbonates such as sodium carbonate. The alkaline substance may be used as an aqueous solution to adjust the pH.

[0045] The solid-liquid separation in washing the yeast cell walls may include centrifugation while adding water. The water may be added using water alone or an alkaline aqueous solution containing an alkaline substance. The alkaline aqueous solution may have a pH of, for example, 8 to 14, preferably 9.5 to 12.

[0046] The washing temperature in the washing step may be, for example, 4° C. or higher and 95° C. or lower, and preferably may be within a temperature range of 20° C. or higher and 50° C. or lower. Furthermore, washing may be carried out, for example, until the liquid obtained by solid-liquid separation by centrifugation becomes colorless or light-colored, or transparent.

[0047] The first treated product obtained in the washing step may be water-insoluble or may be a water-insoluble solid, or may be in the form of a slurry containing yeast cell walls, which are water-insoluble solids.

[0048] Heat Treatment Step In the heat treatment step, the first treated product is heat-treated under alkaline conditions to obtain a second treated product. The heat treatment under alkaline conditions may be alkaline hydrolysis of the first treated product. The heat treatment step may include obtaining a mixture containing the first treated product, an alkaline substance, and a liquid medium, heating the mixture to a predetermined temperature, and maintaining the mixture at the predetermined temperature for a predetermined time. In the heat treatment step, the mixture may be stirred or left to stand. Furthermore, when maintaining the mixture at the predetermined temperature, a temperature fluctuation of, for example, ±10°C, preferably about ±5°C, is permitted.

[0049] The alkaline conditions for heat-treating the first-processed product may be, for example, pH 8 to 14, preferably pH 9 or higher, and preferably pH 12 or lower. Heat-treating under alkaline conditions with a relatively low pH tends to produce a glucan composition with a more excellent immunostimulatory effect. For example, when the first-processed product is a slurry containing yeast cell walls, the pH of the first-processed product can be adjusted to the desired pH by adding the alkaline substance described above to the slurry. The addition method may be batchwise or continuous. The amount of alkaline substance added to adjust the pH to the above range may be, for example, 0.01% by mass to 1% by mass, preferably 0.05% by mass or higher, and preferably 0.5% by mass or lower, relative to the mixture.

[0050] The temperature of the heat treatment may be, for example, 60° C. or higher and 120° C. or lower, preferably 70° C. or higher or 75° C. or higher, and preferably 110° C. or lower or 95° C. or lower. The time of the heat treatment may be, for example, 3 hours or higher and 24 hours or lower, preferably 12 hours or higher and preferably 20 hours or lower. The atmosphere of the heat treatment may be, for example, air.

[0051] The second treated product obtained in the heat treatment step may be in the form of a slurry containing water-insoluble solids, for example, and may have a solid content of 10% by mass or more and 20% by mass or less.

[0052] Recovery Step In the recovery step, a third processed product containing solids is recovered from the second processed product obtained in the heat treatment step. The second processed product to be subjected to the recovery step may be neutralized or may be in an alkaline state. The recovery of solids from the second processed product can be carried out by subjecting the second processed product to solid-liquid separation. For solid-liquid separation, for example, centrifugation, a method using a filter, an ultrafiltration membrane, etc. can be used. For centrifugation, a yeast separator, a nozzle-type centrifuge, an intermittent discharge centrifuge, a Sharpless centrifuge, etc. can be used.

[0053] The third processed product may be recovered as a wet cake containing water or as a slurry containing solids. The solid content of the third processed product may be, for example, 5% by mass or more, preferably 10% by mass or more, or more than 20% by mass.

[0054] The method for producing a glucan composition may further include a drying step of drying the third-treated product. A spray dryer, freeze dryer, drum dryer, or the like may be used for the drying step. The method for producing a glucan composition may also include a sterilization step of sterilizing the third-treated product before the drying step. The sterilization step can be carried out, for example, by heat treatment at 120°C or higher for 10 to 60 seconds using a UHT sterilizer, but this can be adjusted appropriately to meet the desired quality.

[0055] The glucan composition obtained by the method for producing a glucan composition has an excellent immunostimulating effect and may therefore be an immunostimulator.Furthermore, the glucan composition has an excellent interleukin production promoting effect and may therefore be an interleukin production promoter, preferably an interleukin-8 production promoter.

[0056] The method for producing a glucan composition may also include obtaining a yeast-derived water-soluble polysaccharide in addition to obtaining the glucan composition. The yeast-derived water-soluble polysaccharide can be recovered from the supernatant solution separated from the second processed product. Specifically, the yeast-derived water-soluble polysaccharide can be obtained by a method including heat-treating the supernatant solution under acidic conditions, removing the coagulated product formed by the heat treatment, and drying the solution from which the coagulated product has been removed.

[0057] The resulting yeast-derived water-soluble polysaccharide has excellent water solubility, low viscosity in an aqueous solution, and a high mannan content. That is, the method for producing a glucan composition may include the method for producing a mannan composition. The mannan content in the yeast-derived water-soluble polysaccharide may be, for example, 40% by mass or more and 80% by mass or less.

[0058] The heat treatment of the supernatant solution under acidic conditions may be carried out under acidic conditions, for example, at a pH of 2.5 to 5, preferably 3.5 to 4.5. The heat treatment temperature may be, for example, 90° C. to 120° C., preferably 95° C. to 115° C. The heat treatment time may be, for example, 15 to 120 seconds, preferably 30 to 90 seconds.

[0059] The coagulum formed by the heat treatment can be removed by, for example, centrifugation, diatomaceous earth filtration, or ultrafiltration membrane, and the solution from which the coagulum has been removed can be dried by a spray dryer, freeze dryer, drum dryer, or the like.

[0060] The immunostimulating method may include administering an immunostimulating agent containing the glucan composition described above to a subject. The subject may be a vertebrate, an invertebrate, or the like. Details of the subject and the dosage are as described above.

[0061] Other aspects of the present invention include the use of a glucan composition in the production of an immunostimulant used in an immunostimulating method, the use of a glucan composition in an immunostimulating method, and a glucan composition for use in an immunostimulating method.

[0062] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

[0063] Example 1 Yeast cell walls (solid content 15%, derived from yeast autolysate) derived from brewer's yeast belonging to the genus Saccharomyces, produced at the Tochigi Koganei Plant of Asahi Group Foods Co., Ltd., were prepared, and a yeast glucan composition was obtained according to the following method.

[0064] Using a yeast separator, the yeast cell walls were washed under alkaline conditions while adding water and sodium hydroxide, yielding a first processed product with a pH adjusted to 10 to 11. The mixture was heat-treated at a temperature of 80°C to 90°C for 17 hours to yield a second processed product. The resulting second processed product was centrifuged to yield a third processed product as a heavy liquid. The resulting third processed product was dried in a drum dryer to yield the glucan composition (YG35) of Example 1.

[0065] Comparative Example 1 The yeast cell walls prepared in Example 1 were dried to prepare a glucan composition (YCW) of Comparative Example 1.

[0066] Comparative Example 2 The glucan composition (YG35) of Example 1 was purified with protease and then freeze-dried to give the glucan composition (YG45) of Comparative Example 2.

[0067] Comparative Example 3 The glucan composition (YG45) of Comparative Example 2 was purified using an organic solvent to remove lipids, and then freeze-dried to give the glucan composition (YG55) of Comparative Example 3.

[0068] The glucan composition obtained above was subjected to component analysis. The results are shown in Table 1. In Table 1, the contents of β-glucan, α-glucan, and mannan are the contents as part of carbohydrates relative to the entire glucan composition, and the glycogen content is the content as part of α-glucan relative to the entire glucan composition.

[0069]

[0070] Each of the glucan compositions obtained above was ultrasonically treated for 3 minutes while ice-cooling to obtain a dispersion. Porcine-derived pepsin was added to each of the obtained dispersions, and the dispersions were treated with pepsin at 39°C for 2 hours. Porcine-derived pancreatin was then added, and the dispersions were treated with pancreatin at 39°C for 4 hours to prepare β-glucan samples.

[0071] (1) Isolation of neutrophils Peripheral blood was collected from the jugular vein of six 10-week-old weaned piglets into heparinized tubes. Neutrophils were then separated from lymphocytes, monocytes, platelets, etc. by discontinuous density gradient centrifugation using 68% and 75% Percoll solutions. Erythrocytes were lysed with 74.7% ammonium chloride, and centrifuged at 350 × g at 18 °C for 10 minutes. The precipitate was washed three times with RPMI 1640 medium and then separated into 100 ml of ... ６ The cells were suspended at 1000 cells / ml.

[0072] (2) Measurement of IL-8 production amount Isolated neutrophils were suspended in RPMI 1640 medium, and 2 × 10 ５ Similarly, peripheral blood mononuclear cells (PBMCs) were seeded at 2 × 10 cells / well in a 24-well multiwell plate. ７ The cells were seeded at 100 cells / well. ２ After incubation at 37°C for 2 hours, neutrophils or monocytes from PBMCs were allowed to adhere, and the supernatant was removed. Each β-glucan sample was added to a concentration of 20 μg / ml, and incubation was continued for 24 hours. The supernatant was then collected and the amount of IL-8 produced was measured using a commercially available ELISA kit.

[0073] Figure 1 shows the results of using the glucan compositions of Example 1 and Comparative Example 1. A significant difference (p<0.01) was observed by t-test. Figure 2 shows the results of using the glucan compositions of Example 1 and Comparative Examples 2 and 3. A one-way analysis of variance was performed, and a significant difference was observed overall (p<0.05). Therefore, a multiple comparison of all groups using the Bonferroni method was performed, and a significant difference (p<0.01) was observed between Example 1 and Comparative Example 2, and a significant difference (p<0.05) was observed between Example 1 and Comparative Example 3.

[0074] From the above, it is clear that the glucan composition (YG35) containing approximately 35% by mass of β-glucan exhibits a high immunostimulating effect. It is also clear that the glucan composition (YG35) exhibits a significantly higher immunostimulating effect than the more purified glucan compositions (YG45) and (YG55).

[0075] The disclosure of Japanese Patent Application No. 2021-086394 (filing date: May 21, 2021) is incorporated herein by reference in its entirety. All documents, patent applications, and technical standards described herein are incorporated herein by reference to the same extent as if each individual document, patent application, and technical standard was specifically and individually indicated to be incorporated by reference.

Claims

1. An immunostimulant comprising β-glucan derived from yeast cell walls, the β-glucan content being 25% by mass or more and 45% by mass or less.

2. The immunostimulant according to claim 1, further comprising mannan, the content of the mannan in the immunostimulant being 5% by mass or more and 20% by mass or less.

3. The immunostimulant according to claim 2, wherein the content ratio of said mannan to said β-glucan is 0.1 or more and less than 1.

4. The immunostimulant according to claim 1, further comprising α-glucan, the content of the α-glucan in the immunostimulant being 7% by mass or more and 12% by mass or less.

5. The immunostimulant according to claim 4, wherein the content ratio of said α-glucan to said β-glucan is 0.15 or more and 0.55 or less.

6. The immunostimulant according to claim 4 or 5, wherein the α-glucan contains glycogen, and the content of the glycogen in the immunostimulant is 4% by mass or more and 10% by mass or less.

7. The immunostimulant according to claim 6, wherein the content ratio of said glycogen to said β-glucan is 0.16 or more and 0.5 or less.

8. An interleukin-8 production promoter which contains β-glucan derived from yeast cell walls and has a β-glucan content of 25% by mass or more and 45% by mass or less.

9. The method includes washing yeast cell walls under alkaline conditions to obtain a first treated product, heat treating the first treated product under alkaline conditions to obtain a second treated product, and recovering a third treated product as a solid content from the second treated product, A method for producing a glucan composition, wherein the third processed product has a β-glucan content of 25% by mass or more and 45% by mass or less.

10. The method according to claim 9, wherein the heat treatment under alkaline conditions is carried out under conditions of pH 8 to 14 and 70° C. to 110° C.

11. The method according to claim 9 , wherein the yeast cell walls comprise cell walls derived from brewer's yeast or baker's yeast.

12. The method according to claim 9, wherein the washing under alkaline conditions comprises adjusting the pH of the washing solution containing the yeast cell walls to a value between 8 and 14, and performing solid-liquid separation using a centrifuge while adding water.

13. The method according to claim 9 , wherein the third processed product is recovered by subjecting the second processed product to solid-liquid separation by centrifugation.

14. The method according to any one of claims 9 to 13, wherein the glucan composition is an immunostimulant.

15. The method according to any one of claims 9 to 13, wherein the glucan composition is an interleukin 8 production promoter.