WO2020075424A1 - 免疫賦活剤及び感染予防方法 - Google Patents
免疫賦活剤及び感染予防方法 Download PDFInfo
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- WO2020075424A1 WO2020075424A1 PCT/JP2019/034989 JP2019034989W WO2020075424A1 WO 2020075424 A1 WO2020075424 A1 WO 2020075424A1 JP 2019034989 W JP2019034989 W JP 2019034989W WO 2020075424 A1 WO2020075424 A1 WO 2020075424A1
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- glucan
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- yeast
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- immunostimulant
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- A23K20/158—Fatty acids; Fats; Products containing oils or fats
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/163—Sugars; Polysaccharides
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- A—HUMAN NECESSITIES
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- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
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- A—HUMAN NECESSITIES
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- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/716—Glucans
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- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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- A61K36/06—Fungi, e.g. yeasts
- A61K36/062—Ascomycota
- A61K36/064—Saccharomycetales, e.g. baker's yeast
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- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/14—Yeasts or derivatives thereof
- A23L33/145—Extracts
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/324—Foods, ingredients or supplements having a functional effect on health having an effect on the immune system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/37—Extraction at elevated pressure or temperature, e.g. pressurized solvent extraction [PSE], supercritical carbon dioxide extraction or subcritical water extraction
Definitions
- the present invention relates to an immunostimulant and a method for preventing infection.
- Japanese Patent Publication No. 2017-511122 discloses a method of treating a yeast cell wall to obtain ⁇ -glucan soluble in dimethylsulfoxide.
- JP-A-2009-263333 describes a composition containing selenium-enriched yeast and ⁇ -glucan and is said to have an immunostimulatory action.
- JP 2004-099580 A describes an immune enhancing composition prepared by subjecting yeast cells to autolysis.
- Japanese Unexamined Patent Publication No. 2003-169607 describes a feed for breeding fish and shellfish to which a yeast-decomposing composition obtained by autolyzing yeast is added, and it is said that the immune activity is improved.
- an aspect of the present invention is to provide an immunostimulant containing glucan and having an immunostimulatory effect.
- the first aspect of the present invention comprises a glucan derived from a yeast cell wall and a lipid, the total content of the glucan and the lipid is 80% by mass or more, and the content ratio of the lipid to the glucan is 0.1 or more and 0.4 or less. And is a water-insoluble immunostimulant.
- the content ratio of ⁇ -glucan to ⁇ -glucan may be 0.2 or more and 0.85 or less.
- the immunostimulant may contain mannan, and the content ratio of mannan may be 5% by mass or less.
- the immunostimulant may be used against fish.
- the second aspect is a food or drink or feed containing the immunostimulant.
- a third aspect is an agent for preventing infection of fish, which contains glucan and lipid and has a total content of glucan and lipid of 80% by mass or more.
- a fourth aspect is a method for preventing infection, which comprises administering the immunostimulant to a subject.
- a fifth aspect is to prepare a composition containing the yeast cell wall that has been subjected to autolysis, obtain a hydrolyzate by subjecting the composition containing the yeast cell wall to alkaline hydrolysis, and recover glucan from the hydrolyzate.
- a method for producing a yeast-derived glucan comprising:
- a composition containing a yeast cell wall is obtained by a method including autolyzing yeast to obtain an autolysate, and subjecting the autolysate to centrifugation.
- an alkali hydrolysis process is performed by heating in the presence of an alkali metal hydroxide.
- the yeast-derived glucan has a mannan content of 5% by mass or less.
- the yeast-derived glucan has a protein content of 10% by mass or less.
- the yeast-derived glucan has a coefficient of variation in glucan content of 10% or less.
- an immunostimulant containing glucan and having an immunostimulatory effect.
- 5 is a graph showing the ability of porcine leukocytes to promote the production of reactive oxygen species.
- 3 is a graph showing the ability to promote the production of reactive oxygen species on human neutrophil-like cells. It is a graph which shows the survival rate of the Vibrio disease infection test with respect to rainbow trout.
- the term “process” is included in this term as long as the intended purpose of the process is achieved not only as an independent process but also when it cannot be clearly distinguished from other processes. .
- the content of each component in the composition when there are a plurality of substances corresponding to each component in the composition, unless otherwise specified, means the total amount of the plurality of substances present in the composition .
- the content of each component is a value in terms of dry matter of the immunostimulant, and may be an average value.
- the content is taken as an average value in consideration of variations between manufacturing lots, and is, for example, an arithmetic average value of 6 or more samples arbitrarily selected.
- the upper limit of the number of samples for calculating the average value is, for example, 20 or less.
- the immunostimulant contains glucan and lipid, and the total content of glucan and lipid is 80% by mass or more.
- the immunostimulant according to the present embodiment has good quality stability and a clear immunostimulatory effect. This not only is expected to suppress the morbidity and progression of infectious diseases in livestock, humans, cultured fish, etc., but can also provide a means for reducing the threat of drug-resistant bacteria development.
- the glucan and lipid constituting the immunostimulant are preferably derived from, for example, the yeast cell wall and obtained by hydrolyzing the yeast cell wall.
- the hydrolysis treatment includes alkali hydrolysis and acid hydrolysis.
- the glucan that constitutes the immunostimulant is a polymer in which D-glucose is linked by a glycoside bond, and includes ⁇ -glucan and ⁇ -glucan.
- ⁇ -Glucan has, for example, a ⁇ -1,3-bonded straight-chain main chain skeleton and a ⁇ -1,6-bonded side chain for branching.
- the ⁇ -glucan has, for example, a linear main chain skeleton of ⁇ -1,4 bond and a side chain branched by ⁇ -1,6 bond.
- 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, for example, 90% by mass or less, preferably 85% by mass or less. It is not more than 80% by mass, more preferably not more than 80% by mass.
- the content of ⁇ -glucan in the immunopotentiator 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 ⁇ -glucan is, for example, 10% by mass or more, preferably 15% by mass or more, or 20% by mass or more, and for example, 40% by mass or less, preferably 35% by mass or less. Or 30% by mass or less.
- the ratio of the ⁇ -glucan content to the ⁇ -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 It is 0.45 or more, or 0.5 or more, and preferably 0.8 or less, 0.7 or less, or 0.6 or less.
- the content ratio of ⁇ -glucan to ⁇ -glucan is within the above range, the immunostimulatory activity tends to be further enhanced.
- the lipid in the immunostimulant includes a simple lipid formed by the ester bond of alcohol and a fatty acid, a complex lipid that is a lipid containing phosphate, sugar, protein, etc. in the molecule, and a simple lipid or a complex lipid by hydrolysis. Included are derived lipids that are derived.
- the total content of lipids 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 mass% or less. When the lipid content is within the above range, the immunostimulatory activity tends to be enhanced.
- the total content of glucan and lipid in the immunostimulant is 80% by mass or more, preferably 82% by mass or more. Further, for example, it is 95 mass% or less, preferably 90 mass% or less, or 88 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 to the total content of glucans in the immunostimulant 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, It is 0.3 or less, preferably 0.25 or less, or 0.2 or less.
- the content ratio of lipid to glucan is within the above range, the immunostimulatory activity tends to be further enhanced.
- the immunostimulant may further contain mannan in addition to glucan and lipid.
- Mannan is a polysaccharide whose main constituent unit is D-mannose.
- 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 for example, 0.1% by mass or more. And preferably 0.2% by mass or more, or 0.3% by mass or more.
- the mannan content is in 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 It is 0.02 or less, or 0.018 or less.
- the immunostimulant may further contain protein in addition to glucan and lipid.
- the protein content is, for example, 10% by mass or less, preferably 8% by mass or less, or 6% by mass or less, and for example, 1% by mass or more, preferably Is 2% by mass or more, or 3% by mass or more.
- the immunostimulatory activity tends to be further enhanced.
- the ratio of the total content of mannan and protein to the total content of glucan in the immunostimulant is, for example, 0.01 or more and 0.2 or less, preferably 0.02 or more, or 0.05 or more, and It is preferably 0.1 or less, or 0.09 or less.
- the immunostimulant may further contain ash.
- the ash content is, for example, 10 mass% or less, preferably 6 mass% or less, or 4 mass% or less, and for example, 1 mass% or more, preferably Is 1.5% by mass or more, or 2% by mass or more.
- the immunostimulatory activity tends to be further enhanced.
- the content of each component of the immunostimulant can be measured by a conventional method.
- the content of glucan can be measured by quantifying glucose produced by hydrolyzing glucan.
- the content of lipid can be quantified by an acid decomposition method.
- Immunosuppressant has excellent quality stability.
- the stability of the quality of the immunostimulant can be evaluated, for example, by the variation in the content rate of each component due to the difference in the production lot. Specifically, the variation coefficient (%) of the content rate of each component is evaluated.
- the coefficient of variation (%) is calculated by calculating the content ratio of each component of the immunostimulant for 6 or more arbitrarily selected samples, calculating the arithmetic mean value and standard deviation of the content ratio, and calculating the standard deviation. Calculated as a percentage of the value divided by the average value.
- the coefficient of variation (%) of the total content of glucan in the immunostimulant is, for example, 10% or less, preferably 8% or less, or less than 5%.
- the coefficient of variation (%) of 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, or less than 5%. The lower limit of the coefficient of variation is 0%, but it is substantially larger than 0%.
- the immunostimulant may further contain a known component having an immunomodulatory action in addition to the main components glucan and lipid.
- a known component having an immunomodulating action examples include bean tea extract, fucoidan, arabinoxylan, lactoferrin, catechin, chitosan, chitosan oligosaccharide, chitin oligosaccharide, L-ascorbic acid, coenzyme Q 10 (including reduced form) and the like. be able to.
- the immunostimulant if necessary, usually any component known to be used in medicines, foods, feeds, etc., for example, water, fats, sugars, vitamins, sweeteners, seasonings, sourness.
- Preservatives, preservatives, fragrances, colorants, excipients, fillers, binders, thickeners, stabilizers, emulsifiers, pH adjusters and the like can be added.
- the form of the immunostimulant may be any of solids such as powder and granules, liquid, paste and the like.
- the dosage form of the immunostimulant can be appropriately selected depending on the administration method, administration subject, and the like.
- examples of the dosage form include tablets, capsules, granules, troches, powders, liquid preparations and the like for oral administration.
- These formulations can be produced according to a known method using additives such as an excipient, a lubricant, a binder, a disintegrant, a stabilizer, a flavoring agent and a diluent.
- an immunostimulant may be contained to form food compositions such as health foods and health supplements, beverage compositions, feeds and the like.
- an immunostimulant When an immunostimulant is administered to an administration subject such as a vertebrate, it exerts an immunostimulatory action on the subject. This can suppress the onset of infectious disease in the subject. That is, the immunostimulant can be applied to a method for preventing a target infectious disease.
- the vertebrate to be administered include mammals, birds, fish and the like. Mammals may include humans and may be non-human mammals. Examples of mammals other than humans include pets such as pigs, cows, horses, sheep, monkeys, dogs, and cats. Examples of birds include meat chickens, layer chickens, turkeys, ducks, pigeons and the like. Examples of the fish include salmon, trout, yamame trout, charr, salmon, etc. salmonidae, carp, crucian carp, tilapia, catfish, perch, yellowtail, amberjack, yellowtail, flounder, Thailand, tuna, eel and the like.
- an amount of 1 mg to 500 mg (dry weight) / kg body weight per time can be orally administered once to several times a day.
- 0.1 to 1 g can be added per 100 g of food and drink and feed.
- the immunostimulant When the immunostimulant is administered as a feed, for example, for mammals, birds, and fish, a mixture of 0.001% by mass or more and 1% by mass or less with respect to the food to be administered is mixed once to several times a day. It can be administered once.
- the immunostimulant can be produced, for example, by the following production method using yeast cell wall as a raw material.
- the method for producing an immunopotentiator includes, for example, a hydrolysis step of hydrolyzing a yeast cell wall with an aqueous acid solution or an aqueous alkali solution to obtain a hydrolyzate, and a recovery step of recovering a glucan-containing composition from the hydrolyzate.
- the immunostimulant may be a composition containing yeast-derived glucan.
- the yeast cell wall provided for the hydrolysis step here may be pretreated in the pretreatment step.
- the pretreatment step is an auto-digestion step of self-digesting yeast as a raw material to obtain an auto-digestion product, a hot-water extraction step of hot-water extraction of yeast as a raw material to obtain a hot-water extract, and a yeast as a raw material as an enzyme.
- a pre-decomposition step such as an enzyme treatment step for obtaining an enzyme-treated product by treatment, a first centrifugation step for centrifuging a pre-decomposition product such as an autolysate, a hot water extract, an enzyme-treated product to obtain a yeast cell wall included.
- yeasts such as Saccharomyces, Schizosaccharomyces, Kluyveromyces, Candida, Pichia, and Torulopsis, and at least one selected from the group consisting of these.
- yeasts belonging to the genus Saccharomyces is more preferable.
- yeasts belonging to the genus Saccharomyces include Saccharomyces cerevisiae, S. cerevisiae, S. pastorianus, and S. bayanus, and at least one selected from the group consisting of these. It may be a seed.
- the yeast is also called beer yeast, whiskey yeast, shochu yeast, baker's yeast, wine yeast, sake yeast, bioethanol yeast, etc., depending on its use, and may be at least one selected from the group consisting of these.
- the yeast as a raw material may be used alone or in combination of two or more.
- yeast proteins and the like are decomposed by the yeast's own protease, etc.
- the self-digestion step is performed, for example, for 1 hour or more and 48 hours or less by adjusting the pH to 2 to 7 and the temperature to 40 ° C or more and 65 ° C or less.
- the autolysate is centrifuged to obtain a fraction containing the yeast cell wall as heavy liquid.
- a nozzle type centrifuge, an intermittent discharge type centrifuge, a Sharpless type centrifuge or the like can be used, and as long as the insoluble component in the autolysate can be recovered, the conditions of centrifugation Can be adjusted appropriately.
- the yeast cell wall is hydrolyzed using, for example, an alkaline aqueous solution to obtain a hydrolyzate.
- an alkaline aqueous solution for example, an aqueous solution containing an alkali metal hydroxide such as sodium hydroxide is used.
- the concentration of the alkaline aqueous solution is, for example, 0.01% by mass or more and 10% by mass or less, preferably 0.1% by mass or more and 5% by mass or less, and more preferably 1% by mass or more and 3% by mass or less.
- the temperature of the hydrolysis treatment is, for example, 70 ° C. or higher and 100 ° C. or lower, preferably 85 ° C. or higher and 95 ° C. or lower.
- the time for 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 recovering step includes, for example, a neutralizing step of neutralizing the hydrolyzate to obtain a neutralized product if necessary, and a second centrifugation to centrifuge the neutralized product or the hydrolyzate to obtain a glucan-containing composition. It includes a separation step and a drying step of drying the glucan-containing composition.
- an acidic compound is added to the alkali hydrolyzate to adjust the pH to 6.5 to 7.5 to obtain a neutralized product.
- 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 are used.
- the neutralized product is centrifuged to obtain a glucan-containing composition as a heavy liquid.
- the glucan-containing composition is dried to obtain an immunostimulant.
- 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.
- a sterilization step of sterilizing the glucan-containing composition may be provided before the drying step.
- the sterilization step can be performed, for example, by using a UHT sterilizer for heat treatment at 120 ° C. or higher for 10 seconds to 60 seconds, but can be appropriately adjusted so as to match the desired quality.
- a method for producing yeast-derived glucan according to the present embodiment is a preparatory step of preparing a composition containing a self-digested yeast cell wall, and an alkaline hydrolysis treatment of the composition containing a yeast cell wall.
- the method includes a hydrolysis step of obtaining a hydrolyzate and a recovery step of recovering glucan from the hydrolyzate.
- the yeast-derived glucan obtained by subjecting the yeast cell wall obtained by subjecting the yeast cell to autolysis to alkaline hydrolysis exhibits good quality stability, and variation in quality between production lots is suppressed.
- the yeast-derived glucan produced has sufficient recovery of glucan, but impurities such as mannan and protein are sufficiently removed.
- the method for producing yeast-derived glucan may be a method for purifying yeast-derived glucan.
- a composition containing the yeast cell wall that has been subjected to autolysis is prepared.
- the composition containing the yeast cell wall may be appropriately selected and prepared from commercially available products, or the composition containing the yeast cell wall having a desired constitution may be produced and prepared.
- the yeast as a raw material is as described above.
- the composition containing the yeast cell wall is obtained by, for example, a method including an autolysis step of subjecting a yeast cell as a raw material to autolysis to obtain an autolysis product, and a first centrifugation step of centrifuging the autolysis product. It can be manufactured.
- the details of the autolysis step and the first centrifugation step are as described above.
- the yeast cells as a raw material may be purified before the autolysis step.
- the purification treatment includes, for example, removing contaminants by sieving the yeast cells as a raw material with a vibrating sieve, and washing under alkaline conditions of pH 9 to 11.
- the yeast cell wall is hydrolyzed using an alkaline aqueous solution to obtain a hydrolyzate.
- the details of the hydrolysis step are as described above.
- glucan is recovered from the hydrolyzate.
- the glucan obtained in the collecting step is obtained, for example, as a glucan-containing composition.
- the details of the recovery process are as described above.
- the yeast-derived glucan production method achieves good glucan recovery rates and excellent mannan and protein removal rates.
- the glucan recovery rate is calculated by dividing the total amount of glucan contained in the obtained yeast-derived glucan by the total amount of glucan contained in the composition containing the yeast cell wall subjected to autolysis. Further, the removal rate of mannan, the total amount of mannan contained in the composition containing the yeast cell wall subjected to self-digestion treatment, the removal amount obtained by subtracting the total amount of mannan contained in the obtained yeast-derived glucan, the self-digestion treatment It is calculated by dividing by the total amount of mannan contained in the composition containing the yeast cell wall. The same applies to the removal rate of proteins and the like.
- 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 glucan recovery rate 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 mannan removal rate is, for example, 100% or less, preferably less than 100%.
- the protein removal rate 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 protein removal rate is, for example, 100% or less, preferably less than 100%, or 98% or less.
- the present invention includes an immunostimulatory method comprising administering an immunostimulant or a yeast-derived glucan to a subject.
- an immunostimulant or a yeast-derived glucan in the production of a composition used in an immunostimulatory method or a method for preventing an infectious disease, an immunostimulant in an immunostimulatory method or a method for preventing an infectious disease.
- it also includes an immunostimulant or a yeast-derived glucan used in the use of a yeast-derived glucan, an immunostimulatory method or a method for preventing an infectious disease.
- the subject of the immunostimulatory method or the infectious disease prevention method may be the vertebrate described above, and includes mammals, birds, fish and the like.
- Tochigi Koganei factory produced yeast cell wall derived from brewer's yeast belonging to the genus Saccharomyces (solid content 15%; derived from yeast autolysate), which was subjected to hydrolysis treatment I served. Specifically, sodium hydroxide is added to a slurry containing yeast cell walls so that the final concentration is about 1.5% by mass or about 2.0% by mass, and the mixture is heated at 90 ° C. for hydrolysis for 4 hours. Processed.
- the treated yeast cell wall slurry was adjusted to pH 7.0 with hydrochloric acid and then subjected to batch type centrifugation at 8,000 g ⁇ 10 minutes using a high-speed cooling centrifuge (Avanti J-26S XP manufactured by BECKMAN COULTER). 1.5 L of distilled water was added to the precipitate, and the precipitate was suspended and then centrifuged under the above conditions. This operation was repeated 4 times, and the precipitate was dried for 2 nights or more with a freeze dryer (FDU-2100 manufactured by EYELA) to obtain an immunostimulant.
- a freeze dryer FDU-2100 manufactured by EYELA
- Tochigi Koganei factory produced yeast cell wall derived from brewer's yeast belonging to the genus Saccharomyces (solid content 15%; derived from yeast autolysate), which was subjected to hydrolysis treatment I served. Specifically, sodium hydroxide is added to a slurry containing yeast cell walls so that the final concentration is about 1.5% by mass or about 2.0% by mass, and the mixture is heated at 90 ° C. for hydrolysis for 4 hours. Processed.
- the yeast cell wall slurry after the treatment was adjusted to pH 7.0 with hydrochloric acid, and then solid-liquid separated while adding water with a nozzle type continuous centrifuge (FEUX512T-31C-50 and FEUX412U-31C-50 manufactured by Alfa Laval).
- the heavy liquid was sterilized under the conditions of 130 ° C. for 40 seconds and then dried in a drum dryer to obtain an immunostimulant.
- Glucan analysis method 1 About 0.6 g of the sample was weighed, 4 mL of 72 (w / w)% sulfuric acid was added, and the mixture was stirred at room temperature for 1 hour. Thereafter, the sulfuric acid concentration was adjusted to 4 (w / w)% using Milli-Q (registered trademark) water, and the mixture was reacted at 121 ° C. for 1 hour. After cooling, it was neutralized with an aqueous sodium hydroxide solution. After constant volume, filtration was performed and the filtrate was subjected to HPLC.
- the injection volume was 0 mL / min and the injection volume was 2 ⁇ L.
- As a reaction solution a mixed solution of 1 (w / v)% arginine and 3 (w / v)% boric acid was added at 0.7 mL / min. And then reacted at 150 ° C., and glucose was detected using a fluorescence detector (excitation wavelength 320 nm, measurement wavelength 430 nm). The total glucose content was calculated by multiplying the obtained glucose content by 0.9.
- Glucan analysis method 2 Approximately 0.5 g of the sample was weighed, 25 ml of 0.08 mol / l phosphate buffer (pH 6.0) was added, and 0.1 ml of Termamyl 120 L (Novozymes) was added and reacted in a boiling water bath for 30 minutes. . After cooling, the pH was adjusted to 7.5 ⁇ 0.1 using a 0.275 mol / l sodium hydroxide solution, and a protease (Sigma Aldrich, P-5380) was allowed to act at 60 ° C for 30 minutes.
- the content rate of ⁇ -glucan was calculated by subtracting the content rate of ⁇ -glucan from the total content rate of glucan obtained by analysis method 1.
- Lipid analysis method The amount was determined by the acid decomposition method. To the collected sample, 2 ml of ethanol and 10 ml of concentrated hydrochloric acid were added, and the sample was decomposed in a thermostat at 70 ° C to 80 ° C for 30 minutes to 40 minutes. Thereafter, the sample was transferred to a Majorian tube, ethanol and diethyl ether were mixed, and petroleum ether was further mixed. The ether layer was recovered, and a mixed solution of diethyl ether / petroleum ether was further added to the aqueous layer for partitioning. The ether layer was recovered, the aqueous layer was again partitioned using diethyl ether and petroleum ether, and the ether layer was recovered. After distilling off diethyl ether / petroleum ether, it was dried at 105 ° C. for 1 hour, and the weight difference before and after drying was taken as the lipid amount, and the lipid content was calculated from the sampled amount.
- Mannan analysis method About 0.6 g of a sample was weighed, 4 ml of 72 (w / w)% sulfuric acid was added, and the mixture was stirred at room temperature for 1 hour. Then, Milli-Q (registered trademark) water was added so that the sulfuric acid concentration became 4 (w / w)%, and hydrolysis was performed at 121 ° C. for 1 hour. After allowing to cool, the mixture was neutralized and adjusted to a constant volume, and the content of mannose in the filtrate was quantified in the same manner as in the above-mentioned glucan analysis method. The mannose concentration in the sample was calculated and multiplied by 0.9 to calculate the mannan content.
- Protein analysis method The content of protein in the sample was quantified by the combustion method.
- a total nitrogen measuring device (Sumika Chemical Analysis Center) was used for the analysis.
- a sample was taken in a quartz boat, the temperature of the reaction furnace was set to 870 ° C. or higher, the temperature of the reduction furnace was set to 600 ° C., the temperature of the detector was set to 100 ° C., and the temperature of the column was set to 70 ° C. for analysis.
- the detected nitrogen gas was quantified and multiplied by a nitrogen / protein conversion coefficient of 6.25 to calculate the protein content.
- the content of ash in the sample was quantified by the direct ashing method.
- a sample was taken in a porcelain crucible and pre-ashed. Then, ashing was performed at 550 ° C. After allowing to cool in a silica gel desiccator, it was weighed. The difference in weight before and after ashing was taken as the ash content, and the ash content was calculated from the sampled amount.
- Table 1 shows the results of component analysis of 14 samples of the immunostimulant obtained by the above production method.
- the content of each component shown in Table 1 is a dry matter conversion value of the immunostimulant calculated on a mass basis.
- the content of glucan in Table 1 is the total content of ⁇ -glucan and ⁇ -glucan, and so on.
- the immunostimulant had an average total content of lipids and glucan of 80% by mass or more, and the content variation among samples was small.
- the average value of the total content of lipids and glucan in the commercial product A was less than 80% by mass, and the variation among the samples was large.
- the immunostimulant obtained above and the commercially available product A were evaluated for their ability to promote the production of reactive oxygen species (ROS) against white blood cells.
- ROS reactive oxygen species
- Monocytes were fractionated from peripheral blood collected from 10-week-old weaned piglets. Specifically, peripheral blood mononuclear cells (PBMC) suspended in RPMI1640 medium containing 10% fetal bovine serum were seeded in a 96-well plate at 2 ⁇ 10 6 cells / well, and then cultured for 2 hours. The monocytes were attached and fixed in the wells by. After removing the culture supernatant and washing with HBSS (Hanks' Balanced Salt Solution) to remove lymphocytes, the immunostimulant (# 5, # 6) or commercial product A was added at a final concentration of 100 ⁇ g / mL to 400 ⁇ g / mL.
- HBSS Horts' Balanced Salt Solution
- the luminescent reagent (luminol) and HBSS were added to each well so that the amount of the solution became mL. Then, the integrated luminescence amount for 120 minutes was measured with a luminometer (ThermoFisher Scientific). Only the luminescent reagent (luminol) and HBSS were added to the negative control, and the relative luminescence unit (RLU) was evaluated as the ROS production amount. For the positive control, phorbol 12-myristate 13-acetate (PMA) was added together with the luminescent reagent (luminol) and HBSS so that the concentration was 50 ⁇ g / mL. The results are shown in FIG.
- Human leukocyte cell line HL- according to the method of Collins et al. (Collins SJ, Ruscetti FW, Gallagher RE, Gallo RC, Proc. Natl. Acad. Sci. USA, 75, 2458-2462, 1978). 60 was cultured in RPMI1640 medium containing 1.25 vol% dimethyl sulfoxide and 10% fetal bovine serum for 6 days to induce neutrophil-like differentiation. Neutrophil-like HL-60 suspended in the above-mentioned medium was seeded on a 96-well plate at 4 ⁇ 10 5 cells / well, and then adhered and fixed in the well by culturing for 1 hour to give an immunostimulant.
- the immunostimulant # 14 of the present invention produced more ROS than the commercial product A at all addition concentrations from 100 ⁇ g / mL to 800 ⁇ g / mL.
- the total content of glucan and lipid in the immunostimulant # 14 used at this time was 83.8% by mass.
- Vibrio Disease Infection Test of Rainbow Trout Twenty-five rainbow trout with an average body weight of 2 g were respectively bred with a feed spread so that the final concentration of the immunostimulant # 4 or the commercial product A was 0.2% by mass. Seven days after the start of breeding, 0.05 mL of Vibrio anguillarum bacterium diluted with PBS to 9 ⁇ 10 4 cfu / mL was intraperitoneally injected into the rainbow trout to perform infection treatment. After that, 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 the survival rate (%), and the horizontal axis represents the number of days elapsed after infection treatment.
- the survival rate on the 7th day after injection was 64% for the negative control (untreated), 76% for the immunostimulant administration group, and 68% for the commercial product A administration group.
- the total content of glucan and lipid in the immunostimulant # 4 used at this time was 81.3% by mass.
- the immunostimulant of the present invention showed higher survival rate than the commercially available immunostimulant.
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Abstract
Description
免疫賦活剤は、グルカンと脂質とを含み、グルカンと脂質の総含有率が80質量%以上である。本実施形態に係る免疫賦活剤は、良好な品質安定性と明確な免疫賦活効果を有する。これにより、家畜、ヒト、養殖魚等における感染症の罹患、進行を抑制することが期待されるだけでなく、薬剤耐性菌発生の脅威を軽減する手段を提供しうる。免疫賦活剤を構成するグルカンと脂質は、例えば、酵母細胞壁に由来し、酵母細胞壁を加水分解処理して得られるものであることが好ましい。加水分解処理にはアルカリ加水分解、酸加水分解が含まれる。
免疫賦活剤は、例えば、酵母細胞壁を原料とする以下のような製造方法で製造することができる。免疫賦活剤の製造方法は、例えば、酵母細胞壁を酸水溶液又はアルカリ水溶液で加水分解処理して加水分解物を得る加水分解工程と、加水分解物からグルカン含有組成物を回収する回収工程とを含む。すなわち、免疫賦活剤は酵母由来グルカンを含む組成物であってよい。
本実施形態に係る酵母由来グルカンの製造方法は、自己消化処理された酵母細胞壁を含む組成物を準備する準備工程と、酵母細胞壁を含む組成物をアルカリ加水分解処理して加水分解物を得る加水分解工程と、加水分解物からグルカンを回収する回収工程と、を含む。酵母菌体を自己消化処理して得られる酵母細胞壁をアルカリ加水分解して得られる酵母由来グルカンは、良好な品質安定性を示し、製造ロット毎の品質のばらつきが抑制される。また、製造される酵母由来グルカンは、グルカンの回収率が良好でありながら、マンナン、タンパク質等の不純物が充分に除去される。酵母由来グルカンの製造方法は、酵母由来グルカンの精製方法であってよい。
アサヒグループ食品(株)栃木小金井工場にて製造したサッカロマイセス(Saccharomyces)属に属するビール酵母に由来する酵母細胞壁(固形分15%;酵母の自己消化物由来)を準備し、これを加水分解処理に供した。具体的には、酵母細胞壁を含むスラリーに、最終濃度が約1.5質量%、又は約2.0質量%となるように水酸化ナトリウムを添加し、90℃に加熱して4時間加水分解処理した。処理後の酵母細胞壁スラリーを塩酸でpH7.0に調整後、高速冷却遠心機(BECKMAN COULTER社製 Avanti J-26S XP)を用い、8,000g×10分間のバッチ式遠心分離を行った。沈殿に1.5Lの蒸留水を添加し、懸濁した後に上述の条件で遠心分離を行った。本操作を4回繰り返し、沈殿を凍結乾燥機(EYELA社製FDU-2100)にて2晩以上乾燥して免疫賦活剤を得た。
アサヒグループ食品(株)栃木小金井工場にて製造したサッカロマイセス(Saccharomyces)属に属するビール酵母に由来する酵母細胞壁(固形分15%;酵母の自己消化物由来)を準備し、これを加水分解処理に供した。具体的には、酵母細胞壁を含むスラリーに、最終濃度が約1.5質量%、又は約2.0質量%となるように水酸化ナトリウムを添加し、90℃に加熱して4時間加水分解処理した。処理後の酵母細胞壁スラリーを塩酸でpH7.0に調整後、ノズル式連続遠心分離機(アルファラバル社製FEUX512T-31C-50およびFEUX412U-31C-50)により加水しながら固液分離し、得られた重液を130℃40秒の条件で殺菌し、次いでドラム乾燥機にて乾燥して免疫賦活剤を得た。
試料を約0.6g秤量し、72(w/w)%硫酸を4mL添加後、室温にて1時間撹拌した。その後、Milli-Q(登録商標)水を用いて硫酸濃度を4(w/w)%に調整し、121℃で1時間反応させた。冷却後に水酸化ナトリウム水溶液を用いて中和した。定容後にろ過を行い、ろ液をHPLCに供した。カラムはWakopak(登録商標)Wakosil(登録商標)5NH2(φ4.6mm×250mm、富士フイルム和光純薬工業株式会社)、カラム温度は室温、移動相はアセトニトリル:水=75:25、流量1.0mL/分、注入量は2μLとした。反応液として1(w/v)%アルギニンおよび3(w/v)%ホウ酸混合溶液を0.7mL/min.で流し、150℃で反応させ、蛍光検出器を用いてグルコースを検出した(励起波長320nm、測定波長430nm)。得られたグルコース含有率に0.9を乗じてグルカンの総含有率を算出した。
試料を約0.5g秤量し、0.08mol/lリン酸緩衝液(pH6.0)を25ml添加し、ターマミル120L(Novozymes社)を0.1ml添加し、沸騰水浴中で30分間反応させた。放冷後、0.275mol/lの水酸化ナトリウム溶液を用いてpHを7.5±0.1に調整し、プロテアーゼ(シグマアルドリッチ社、P-5380)を60℃で30分間作用させた。放冷後、0.325mol/lの塩酸を用いてpHを4.3±0.1に調整し、アミログルコシダーゼ(シグマアルドリッチ社、A-9913)を60℃で30分間作用させた。酵素処理後の液に4倍量の95%エタノールを添加し、1時以上放置した。珪藻土を入れたろ過器に、液を流し入れ、吸引濾過を行った。残渣をエタノール、アセトンで洗浄した。残渣を回収し、72(w/w)%硫酸を5ml添加し、20℃で4時間分解した。4(w/w)%になるよう水を添加し、沸騰水浴中で2時間分解した。放冷後に中和、定容、ろ過を行った。ろ液中のグルコースをグルコースオキシダーゼ法により定量し、グルコース濃度を求め、0.9を乗じ、β-グルカンの含有率を算出した。
酸分解法により定量した。採取した試料に2mlのエタノールと10mlの濃塩酸を添加し、70℃から80℃の恒温槽で30分間から40分間分解処理した。その後、試料をマジョニア管に移し、エタノールとジエチルエーテルを混合し、さらに石油エーテルを混合した。エーテル層を回収し、水層にさらにジエチルエーテル・石油エーテルの混合液を添加して分配を行った。エーテル層を回収し、水層は再度ジエチルエーテルと石油エーテルを用いて分配を行ってエーテル層を回収した。ジエチルエーテル・石油エーテルを留去後、105℃で1時間乾燥させて、乾燥前後の重量差を脂質量とし、試料採取量から脂質の含有率を算出した。
試料を約0.6g秤量し、72(w/w)%硫酸を4ml添加し、室温で1時間撹拌した。その後、硫酸濃度が4(w/w)%になるようMilli-Q(登録商標)水を添加し、121℃で1時間加水分解を行った。放冷後に中和、定容し、ろ液中のマンノースの含有量を、上述したグルカンの分析方法と同様にして定量した。試料中のマンノース濃度を算出し、0.9を乗じて、マンナン含有率を算出した。
試料中のタンパク質の含有率は、燃焼法にて定量した。分析には全窒素測定装置(住化分析センター)を用いた。試料を石英ボートに採取し、反応炉の温度を870℃以上、還元炉の温度を600℃、検出器の温度を100℃、カラムの温度を70℃に設定し、分析を行った。検出された窒素ガスを定量し、窒素・タンパク質換算係数6.25を乗じ、タンパク質含有率を算出した。
試料中の灰分の含有率は、直接灰化法により定量した。磁製るつぼに試料を採取し、予備灰化を行った。その後、550℃にて灰化を行った。シリカゲルデシケーター内で放冷後、秤量した。灰化前後の重量差を灰分量とし、試料採取量から灰分含有率を算出した。
10週齢の離乳子豚より採取した末梢血から単球を分取した。具体的には、10%ウシ胎児血清入りRPMI1640培地で懸濁させた末梢血単核細胞(PBMC)を2×106個/ウェルとなるように96ウェルプレートに播種した後、2時間の培養によりウェル内に単球を付着固定した。培養上清を除去し、HBSS(Hanks’Balanced Salt Solution)で洗浄してリンパ球を除去した後、免疫賦活剤(#5、#6)又は市販品Aを最終濃度が100μg/mLから400μg/mLとなるように発光試薬(ルミノール)とHBSSとともに各ウェルに添加した。その後、ルミノメーター(ThermoFisher Scientific社)で120分間の積算発光量を測定した。陰性対照には発光試薬(ルミノール)とHBSSのみを添加し、相対発光単位(RLU)をROS産生量として評価した。陽性対照にはホルボール12-ミリステート13-アセテート(PMA)を50μg/mLとなるように発光試薬(ルミノール)とHBSSとともに添加した。結果を図1に示す。
Collinsらの方法(Collins SJ,Ruscetti FW,Gallagher RE,Gallo RC,Proc.Natl.Acad.Sci.USA,75,2458-2462,1978)に従い、ヒト白血病細胞株HL-60を1.25体積%のジメチルスルホキシドと10%ウシ胎児血清入りRPMI1640培地で6日間培養して好中球様に分化誘導した。前述の培地で懸濁させた好中球様HL-60を4×105個/ウェルとなるように96ウェルプレートに播種した後、1時間の培養によりウェル内に付着固定し、免疫賦活剤#14又は市販品Aを最終濃度が100μg/mLから800μg/mLとなるように発光試薬(ルミノール、ナカライテスク社製)とHBSSとともに各ウェルに添加した。その後、ルミノメーターで120分間の積算発光量を測定した。陰性対照には発光試薬(ルミノール)とHBSSのみを添加し、相対発光単位(RLU)をROS産生量として評価した。結果を図2に示す。
平均体重2gのニジマス25匹を、免疫賦活剤#4又は市販品Aの最終濃度が0.2質量%となるように展着した飼料でそれぞれ飼育した。飼育開始から7日後にPBSにより9×104cfu/mLに希釈したVibrio anguillarum菌をニジマス腹腔内に0.05mL注射して感染処理した。その後、10日間にわたって飼育を継続し、1日毎に生存率を算出した。結果を図3に示す。図3において縦軸は生存率(%)、横軸は感染処理後の経過日数を示す。
Claims (13)
- 酵母細胞壁に由来するグルカンと脂質とを含み、グルカンと脂質の総含有率が80質量%以上であり、グルカンに対する脂質の含有比が0.1以上0.4以下であり、水不溶性である免疫賦活剤。
- β-グルカンに対するα-グルカンの含有比が、0.2以上0.85以下である請求項1に記載の免疫賦活剤。
- マンナンを含み、マンナンの含有率が5質量%以下である請求項1又は2に記載の免疫賦活剤。
- 魚類に対して用いられる請求項1から3のいずれか1項に記載の免疫賦活剤。
- 請求項1から4のいずれか1項に記載の免疫賦活剤を含む飲食品又は飼料。
- グルカンと脂質とを含み、グルカンと脂質の総含有率が80質量%以上である魚類に対する感染予防剤。
- 請求項1から4のいずれか1項に記載の免疫賦活剤を、対象に投与することを含む感染予防方法。
- 自己消化処理された酵母細胞壁を含む組成物を準備することと、
前記酵母細胞壁を含む組成物をアルカリ加水分解処理して加水分解物を得ることと、
前記加水分解物からグルカンを回収することと、を含む酵母由来グルカンの製造方法。 - 前記酵母細胞壁を含む組成物は、酵母を自己消化処理して自己消化物を得ることと、
前記自己消化物を遠心分離処理することと、を含む方法で得られる請求項8に記載の製造方法。 - 前記アルカリ加水分解処理は、アルカリ金属水酸化物の存在下に加熱して行われる請求項8又は9に記載の製造方法。
- 前記酵母由来グルカンは、マンナンの含有率が5質量%以下である請求項8から10のいずれか1項に記載の製造方法。
- 前記酵母由来グルカンは、タンパク質の含有率が10質量%以下である請求項8から11のいずれか1項に記載の製造方法。
- 前記酵母由来グルカンは、グルカン含有率の変動係数が10%以下である請求項8から12のいずれか1項に記載の製造方法。
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WO2022244580A1 (ja) * | 2021-05-21 | 2022-11-24 | アサヒグループホールディングス株式会社 | 免疫賦活剤及びグルカン組成物の製造方法 |
CN115669799A (zh) * | 2021-07-21 | 2023-02-03 | 安琪酵母股份有限公司 | 可替代抗生素和动物蛋白的酵母水解物及制备方法和应用 |
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CN115895901A (zh) * | 2021-08-11 | 2023-04-04 | 安琪酵母股份有限公司 | 一种高免疫型酵母细胞壁及制备方法和应用 |
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- 2019-09-05 WO PCT/JP2019/034989 patent/WO2020075424A1/ja active Application Filing
- 2019-09-05 US US17/279,293 patent/US20220211786A1/en active Pending
- 2019-09-05 EP EP19870358.9A patent/EP3865138A4/en active Pending
- 2019-09-05 BR BR112021006128-2A patent/BR112021006128A2/pt unknown
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2022244580A1 (ja) * | 2021-05-21 | 2022-11-24 | アサヒグループホールディングス株式会社 | 免疫賦活剤及びグルカン組成物の製造方法 |
CN115669799A (zh) * | 2021-07-21 | 2023-02-03 | 安琪酵母股份有限公司 | 可替代抗生素和动物蛋白的酵母水解物及制备方法和应用 |
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TW202027757A (zh) | 2020-08-01 |
BR112021006128A2 (pt) | 2021-07-20 |
EP3865138A1 (en) | 2021-08-18 |
JP6530846B1 (ja) | 2019-06-12 |
US20220211786A1 (en) | 2022-07-07 |
CN112805013A (zh) | 2021-05-14 |
JP2020059667A (ja) | 2020-04-16 |
EP3865138A4 (en) | 2022-08-24 |
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