阅读说明：本技术 免疫刺激剂及预防感染的方法 (Immunostimulant and method for preventing infection ) 是由 白井建史 吉田淳平 于 2019-09-05 设计创作，主要内容包括：本发明提供含有葡聚糖的具有免疫刺激效果的免疫刺激剂。免疫刺激剂为水不溶性,其包含源自酵母细胞壁的葡聚糖和脂质,且葡聚糖和脂质的总含有率为80质量％以上,脂质相对于葡聚糖的含有比为0.1以上且0.4以下。(The present invention provides an immunostimulant having an immunostimulating effect, which contains glucan. The immunostimulant is water-insoluble, and comprises glucan derived from yeast cell wall and lipid, wherein the total content of glucan and lipid is 80% by mass or more, and the content ratio of lipid to glucan is 0.1 to 0.4.)

1. An immunostimulant which is water-insoluble and comprises glucan derived from a yeast cell wall and a lipid, wherein the total content of glucan and lipid is 80% by mass or more, and the content ratio of lipid to glucan is 0.1 to 0.4.

2. The immunostimulant according to claim 1, wherein the content ratio of α -glucan to β -glucan is 0.2 or more and 0.85 or less.

3. The immunostimulant according to claim 1 or 2, which comprises mannan, and the content of mannan is 5% by mass or less.

4. The immunostimulant according to any one of claims 1 to 3, which is used for fish.

5. A drink, food or feed comprising the immunostimulant according to any one of claims 1 to 4.

6. An agent for preventing infection in fish, which comprises glucan and lipid and has a total glucan and lipid content of 80% by mass or more.

7. A method of preventing an infection comprising administering the immunostimulant of any one of claims 1 to 4 to a subject.

8. A method for producing yeast-derived glucan, comprising:

preparing an autolysed composition comprising yeast cell walls;

subjecting the composition comprising yeast cell walls to an alkaline hydrolysis treatment to obtain a hydrolysate; and

recovering glucan from the hydrolysate.

9. The production method according to claim 8, wherein the composition comprising yeast cell walls is obtained by a method comprising subjecting yeast to an autolysis treatment to obtain an autolysate, and subjecting the autolysate to a centrifugal separation treatment.

10. The production method according to claim 8 or 9, wherein the alkali hydrolysis treatment is carried out by heating in the presence of an alkali metal hydroxide.

11. The production method according to any one of claims 8 to 10, wherein the content of mannan of the yeast-derived glucan is 5% by mass or less.

12. The production method according to any one of claims 8 to 11, wherein the content of the protein in the yeast-derived glucan is 10% by mass or less.

13. The production method according to any one of claims 8 to 12, wherein the coefficient of variation of the glucan content of the yeast-derived glucan is 10% or less.

Technical Field

The present invention relates to immunostimulants and methods of preventing infection.

Background

In recent years, there is a fear that drug-resistant bacteria spread all over the world. The World Health Organization (WHO) points out that the abuse of antibiotics for livestock is one of the causes, and the animal industry world is seeking alternatives to antibiotics, i.e., solutions to infections. Enhancement of the immune function originally possessed by a living body is effective as a countermeasure against infection, and is expected as β -glucan having a high material recognition degree and an immunostimulating effect.

In connection with the above, a method of treating yeast cell walls to obtain dimethyl sulfoxide-soluble β -glucan is described in Japanese patent application laid-open No. 2017-511122. Japanese patent application laid-open No. 2009-263333 describes a composition comprising selenium-fortified yeast and beta-glucan, which is thought to have an immunostimulating effect. Japanese patent application laid-open No. 2004-099580 discloses an immunopotentiating composition prepared by subjecting yeast cells to an autolysis treatment. Japanese patent laid-open No. 2003-169607 discloses a fish/shellfish feeding feed to which a yeast decomposition composition obtained by autolyzing yeast is added, and it is considered that the immunological activity is improved.

Summary of The Invention

Problems to be solved by the invention

Materials containing β -glucan are widely distributed in the market, but have problems that the quality varies greatly depending on products, and that many products cannot guarantee functionality. Accordingly, an object of one embodiment of the present invention is to provide an immunostimulant containing glucan and having an immunostimulating effect.

Means for solving the problems

The first embodiment of the present invention is an immunostimulant which is water-insoluble and contains glucan derived from yeast cell walls and lipid, and the total content of glucan and lipid is 80 mass% or more, and the content ratio of lipid to glucan is 0.1 or more and 0.4 or less. In one embodiment, the content ratio of α -glucan to β -glucan may be 0.2 or more and 0.85 or less. In one embodiment, the immunostimulant comprises mannan, and the content of mannan may be 5% by mass or less. In one embodiment, the fish may be administered an immunostimulant.

The second embodiment is a beverage, food or feed comprising the above immunostimulant. The third embodiment is an agent for preventing infection in fish, which comprises glucan and lipid and has a total glucan and lipid content of 80 mass% or more. A fourth embodiment is a method of preventing an infection comprising administering the above-described immunostimulant to a subject.

The fifth embodiment is a method for producing yeast-derived glucan, comprising: preparing an autolysed composition comprising yeast cell walls, subjecting the composition comprising yeast cell walls to an alkaline hydrolysis treatment to obtain a hydrolysate, and recovering the glucan from the hydrolysate.

In one embodiment, the composition comprising yeast cell walls is obtained by a method comprising subjecting yeast to an autolysis treatment to obtain an autolysate, and subjecting the autolysate to a centrifugation treatment. In one embodiment, the alkaline hydrolysis treatment is carried out by heating in the presence of an alkali metal hydroxide. In one embodiment, the content of mannan in yeast-derived glucan is 5% by mass or less. In one embodiment, the content of the yeast-derived glucan protein is 10% by mass or less. In one embodiment, the coefficient of variation of the glucan content of yeast-derived glucan is 10% or less.

Effects of the invention

According to an embodiment of the present invention, an immunostimulant containing glucan and having an immunostimulating effect can be provided.

Brief Description of Drawings

FIG. 1 is a graph showing the ability to promote the production of reactive oxygen species from porcine leukocytes.

FIG. 2 is a graph showing the ability to promote the production of reactive oxygen species by human neutrophil-like cells.

FIG. 3 is a graph showing the survival rate of vibriosis infection test for rainbow trout.

Detailed Description

In the present specification, the term "step" does not refer to an independent step, but is included in the present word as long as the intended purpose of the step is achieved even in the case where it cannot be clearly distinguished from other steps. In addition, the content of each component in the composition means a total amount of a plurality of substances present in the composition in the case where the plurality of substances corresponding to each component is present in the composition, unless otherwise specified. Further, the content of each component is a value converted from a dry matter of the immunostimulant, and may be an average value. The reason why the content is set as an average value here is to take into account the difference between the preparation lots, and is, for example, an arithmetic average value of arbitrarily selected 6 or more samples. The upper limit of the number of samples for calculating the average value is, for example, 20 or less. Embodiments of the present invention are described in detail below. However, the embodiments shown below are exemplified to embody the technical idea of the present invention, and the present invention is not limited to the immunostimulants and the like shown below.

Immunostimulant

The immunostimulant comprises dextran and lipid, and the total content of dextran and lipid is 80% by mass or more. The immunostimulant of the present embodiment has good quality stability and a definite immunostimulating effect. Thus, it is expected to provide a method for reducing the threat of drug-resistant bacteria, as well as inhibiting the development and progression of infections in livestock, humans, farmed fish, and the like. Preferably, the glucans and lipids constituting the immunostimulant are, for example, those derived from yeast cell walls obtained by hydrolytic treatment of yeast cell walls. The hydrolysis treatment comprises alkaline hydrolysis and acid hydrolysis.

The glucan constituting the immunostimulant is a polymer in which D-glucose is linked by glycosidic bonds, and includes β -glucan and α -glucan. The β -glucan has, for example, a linear main chain skeleton of β -1,3 linkages and branched side chains of β -1,6 linkages. The α -glucan has, for example, a linear main chain skeleton of α -1,4 linkages and branched side chains of α -1,6 linkages.

The total content of glucan in the immunostimulant is, for example, 60% by mass or more, preferably 65% by mass or more, more preferably 70% by mass or more, and is, for example, 90% by mass or less, preferably 85% by mass or less, more preferably 80% by mass or less.

The content of β -glucan in the immunostimulant is, for example, 30% by mass or more, preferably 35% by mass or more or 40% by mass or more, and for example 65% by mass or less, preferably 60% by mass or less or 50% by mass or less. The content of α -glucan is, for example, 10 mass% or more, preferably 15 mass% or more or 20 mass% or more, and is, for example, 40 mass% or less, preferably 35 mass% or less or 30 mass% or less.

The ratio of the α -glucan content to the β -glucan content in the immunostimulant is, for example, 0.2 or more and 0.85 or less, preferably 0.25 or more, 0.3 or more, 0.4 or more, 0.45 or more, or 0.5 or more, and preferably 0.8 or less, 0.7 or less, or 0.6 or less. When the content ratio of α -glucan to β -glucan is in the above range, the immunostimulatory activity tends to be further enhanced.

The lipid in the immunostimulant comprises simple lipid formed by ester bonding of alcohol and fatty acid; complex lipids that are lipids containing phosphate, sugar, protein, and the like in the molecule; and derived lipids derived from simple lipids or complex lipids by hydrolysis. The total content of the lipid in the immunostimulant is, for example, 3% by mass or more, preferably 5% by mass or more or 10% by mass or more, and for example 20% by mass or less, preferably 16% by mass or less or 14% by mass or less. When the content of the lipid is within the above range, the immunostimulatory activity tends to be further enhanced.

The total content of glucan and lipid in the immunostimulant is 80% by mass or more, preferably 82% by mass or more. And is, for example, 95% by mass or less, preferably 90% by mass or less or 88% by mass or less. When the total content of glucan and lipid is within the above range, the immunostimulatory activity tends to be further enhanced.

The ratio of the total content of lipids in the immunostimulant to the total content of glucan is, for example, 0.1 or more and 0.4 or less, preferably 0.12 or more or 0.15 or more, and, for example, 0.3 or less, preferably 0.25 or less or 0.2 or less. When the content ratio of the lipid to the glucan is in the above range, the immunostimulatory activity tends to be further enhanced.

The immunostimulant may also further comprise mannan in addition to the glucan and lipid. Mannan is a polysaccharide having D-mannose as a main structural unit. In the case where the immunostimulant comprises mannan, the content of mannan is, for example, 5% by mass or less, preferably 3% by mass or less or 1.2% by mass or less, and is, for example, 0.1% by mass or more, preferably 0.2% by mass or more or 0.3% by mass or more. When the content of mannan is within the above range, the immunostimulatory activity tends to be further enhanced. The ratio of the total content of mannan to the total content of glucan in the immunostimulant is, for example, 0.004 or more and 0.05 or less, preferably 0.005 or more or 0.008 or more, and preferably 0.02 or less or 0.018 or less.

The immunostimulant may also further comprise a protein in addition to the glucan and the lipid. In the case where the immunostimulant comprises a protein, the content of the protein is, for example, 10% by mass or less, preferably 8% by mass or less or 6% by mass or less, and is, for example, 1% by mass or more, preferably 2% by mass or more or 3% by mass or more. When the content of the protein is within the above range, the immunostimulatory activity tends to be further enhanced.

The ratio of the total content of mannan and protein in the immunostimulant to the total content of glucan is, for example, 0.01 or more and 0.2 or less, preferably 0.02 or more or 0.05 or more, and preferably 0.1 or less or 0.09 or less.

The immunostimulant may also further comprise ash. In the case where the immunostimulant contains ash, the content of ash is, for example, 10 mass% or less, preferably 6 mass% or less or 4 mass% or less, and is, for example, 1 mass% or more, preferably 1.5 mass% or more or 2 mass% or more. When the ash content is within the above range, the immunostimulating activity tends to be further enhanced.

The content of each component of the immunostimulant can be determined by conventional methods. For example, the content of glucan can be measured by quantifying glucose produced by hydrolyzing glucan. The content of lipid can be determined by an acid decomposition method.

The immunostimulant has excellent quality stability. The stability of the quality of the immunostimulant can be evaluated, for example, by the variation in the content of each component due to the difference in the preparation lot. Specifically, the evaluation was performed by the coefficient of variation (%) of the content of each component. The coefficient of variation (%) is calculated as a percentage of a value obtained by dividing the standard deviation by the arithmetic mean by measuring the content of each component of the immunostimulant for an arbitrarily selected 6 or more samples and calculating the arithmetic mean and standard deviation of the content. The coefficient of variation (%) in the total glucan content in the immunostimulant is, for example, 10% or less, preferably 8% or less than 5%. The coefficient of variation (%) in the lipid content is, for example, 20% or less, preferably 15% or less or 8% or less. Further, the coefficient of variation (%) of the total content of glucan and lipid is, for example, 6% or less, preferably 5% or less than 5%. The lower limit of the coefficient of variation is 0%, but it is substantially greater than 0%.

The immunostimulant may further comprise a known component having an immunomodulating effect in addition to the glucan and the lipid as main componentsAnd (4) dividing. Examples of the component having an immunoregulatory effect include: sweet tea extract, fucoidin, arabinoxylan, lactoferrin, catechin, chitosan oligosaccharide, chitin oligosaccharide, L-ascorbic acid, and coenzyme Q₁₀(including reduced forms), and the like.

The immunostimulant may contain, as necessary, any component generally known to be used in medicines, foods, feeds, and the like, for example, water, fats and oils, saccharides, vitamins, sweeteners, seasonings, acidulants, preservatives, flavors, pigments, excipients, extenders, binders, thickeners, stabilizers, emulsifiers, pH adjusters, and the like.

The immunostimulant may be in the form of a solid such as powder or granule, a liquid, or a paste. The formulation of the immunostimulant may be appropriately selected depending on the administration method, the subject to be administered, and the like. Examples of the dosage form include orally administered preparations such as tablets, capsules, granules, dragees, powders, and liquids. These preparations can be prepared by a known method using additives such as excipients, lubricants, binders, disintegrants, stabilizers, flavoring agents, and diluents. Further, the immunostimulant may be contained to constitute a food composition such as a health food or health supplementary food, a drink composition, a feed, or the like.

When an immunostimulant is administered to a subject such as a vertebrate, an immunostimulant effect is exhibited in the subject. This can suppress the onset of infection in a subject. That is, the immunostimulant can be applied to a method for preventing infection of a subject. Examples of the vertebrate to be administered include mammals, birds, and fishes. The mammal may include a human, or may be a mammal other than a human. Examples of the mammals other than humans include pet animals such as dogs and cats, in addition to pigs, cows, horses, sheep, and monkeys. Examples of the birds include broilers, layers, turkeys, ducks, and pigeons. Further, the fish may, for example, be salmon, trout (Oncorhynchus masou), salmonids, and taimen, and other salmonids, carps, crucian carps, tilapia, catfish, japanese perch, yellowtail, flatfish, sea bream, tuna, and eel.

In the case where the immunostimulant is administered as a drug or a drink, food, for example, it may be orally administered 1 time to several times in an amount of 1mg to 500mg (dry weight)/kg body weight per time for 1 day. In addition, in the case of using as a drink, food, feed, or the like, 0.1g to 1g may be added to 100g of the drink, food, feed, or the like.

When the immunostimulant is administered as a feed, for example, in mammals, birds, and fishes, the obtained bait may be mixed in an amount of 0.001 mass% or more and 1 mass% or less with respect to the amount of the bait to be administered, and administered 1 to several times a day.

Method for preparing immunostimulant

The immunostimulant can be produced, for example, by the following production method using yeast cell walls as a raw material. The method for producing the immunostimulant comprises, for example, a hydrolysis step of hydrolyzing yeast cell walls with an aqueous acid solution or an aqueous alkali solution to obtain a hydrolysate, and a recovery step of recovering a composition containing glucan from the hydrolysate. That is, the immunostimulant may be a composition comprising yeast-derived glucan.

Here, the yeast cell walls for the hydrolysis step may also be pretreated by a pretreatment step. The pretreatment step includes a preliminary decomposition step such as an autolysis step of autolyzing the yeast to be the raw material to obtain an autolysate, a hot water extraction step of extracting the yeast to be the raw material with hot water to obtain a hot water extract, an enzyme treatment step of treating the yeast to be the raw material with an enzyme to obtain an enzyme-treated product, and a 1 st centrifugal separation step of subjecting the preliminary decomposition product such as the autolysate, the hot water extract, and the enzyme-treated product to centrifugal separation to obtain a yeast cell wall.

Examples of the yeast to be used as the raw material include: yeasts such as Saccharomyces (Saccharomyces), Schizosaccharomyces (Schizosaccharomyces), Kluyveromyces (Kluyberomyces), Candida (Candida), Pichia (Pichia), Torulopsis (Torulopsis), preferably at least 1 selected from the group consisting of these, more preferably Saccharomyces. Examples of the saccharomyces yeast include saccharomyces cerevisiae (s.cerevisiae), saccharomyces pastorianus (s.pastorianus), saccharomyces bayanus (s.bayanus), and the like, and at least 1 kind selected from the group consisting of these may be used. The yeast may be at least 1 kind selected from the group consisting of brewers 'yeast, whisky yeast, distillers' yeast, baker's yeast, wine's yeast, sake 'yeast, and bioethanol' yeast, depending on the use thereof. The starting yeast may be used alone in 1 kind, or 2 or more kinds may be used in combination.

In the autolysis step, proteins of yeast and the like are decomposed by protease of the yeast itself to obtain an autolysate. The autolysis step is performed, for example, by adjusting the pH to 2 to 7, adjusting the temperature to 40 ℃ or higher and 65 ℃ or lower, and performing the autolysis step for 1 hour or longer and 48 hours or shorter. In the 1 st centrifugation step, the autolysate is centrifuged to obtain a fraction comprising yeast cell walls in the form of a heavy liquid. For the centrifugal separation, for example, in industrial production, a nozzle type centrifuge, a batch discharge type centrifuge, a sharp (Sharples) type centrifuge, or the like can be used, and conditions for the centrifugal separation can be appropriately adjusted as long as insoluble components in the dissolved matter can be recovered.

In the hydrolysis step, the yeast cell wall is subjected to hydrolysis treatment using, for example, an aqueous alkali solution to obtain a hydrolysate. As the aqueous alkali solution, for example, an aqueous solution containing an alkali metal hydroxide such as sodium hydroxide is used. The concentration of the aqueous alkali solution is, for example, 0.01 mass% or more and 10 mass% or less, preferably 0.1 mass% or more and 5 mass% or less, and more preferably 1 mass% or more and 3 mass% or less. The temperature of the hydrolysis treatment is, for example, 70 ℃ or more and 100 ℃ or less, preferably 85 ℃ or more and 95 ℃ or less. The time of the hydrolysis treatment is, for example, 1 hour or more and 48 hours or less, preferably 4 hours or more and 24 hours or less.

The recovery step includes, for example, a neutralization step of neutralizing the hydrolysate to obtain a neutralized product, a 2 nd centrifugation step of centrifuging the neutralized product or the hydrolysate to obtain a glucan-containing composition, and a drying step of drying the glucan-containing composition, if necessary. By subjecting yeast cell walls obtained from yeast autolysates or the like to alkaline hydrolysis treatment, an immunostimulant comprising glucan and lipid can be produced with stable quality.

In the neutralization step, an acidic compound is added to the basic hydrolysate to adjust the pH to 6.5 to 7.5, obtaining a neutralized product. As the acidic compound, inorganic acids such as hydrochloric acid, nitric acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid and acetic acid can be used. In the 2 nd centrifugation step, the neutralized product is centrifuged to obtain a composition containing glucan in the form of a heavy liquid. For the centrifugal separation, for example, in industrial production, a nozzle type centrifuge, a batch discharge type centrifuge, a sharp (Sharples) type centrifuge, or the like can be used, and conditions for the centrifugal separation can be appropriately adjusted as long as insoluble components in the neutralized product or the hydrolysate can be recovered. In the drying step, the composition containing glucan is dried to obtain the immunostimulant. As the drying conditions, a spray dryer, a freeze dryer, a drum dryer, or the like can be used in industrial production, and the conditions can be appropriately adjusted. Before the drying step, a sterilization step of sterilizing the composition containing glucan may be provided. The sterilization step may be performed, for example, by performing heat treatment at 120 ℃ or higher for 10 seconds to 60 seconds using a UHT sterilizer, and may be appropriately adjusted so as to meet the target quality.

Method for producing yeast-derived glucan

The method for producing yeast-derived glucan of the present embodiment includes: a preparation step of preparing an autolysed composition comprising yeast cell walls; a hydrolysis step of subjecting a composition comprising yeast cell walls to an alkaline hydrolysis treatment to obtain a hydrolysate; and a recovery step of recovering glucan from the hydrolysate. Yeast-derived glucans obtained by alkaline hydrolysis of yeast cell walls obtained by autolysis of yeast cells exhibit good quality stability, and the difference in quality between production batches is suppressed. In addition, the recovery rate of glucan was good for the yeast-derived glucan produced, and impurities such as mannan and protein were sufficiently removed. The yeast-derived glucan may be produced by a method of purifying yeast-derived glucan.

In the preparation step, a composition comprising yeast cell walls that has been subjected to autolysis treatment is prepared. The composition containing yeast cell walls may be prepared by appropriately selecting it from commercially available products, or may be prepared by preparing a composition containing yeast cell walls having a desired composition. The yeast used as the raw material is as described above.

A composition containing a yeast cell wall can be prepared, for example, by a method comprising an autolysis step of subjecting a raw material yeast to autolysis treatment to obtain an autolysate and a 1 st centrifugation step of subjecting the autolysate to centrifugation treatment. Details of the autolysis step and the 1 st centrifugation step are as described above.

In the method of obtaining a composition containing a yeast cell wall, the yeast cells as a raw material may be purified before the autolysis step. The purification treatment includes, for example, removing impurities by sieving yeast cells as a raw material with a vibrating screen, washing under an alkaline condition of pH 9 to 11, and the like.

In the hydrolysis step, the yeast cell walls are subjected to hydrolysis treatment using an aqueous alkali solution to obtain a hydrolysate. Details regarding the hydrolysis step are as described above.

In the recovery step, glucan is recovered from the hydrolysate. The glucan obtained in the recovery step is obtained, for example, in the form of a glucan-containing composition. Details of the recovery step are as described above.

The method for producing yeast-derived glucan achieves a good recovery rate of glucan and is excellent in removal rate of mannan, protein and the like. The recovery rate of glucan was calculated by: the total amount of glucan contained in the yeast-derived glucan obtained was divided by the total amount of glucan contained in the autolysed yeast cell wall-containing composition. In addition, the removal rate of mannan was calculated by: the amount of removal obtained by subtracting the total amount of mannan contained in the yeast-derived glucan obtained from the total amount of mannan contained in the autolyzed yeast cell wall-containing composition was calculated by dividing the amount of removal by the total amount of mannan contained in the autolyzed yeast cell wall-containing composition. The removal rate of proteins and the like is also the same.

The recovery rate of glucan in the method for producing yeast-derived glucan is, for example, 65% or more, preferably 70% or more or 75% or more. The upper limit of the recovery rate of glucan is, for example, 95% or less, preferably 90% or less or 85% or less.

The removal rate of mannan in the method for producing yeast-derived glucan is, for example, 85% or more, preferably 90% or more or 95% or more. The upper limit of the removal rate of mannan is, for example, 100% or less, preferably less than 100%.

The removal rate of protein in the method for producing yeast-derived glucan is, for example, 80% or more, preferably 85% or more or 90% or more. The upper limit of the removal rate of the protein is, for example, 100% or less, and preferably less than 100% or 98% or less.

As another aspect, the invention encompasses an immunostimulating method comprising administering an immunostimulant or yeast-derived glucan to a subject. In addition, as another aspect, the present invention also encompasses the use of an immunostimulant or yeast-derived glucan in the preparation of a composition for use in an immunostimulating method or a method for preventing infection; use of an immunostimulant or a yeast-derived glucan in an immunostimulating method or a method of preventing infection; an immunostimulant or yeast-derived glucan for use in an immunostimulating method or a method for preventing infection. The subject of the immunostimulation method or the infection prevention method may be a vertebrate as described above, including a mammal, a bird, a fish, and the like.

Examples

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Method for preparing immunostimulant 1

A yeast cell wall (solid content: 15%; yeast-derived autolysate) of a lager brewing yeast belonging to the genus Saccharomyces (Saccharomyces) prepared at Mitsui Kimura Korea, Inc. from Asahi Group Foods Co., Ltd was prepared and subjected to hydrolysis treatment. Specifically, sodium hydroxide was added to the yeast cell wall-containing slurry so that the final concentration thereof became about 1.5% by mass or about 2.0% by mass, and the resulting mixture was heated to 90 ℃ to conduct hydrolysis for 4 hours. The treated yeast cell wall slurry was adjusted to pH 7.0 with hydrochloric acid, and then subjected to batch centrifugation at 8,000 g.times.10 minutes using a high-speed cooling centrifuge (Avanti J-26S XP manufactured by BECKMAN COULTER Co., Ltd.). 1.5L of distilled water was added to the precipitate, and after suspending, centrifugal separation was performed under the above conditions. The operation was repeated 4 times, and the precipitate was dried with a lyophilizer (FDU-2100 manufactured by EYELA) over 2 nights to obtain an immunostimulant.

Method for preparing immunostimulant 2

A yeast cell wall (solid content: 15%; yeast-derived autolysate) of a lager brewing yeast belonging to the genus Saccharomyces (Saccharomyces) prepared at Mitsui Kimura Korea, Inc. from Asahi Group Foods Co., Ltd was prepared and subjected to hydrolysis treatment. Specifically, sodium hydroxide was added to the yeast cell wall-containing slurry so that the final concentration thereof became about 1.5% by mass or about 2.0% by mass, and the resulting mixture was heated to 90 ℃ to conduct hydrolysis for 4 hours. After the pH of the treated yeast cell wall slurry was adjusted to 7.0 with hydrochloric acid, solid-liquid separation was carried out by a nozzle type continuous centrifuge (FEUX 512T-31C-50 and FEUX412U-31C-50 manufactured by Alfa Laval Co., Ltd.) while adding water, and the obtained heavy liquid was sterilized at 130 ℃ for 40 seconds and then dried by a drum dryer to obtain an immunostimulant.

Dextran assay method 1

About 0.6g of a sample was weighed, and after adding 4mL of 72 (w/w)% sulfuric acid, the mixture was stirred at room temperature for 1 hour. Thereafter, the reaction mixture was adjusted to a sulfuric acid concentration of 4 (w/w)%, using Milli-Q (registered trademark) water, and reacted at 121 ℃ for 1 hour. After cooling, the mixture was neutralized with an aqueous sodium hydroxide solution. After the volume was fixed, the mixture was filtered, and the filtrate was subjected to HPLC. The column is Wakopak (registered trademark) Wakosil (registered trademark) 5NH₂( FUJIFILM Wako Pure Chemical Industries, ltd.), the column temperature is set to room temperature, the mobile phase is set to acetonitrile: 75 parts of water: 25. the flow rate was 1.0 mL/min, and the injection amount was set to 2. mu.L. As a reaction solution, a mixed solution of 1 (w/v)% arginine and 3 (w/v)% boric acid was flowed at 0.7 mL/min, reacted at 150 ℃ and glucose was detected by a fluorescence detector (excitation wavelength 320nm, measurement wavelength 430 nm). The total glucan content was calculated by multiplying the obtained glucose content by 0.9.

Dextran assay 2

About 0.5g of the sample was weighed, and 25ml of 0.08mol/L phosphate buffer (pH 6.0) and 0.1ml of Termamyl 120L (Novozymes corporation) were added to the sample and reacted in a boiling water bath for 30 minutes. After allowing to cool, the pH was adjusted to 7.5. + -. 0.1 using 0.275mol/l sodium hydroxide solution, and protease (Sigma-Aldrich, P-5380) was allowed to act at 60 ℃ for 30 minutes. After allowing to cool, amyloglucosidase (Sigma-Aldrich, A-9913) was allowed to act at 60 ℃ for 30 minutes with pH adjusted to 4.3. + -. 0.1 using 0.325mol/l hydrochloric acid. 4 times of 95% ethanol was added to the enzyme-treated liquid, and the mixture was left to stand for 1 hour or more. The liquid was poured into a filter containing diatomaceous earth and suction-filtered. Washing the residue with ethanol and acetone. The residue was collected, 5ml of 72 (w/w)% sulfuric acid was added thereto, and the mixture was decomposed at 20 ℃ for 4 hours. Water was added to make the sulfuric acid 4 (w/w)%, and the mixture was decomposed in a boiling water bath for 2 hours. After standing and cooling, neutralizing, fixing the volume and filtering. The glucose in the filtrate was quantified by the glucose oxidase method, and the glucose concentration was determined and multiplied by 0.9 to calculate the content of β -glucan.

The content of α -glucan was calculated by subtracting the content of β -glucan from the total content of glucan obtained in analysis method 1.

Method for analyzing lipid

Quantification was performed by acid decomposition. 2ml of ethanol and 10ml of concentrated hydrochloric acid were added to the sample, and the mixture was subjected to decomposition treatment in a thermostatic bath at 70 ℃ to 80 ℃ for 30 minutes to 40 minutes. Thereafter, the sample was transferred to a Morse tube (Mojonnier tube), mixed with ethanol and diethyl ether, and further mixed with petroleum ether. The ether layer was recovered, and a mixed solution of diethyl ether and petroleum ether was further added to the aqueous layer to partition the mixture. The ether layer was recovered and the aqueous layer was partitioned again with diethyl ether and petroleum ether to recover the ether layer. After removing diethyl ether and petroleum ether by distillation, the mixture was dried at 105 ℃ for 1 hour, and the difference in weight between before and after drying was taken as the mass of the lipid, and the content of the lipid was calculated from the amount of the sample obtained.

Mannan analysis method

About 0.6g of a sample was weighed, and 4ml of 72% (w/w)% sulfuric acid was added thereto, followed by stirring at room temperature for 1 hour. Subsequently, Milli-Q (registered trademark) water was added to adjust the sulfuric acid concentration to 4 (w/w)%, and hydrolysis was carried out at 121 ℃ for 1 hour. After leaving to cool, the solution was neutralized and the volume was determined, and the content of mannose in the filtrate was determined in the same manner as in the analysis method of glucan described above. The mannose concentration in the sample was calculated and multiplied by 0.9 to calculate the mannan content.

Method for analyzing protein

The content of protein in the sample was determined by a combustion method. The Analysis was performed using a total nitrogen measuring apparatus (Sumika Chemical Analysis Service). The samples were taken out into a quartz boat, and analyzed while the temperature of the reaction furnace was 870 ℃ or higher, the temperature of the reduction furnace was 600 ℃, the temperature of the detector was 100 ℃, and the temperature of the column was 70 ℃. The content of protein was calculated by quantifying the amount of the detected nitrogen and multiplying the result by a nitrogen/protein conversion factor of 6.25.

Method for analyzing ash

The ash content in the sample was quantified by a direct ashing method. The sample was taken into a magnetic crucible and subjected to preliminary ashing. Thereafter, ashing was performed at 550 ℃. After cooling in a silica gel drier, weighing is carried out. The weight difference before and after ashing was defined as an ash content, and the ash content was calculated from the sample acquisition amount.

The results of analyzing the components of the 14 samples of immunostimulants obtained by the above-mentioned preparation method are shown in Table 1. The content of each component shown in table 1 is a dry matter equivalent of the immunostimulant calculated on a mass basis. The glucan content in table 1 is the total content of α -glucan and β -glucan, and is the same as below.

[ Table 1]

As a reference example, 9 samples of commercial immunostimulant containing β -1,3/1, 6-glucan as a main component (hereinafter, also referred to as "commercial product a") were obtained from different preparation batches, and subjected to component analysis in the same manner. The average value, standard deviation and coefficient of variation (%) of the content of each component are shown in table 2. The content of each component shown in table 2 is a dry matter equivalent on a mass basis.

[ Table 2]

As shown in table 1, the average of the total content of the immunostimulant lipid and glucan was 80 mass% or more, and the difference in content between samples was small. On the other hand, as shown in table 2, the average of the total content of lipids and dextran in the commercial product a was less than 80 mass%, and the difference between samples was large.

Then, the immunostimulant obtained as described above and commercial product a were evaluated for their ability to promote the production of Reactive Oxygen Species (ROS) in leukocytes.

Ability to promote ROS production in porcine leukocytes

Monocytes were collected from peripheral blood collected from weaned piglets at 10 weeks of age. Specifically, Peripheral Blood Mononuclear Cells (PBMCs) suspended in RPMI1640 medium supplemented with 10% fetal bovine serum were seeded in a 96-well plate so as to be 2 × 10⁶After that, monocytes were attached and fixed to the wells by culturing for 2 hours. Removing culture supernatant, washing with HBSS (Hanks balanced salt solution) to remove lymphocytes, and mixing with immunostimulant (#)5. #6) or commercially available A was added to each well together with the luminescent reagent (luminol) and HBSS in such a manner that the final concentration became 100. mu.g/mL to 400. mu.g/mL. Thereafter, the cumulative luminescence amount was measured for 120 minutes by a photometer (ThermoFisher Scientific Co.). In the negative control, only the luminescent reagent (luminol) and HBSS were added, and the Relative Luminescence Unit (RLU) was evaluated as the ROS production amount. In the positive control, phorbol 12-myristate 13-acetate (PMA) was added together with a luminescent reagent (luminol) and HBSS at 50. mu.g/mL. The results are shown in FIG. 1.

As shown in FIG. 1, in two different preparation batches of immunostimulants #5, #6, the amount of ROS produced was greater than that of commercial product A at all concentrations of 100. mu.g/mL to 400. mu.g/mL. The total content of glucan and lipid in the immunostimulant #5 was 85.4 mass%, and the total content of glucan and lipid in the immunostimulant #6 was 83.9 mass%.

Ability to promote ROS production in human leukocytes

Human leukemia cell line HL-60 was induced to differentiate into neutrophil-like cells by culturing for 6 days in RP1640 MI medium supplemented with 1.25% by volume of dimethyl sulfoxide and 10% of fetal bovine serum according to the method of Collins et al (Collins SJ, Rusceti FW, Gallagher RE, Gallo RC, Proc. Natl. Acad. Sci. USA, 75, 2458-2462, 1978). HL-60 neutrophil-like cells suspended in the above medium were cultured at 4X 10⁵After each cell/well was seeded in a 96-well plate, and was attached and fixed in the well by 1 hour culture, immunostimulant #14 or commercially available product a was added to each well together with a luminescent reagent (luminol, manufactured by Nacalai Tesque corporation) and HBSS so that the final concentration became 100 μ g/mL to 800 μ g/mL. Thereafter, the integrated luminescence amount was measured for 120 minutes by a photometer. In the negative control, only the luminescent reagent (luminol) and HBSS were added, and the Relative Luminescence Unit (RLU) was evaluated as the ROS production amount. The results are shown in FIG. 2.

As shown in FIG. 2, the amount of ROS produced by immunostimulant #14 of the present invention was greater than that of commercial product A at all concentrations ranging from 100. mu.g/mL to 800. mu.g/mL. The total content of glucan and lipid in the immunostimulant #14 used in this case was 83.8 mass%.

Vibriosis infection test of rainbow trout

25 rainbow trout having an average body weight of 2g were bred using the feed prepared by dispersing and mixing the immunostimulant #4 or the commercial product A so that the final concentration became 0.2 mass%. 7 days after the start of rearing, the cells were diluted to 9X 10 with PBS⁴0.05mL of Vibrio anguillarum (Vibrio anguillarum) was injected into the abdominal cavity of rainbow trout and subjected to infection treatment. Thereafter, the breeding was continued for 10 days, and the survival rate was calculated every day. The results are shown in FIG. 3. In fig. 3, the vertical axis represents survival rate (%), and the horizontal axis represents the number of days elapsed after infection treatment.

The survival rate at day 7 after injection was 64% for the negative control group (no treatment), 76% for the immunostimulant-administered group, and 68% for the commercial product a-administered group. The total content of glucan and lipid in the immunostimulant #4 used in this case was 81.3% by mass.

As shown in fig. 3, the immunostimulant of the present invention showed higher survival rate compared to the commercially available immunostimulant.

The disclosure of japanese patent application No. 2018-191154 (application date: 2018, 10, 9) is incorporated by reference in its entirety into the present specification. All documents, patent applications, and technical specifications described in the present specification are incorporated in the present specification by reference to the same extent as if each was specifically and individually indicated to be incorporated in the present specification.