Method for producing processed product of mycelia of hericium erinaceus, and method for improving absorption of erinacine a contained in processed product of mycelia of hericium erinaceus into body

Abstract

According to the present invention, the absorption of erinacine A derived from mycelia of Hericium erinaceus into the body is improved.　Provided is a method for producing a processed product of mycelia of Hericium erinaceus, the method comprising subjecting a culture product of mycelia of Hericium erinaceus which has been cultured in a liquid medium to an enzymatic treatment and a heat treatment in this order. Also provided is a method for improving the absorption of erinacine A contained in a processed product of mycelia of Hericium erinaceus into the body, the method comprising subjecting a culture product of mycelia of Hericium erinaceus which has been cultured in a liquid medium to an enzymatic treatment and a heat treatment in this order.

Description

Method for producing processed Hericium erinaceus mycelium product, and method for improving in vivo absorption of erinacine A contained in processed Hericium erinaceus mycelium product

[0001] The present invention relates to a method for producing a processed Hericium erinaceus mycelium product, and a method for improving in vivo absorption of erinacine A contained in the processed Hericium erinaceus mycelium product.

[0002] According to a report by the Cabinet Office of Japan, the total population of Japan was 124.95 million as of October 1, 2022. The population aged 65 years or older is 36.24 million, and the proportion of this group in the total population (aging rate) reached 29%, the highest level in history. It is projected that the aging rate will continue to rise, reaching 33.3% in 2037, and one out of every three Japanese citizens will be aged 65 years or older (Non-Patent Document 1). On the other hand, among elderly people aged 65 years or older, the number of elderly people with dementia is estimated to increase, and it is projected that there will be approximately 7 million elderly people with dementia in 2025, meaning approximately one out of every five elderly people will have dementia (Non-Patent Document 2). It can be said that the issue of dementia is a social problem that requires urgent countermeasures in Japan, where the aging of the population is progressing.

[0003] In the brains of patients with Alzheimer's dementia, which accounts for the highest proportion of all dementia cases, significant loss of basal forebrain cholinergic neurons, on which nerve growth factor (NGF) acts, is observed. For this reason, it has been pointed out that deficiency of NGF or its receptors is associated with Alzheimer's dementia (Non-Patent Documents 3 and 4). Since NGF, which is a protein, cannot pass through the blood-brain barrier, to achieve the effect of NGF, it is necessary either to inject NGF directly into the brain, or instead of administering NGF itself, to administer a substance that can pass through the blood-brain barrier and enhance NGF synthesis through the action of that substance.

[0004] Erinacins are attracting attention as one of the components that have the effect of inducing NGF production. Erinacins are found, for example, in the secondary metabolites produced by the mycelium of Lion's Mane mushroom (Non-Patent Documents 5-8), and are considered to be among the most potent active substances known to date in vitro. Among the erinacins, erinacin A has been reported to increase NGF in the hippocampus of the brain in rat studies (Non-Patent Document 9), and has also been reported to cross the blood-brain barrier in rat studies (Non-Patent Document 10). Furthermore, it has been reported to have an inhibitory effect on cognitive decline in patients with mild Alzheimer's disease in humans, and is attracting increasing attention as a functional component effective for dementia prevention and cognitive function improvement (Non-Patent Document 11).

[0005] Since erinacins are secondary metabolites produced by Lion's Mane mushroom, they are hardly produced under normal culture conditions, and the expression of related genes is suppressed. Therefore, studies have been conducted to improve the production of erinacin A by adding components and mineral sources commonly used in synthetic culture media when culturing Lion's Mane mycelium in liquid (Non-patent documents 12, 13).

[0006] One inexpensive and efficient way to regularly consume erinacins contained in the mycelium of Lion's Mane mushroom obtained in this way is to ingest crushed Lion's Mane mycelium. However, even when ingesting crushed Lion's Mane mycelium, erinacins are poorly absorbed by the body. Therefore, there is a need to develop methods to improve not only the productivity of erinacins through optimization of the culture medium, but also their absorption into the body.

[0007] White Paper on Aging Society, 2023 Edition; Cabinet Office; Research on Future Projections of the Elderly Population with Dementia in Japan (FY2014 Grant-in-Aid for Scientific Research, Special Research Project, Professor Ninomiya, Kyushu University); Mufson, EJ, Ma SY, Cochran EJ, Bennett, DA, Beckett, LA, Jaffar, S., Saragovi, HU, Kordower, JH 2000. Loss of nucleus basalis neurons containing trkA immunoreactivity in individuals with mild cognitive impairment and early Alzheimer's disease, J. Comp. Neurol. Nov 6; 427(1): 19-30. Furukawa, Shoei, Kawagishi, Hirokazu. Physiological significance of nerve growth factor (NGF) and its synthesis-promoting substances. Chemistry and Biology. 1991, 29, 10, p. 640-646. Kawagishi, H., Shimada, A., Shirai, R. ., Okamoto, K., Ojima, F., Sakamoto, H., Ishiguro, Y. and Furukawa, S. 1994. Erinacines A, B and C, strong stimulators of nerve growth factor (NGF)-synthesis, from the mycelia of Hericium erinaceum. Tetrahedron Lett. 35: 1569-1572. Kawagishi, H., Shimada, A., Hosokawa, S., Mori, H., Sakamoto, H., Ishiguro, Y., Sakemi, S., Bordner, J., Kojima, N. and Furukawa, S. 1996. Erinacines E, F, and G, stimulators of nerve growth factor (NGF)-synthesis, from the mycelia of Hericium erinaceum. Tetrahedron Lett. 37: 7399-7402. Kawagishi, H., Simada, A.,Shizuki,K.,Mori,H .,Okamoto,K.,Sakamoto,H. and Furukawa,S. 1996. Erinacine D,a stimulator of NGF-synthesis, from the mycelia of Hericium erinaceum. Heterocycl. Commun. 2: 51-54.Lee,E. W.,Shizuki,K., Hosokawa,S.,Suzuki,M.,Suganuma,H.,Inakuma,T.,Li,J., Ohnishi-Kameyama,M .,Nagata,T.,Furukawa,S. and Kawagishi,H . 2000. Two novel diterpenoids, erinacines H and I from the mycelia of Hericium erinaceum. Biosci. Biotechnol. Biochem. 64: 2402-2405.Shimbo, M., Kawagishi, H. and Yokogoshi, H. 2005. Erinacine A increases catecholamine and nerve growth factor content in the central nervous system of rats. Nutrition Research. 25: 617-623.Tsai, P-C., Wu, Y-K., Hu, J-H., Li, I-C., Lin, T-W., Chen, C-C. and Kuo, C-F. 2021. Preclinical Bioavailability, Tissue Distribution, and Protein Binding Studies of Erinacine A, a Bioactive Compound from Hericium erinaceus Mycelia Using Validated LC-MS / MS Method. Molecules. 26: 4510.Li, I-C., Chang, H-H., Lin, C-H., Chen, W-P., Lu, T-H., Lee, L-Y., Chen, Y-W., Chen, YP., Che., CC. and Lin, DP-C. 2020. Prevention of Early Alzheimer's Disease by Erinacine A-Enriched Hericium erinaceus Mycelia Pilot Double-Blind Placebo-Controlled Study. Frontiers in Aging Neuroscience. 12: 155.Chang,CH., Chen, Y., Yew, XX., Chen, HX., Kim, JX., Chang, CC., Peng, CC. and Peng, RY 2015. Improvement of erinacine A productivity in Hericium erinaceus mycelia and its neuroprotective bioactivity against the glutamate-insulted apoptosis. LWT - Food Science and Technology. 65: 1100-1108.Krzyczkowski, W. and Malinowska, E., Herold F. 2010. Erinacine A biosynthesis in submerged cultivation of Hericium erinaceum: Quantification and improved cultivation. Eng. Life Sci. 10(5): 446-457.

[0008] This invention has been made in view of the above circumstances, and aims to improve the bioavailability of erinacin A derived from the mycelium of Lion's Mane mushroom.

[0009] In view of these circumstances, the present inventors conducted extensive research and found that enzymatic treatment followed by heat treatment of Lion's Mane mushroom mycelial culture obtained by liquid culture significantly improved the bioavailability of erinacin A contained in the processed Lion's Mane mushroom mycelial culture.

[0010] In other words, the present invention provides the following [1] to [6]: [1] A method for producing a processed lion's mane mushroom mycelium, comprising performing an enzyme treatment and a heat treatment in this order on a liquid-cultured lion's mane mushroom mycelium culture. [2] The method for producing a processed lion's mane mushroom mycelium according to [1], wherein the enzyme treatment is an enzyme treatment using an enzyme having at least one of chitinase activity or chitobiase activity. [3] The method for producing a processed lion's mane mushroom mycelium according to [1] or [2], wherein the heat treatment is a heat treatment at 100°C or higher. [4] The method for producing a processed lion's mane mushroom mycelium according to any one of [1] to [3], wherein the chitinase activity of the enzyme used in the enzyme treatment is 700 U / g or more, and / or the chitobiase activity of the enzyme is 60,000 U / g or more. [5] A method for producing a processed lion's mane mushroom mycelium according to any one of [1] to [4], wherein a grinding treatment is performed at least one of the following stages: before or after the enzyme treatment and after the heat treatment. [6] A method for improving the bioavailability of erinacin A contained in a processed lion's mane mushroom mycelium, wherein an enzyme treatment and a heat treatment are performed in this order on a liquid-cultured lion's mane mushroom mycelium culture.

[0011] Furthermore, the present invention provides the following [7] to

[10] : [7] A method for improving the bioavailability of erinasin A contained in a processed lion's mane mushroom mycelium according to [6], wherein the enzyme treatment is an enzyme treatment using an enzyme having at least one of chitinase activity or chitobiase activity. [8] A method for improving the bioavailability of erinasin A contained in a processed lion's mane mushroom mycelium according to [6] or [7], wherein the heat treatment is a heat treatment at 100°C or higher. [9] A method for improving the bioavailability of erinasin A contained in a processed lion's mane mushroom mycelium according to any one of [6] to [8], wherein the chitinase activity of the enzyme used in the enzyme treatment is 700 U / g or more, or the chitobiase activity of the enzyme is 60,000 U / g or more.

[10] A method for improving the bioavailability of erinacin A contained in the processed mycelial product of Lion's Mane mushroom according to any one of [6] to [9], comprising grinding the Lion's Mane mushroom mycelial culture at at least one of the following stages: before or after the enzyme treatment and after the heat treatment.

[0012] According to the present invention, the bioavailability of erinacin A derived from the mycelium of Lion's Mane mushroom can be improved.

[0013] This is a graph showing the measurement results of the erinasin A content in Test Example 1. This is a graph showing the measurement results of the maximum blood erinasin A concentration in Test Example 2. This is a graph showing the measurement results of the erinasin A content in Test Example 3. This is a graph showing the measurement results of the chitinase activity in Test Example 4. This is a graph showing the measurement results of the chitobiase activity in Test Example 4.

[0014] The present invention will be described below based on examples of embodiments for carrying out the present invention. However, the present invention is not limited to the embodiments described below.

[0015] In this specification, "X and / or Y (where X and Y are any configurations)" means at least one of X and Y, and can mean X only, Y only, or X and Y.

[0016] [Method for Producing Processed Lion's Mane Mushroom Mycelium] One embodiment of the present invention is a method for producing processed Lion's Mane mushroom mycelium, for example, by performing enzyme treatment and heat treatment in this order on a liquid-cultured Lion's Mane mushroom mycelium culture (hereinafter sometimes referred to as "this manufacturing method"). "Processed Lion's Mane Mushroom Mycelium" means a product (processed product) obtained through this manufacturing method, and is a processed product that has undergone at least the steps of enzyme treatment and heat treatment in this order. Specifically, for example, it is a product (processed product) obtained through a manufacturing method that includes, in this order, a liquid culture step of culturing Lion's Mane mushroom mycelium in a liquid medium, an enzyme treatment step of treating the Lion's Mane mushroom mycelium culture obtained in the liquid culture step with an enzyme having at least one of chitinase activity or chitobiase activity, and a heat treatment step of heating the Lion's Mane mushroom mycelium culture. This manufacturing method is characterized by the order of enzyme treatment and heat treatment. By including a step of heat treatment after enzyme treatment, the absorption of erinacin A derived from Lion's Mane mushroom mycelium can be improved in the body.

[0017] In this invention, "Lion's Mane Mushroom Mycelial Culture" refers not only to Lion's Mane Mushroom mycelium (which may be referred to as "cultured mycelium") that is separated and recovered from the liquid culture medium after the liquid culture process, but also to a Lion's Mane Mushroom cultured mycelium-containing composition that includes the cultured mycelium and residues of the manufacturing raw materials used in the liquid culture process. For example, in one embodiment of this invention, after a liquid culture process using a liquid culture medium containing solid components, the cultured mycelium separated and recovered from the liquid culture medium by known separation means such as filtration may contain residues of manufacturing raw materials such as the solid components of the culture medium. Therefore, in this invention, "Lion's Mane Mushroom Mycelial Culture" is a concept that also includes a Lion's Mane Mushroom cultured mycelium-containing composition that includes the cultured mycelium and residues of the manufacturing raw materials.

[0018] The details of this manufacturing method are described below.

[0019] <<Liquid Culture Process>> This manufacturing method includes a liquid culture process for cultivating the mycelium of Lion's Mane mushroom. Lion's Mane mushroom (scientific name: Hericium erinaceus), used in the liquid culture process, is a species of edible mushroom belonging to the genus Hericium in the family Hericaceae, and as mentioned above, it is attracting attention for containing components that are effective in preventing and improving dementia. The Lion's Mane mushroom used in this manufacturing method may be either wild or obtained through artificial cultivation. In this manufacturing method, Lion's Mane mycelium is used, and erinacine A is included in the secondary metabolites produced by the Lion's Mane mycelium.

[0020] The liquid culture medium used in the liquid culture step of this manufacturing method is not particularly limited, but can be appropriately prepared using, for example, a carbon source, a nitrogen source, as well as crushed grain husks, sake lees and beer lees, soybean flour, etc., according to conventional methods.

[0021] <Carbon Source, Nitrogen Source> The liquid culture medium is prepared by dissolving any carbon source, nitrogen source, etc., in purified water. Examples of carbon sources include monosaccharides such as fructose and glucose, disaccharides such as sucrose and maltose, natural polymers such as hemicellulose, starch, and corn starch, and oils and fats such as olive oil. Examples of nitrogen sources include inorganic nitrogen sources such as urea, ammonium chloride, ammonium nitrate, and ammonium sulfate, and organic nitrogen sources such as tryptone, yeast extract, meat extract, peptone, and malt extract. Inorganic salts such as potassium phosphate, magnesium sulfate, and sodium chloride may also be added. Among these, it is preferable to use monosaccharides such as glucose, peptone, and organic nitrogen sources such as yeast extract.

[0022] The total content of monosaccharides and disaccharides is not particularly limited, but is preferably 3 w / v% or more, more preferably 4 w / v% or more, and even more preferably 5 w / v% or more, relative to the total volume of the liquid culture medium. On the other hand, although not limited to the following, is preferably 10 w / v% or less, more preferably 8 w / v% or less, and even more preferably 6 w / v% or less.

[0023] [Glucose] The total glucose content is not particularly limited, but is preferably 3 w / v% or more, more preferably 4 w / v% or more, and even more preferably 5 w / v% or more, relative to the total volume of the liquid medium. On the other hand, although not limited to the following, is preferably 10 w / v% or less, more preferably 8 w / v% or less, and even more preferably 6 w / v% or less.

[0024] The total content of the organic nitrogen source is not particularly limited, but is preferably 0.01 w / v% or more, more preferably 0.02 w / v% or more, and even more preferably 0.03 w / v% or more, relative to the total amount of liquid culture medium. On the other hand, although not limited to the following, is preferably 1 w / v% or less, more preferably 0.8 w / v% or less, and even more preferably 0.7 w / v% or less.

[0025] [Peptone] The total peptone content is not particularly limited, but is preferably 0.01 w / v% or more, more preferably 0.02 w / v% or more, and even more preferably 0.03 w / v% or more, relative to the total amount of liquid culture medium. On the other hand, although not limited to the following, is preferably less than 0.4 w / v%, more preferably 0.1 w / v% or less, and even more preferably 0.08 w / v% or less.

[0026] [Yeast Extract] The total content of yeast extract is not particularly limited, but is preferably 0.01 w / v% or more, more preferably 0.02 w / v% or more, and even more preferably 0.03 w / v% or more, relative to the total amount of liquid culture medium. On the other hand, although not limited to the following, is preferably 1 w / v% or less, more preferably 0.8 w / v% or less, and even more preferably 0.7 w / v% or less.

[0027] <Grain husks> The liquid culture medium may contain grain husks. Grain husks are pulverized by grinding the outer layer of grains using methods commonly practiced in the art. The outer layer of grains is also called bran. Specifically, examples include, but are not limited to, at least one pulverized material selected from the group consisting of wheat bran, rice bran, soybean hulls, and corn seed hulls. Examples of wheat bran include, but are not limited to, wheat bran, barley bran, oat bran, rye bran, etc.

[0028] [Wheat bran] Wheat bran is a by-product produced during the milling process of wheat flour. Any wheat bran produced during the normal milling process is acceptable, and there are no particular limitations on the manufacturing method or type of wheat.

[0029] Specifically, wheat bran refers to the outer layer of wheat grain obtained through the milling process, and is mostly composed of the outer layer, but may also contain small amounts of endosperm and germ. In this manufacturing method, wheat bran obtained through a normal milling process, wheat bran in the form of fine powder obtained by further processing such as grinding, or mixtures thereof can be used. The yield of wheat bran is preferably less than 40% by weight, more preferably 20% by weight or less, and particularly preferably 10 to 15% by weight.

[0030] The type of wheat used as raw material is not particularly limited, and examples include white wheat and red wheat. Using these raw materials, wheat bran is obtained through a grinding process and a separation process, for example. In the grinding process, conventionally known methods such as dry grinding, wet grinding, and freeze grinding are used as appropriate. In the separation process, conventionally known methods such as sieving are used for separation. Furthermore, the wheat bran used in this manufacturing method may be defatted wheat bran obtained through a defatting process, or roasted wheat bran obtained through a roasting process.

[0031] The average particle size of wheat bran is not particularly limited, but is preferably 500 μm or less, more preferably less than 400 μm, and even more preferably less than 300 μm. The lower limit is not particularly limited, but is, for example, 10 μm or more.

[0032] The wheat bran used in the liquid culture medium of this manufacturing method may be any commercially available product. For example, "Wheat Bran MP (wheat bran)" from Nippon Flour Mills Co., Ltd. and "Yume Bran" from Kinoshita Flour Milling Co., Ltd. are available.

[0033] [Rice Bran] Rice bran is a by-product produced during the process of milling brown rice into white rice. Any rice bran produced during the normal rice milling process is acceptable, and there are no particular restrictions on the manufacturing method or type of rice.

[0034] Rice bran is specifically the outer layer of brown rice obtained through the milling process, beyond the endosperm. Rice bran is mostly composed of the outer layer, but may also contain small amounts of endosperm and germ. Specifically, for example, the pericarp, seed coat, and starch layer of brown rice, which are by-products when brown rice is milled to produce white rice, can be used as appropriate. In this manufacturing method, rice bran obtained in a normal milling process, rice bran that has been further processed by crushing or other methods to become a fine powder, or a mixture thereof can be used.

[0035] The type of rice used as raw material is not particularly limited, and examples include non-glutinous rice, glutinous rice, and indica rice. Rice bran is obtained in the rice milling process using these raw materials. The rice bran obtained in the rice milling process may be further crushed. In addition, the rice bran used in this manufacturing method may be defatted rice bran obtained through a defatting process, or roasted rice bran obtained through a roasting process.

[0036] Furthermore, as rice bran, the following types can be used as appropriate: "red bran," which is obtained when rice is milled to a weight milling ratio of 90% or more; "medium white bran," which is obtained when rice is milled to a weight milling ratio of 80% or more but less than 90%; and "white bran," which is obtained when rice is milled to a weight milling ratio of less than 80%. The weight milling ratio is the value calculated by the following formula. Also, "white rice" below refers to brown rice after the above milling. (Formula) Weight milling ratio (%) = (Weight of white rice / Weight of brown rice) × 100

[0037] The average particle size of the rice bran is not particularly limited, but is preferably 100 μm or less, more preferably less than 70 μm, and even more preferably less than 50 μm. The lower limit is not particularly limited, but is, for example, 10 μm or more.

[0038] Various commercially available rice bran products may be used as the liquid culture medium in this manufacturing method. Examples include "Edible Rice Bran" manufactured by Lives Co., Ltd. and "Edible Rice Bran Powder" manufactured by Yuuki Foods Co., Ltd.

[0039] [Soybean hull] Soybean hull is a by-product generated in the production process of various soybean products. There are no particular limitations on the production method or the type of soybean, as long as the soybean hull is produced in the production process of ordinary soybean products.

[0040] Soybean hull is, for example, the seed coat portion of soybeans obtained in the dehulling step during the production of soybean powder, soybean oil, defatted soybeans, or processed soybean products such as soy milk and tofu. In the present production method, soybean hull obtained by an ordinary dehulling step, finely powdered soybean hull obtained by further pulverizing said soybean hull, or a mixture of these is used.

[0041] There is no particular limitation on the type of soybean used as a raw material, and examples thereof include yellow soybean, white soybean, green soybean, black soybean, Kurakake soybean, and the like. Using these raw materials, soybean hull can be obtained through, for example, a dehulling step or an oil pressing step. The soybean hull obtained in said step may be further subjected to a pulverization treatment.

[0042] The average particle diameter of soybean hull is not particularly limited, but is preferably 500 µm or less, more preferably less than 400 µm, and still more preferably less than 300 µm. Although there is no particular limitation on the lower limit value, it is, for example, 10 µm or more.

[0043] As the soybean hull used in the liquid medium of the present production method, various commercially available products may be used. Examples include "Mamefuru Bran" manufactured by Showa Sangyo Co., Ltd., and "Soybean Hull" manufactured by Shimizu Flour Mill Co., Ltd.

[0044] [Content of pulverized cereal hull] The total content of pulverized cereal hull is not particularly limited, but is preferably more than 0.25 w / v% relative to the total amount of the liquid medium. The total content of pulverized cereal hull contained in the liquid medium can be appropriately set within the above range, but relative to the total amount of the liquid medium, it is preferably 0.26 w / v% or more, more preferably 0.3 w / v% or more, still more preferably 0.4 w / v% or more, and particularly preferably 0.6 w / v% or more. On the other hand, although not limited to the following, it is preferably 3 w / v% or less, more preferably 2.75 w / v% or less, still more preferably 2.5 w / v% or less, and particularly preferably 2 w / v% or less. Note that "w / v" is synonymous with "g / 100mL".

[0045] The total content of wheat bran can be appropriately set within the above range, but is preferably 0.26 w / v% or more, more preferably 0.3 w / v% or more, still more preferably 0.4 w / v% or more, particularly preferably 0.6 w / v% or more, based on the total amount of the liquid medium. On the other hand, although not limited to the following, it is preferably 3 w / v% or less, more preferably 2.75 w / v% or less, still more preferably 2.5 w / v% or less, particularly preferably 2 w / v% or less.

[0046] The total content of rice bran can be appropriately set within the above range, but is preferably 0.26 w / v% or more, more preferably 0.3 w / v% or more, still more preferably 0.4 w / v% or more, particularly preferably 0.6 w / v% or more, based on the total amount of the liquid medium. On the other hand, although not limited to the following, it is preferably 3 w / v% or less, more preferably 2.75 w / v% or less, still more preferably 2.5 w / v% or less, particularly preferably 2 w / v% or less.

[0047] The total content of soybean hull can be appropriately set within the above range, but is preferably 0.26 w / v% or more, more preferably 0.3 w / v% or more, still more preferably 0.4 w / v% or more, particularly preferably 0.6 w / v% or more, based on the total amount of the liquid medium. On the other hand, although not limited to the following, it is preferably 3 w / v% or less, more preferably 2.75 w / v% or less, still more preferably 2.5 w / v% or less, particularly preferably 2 w / v% or less.

[0048] <Sake Lees> Sake lees is a by-product generated in the production process of Japanese sake. The sake lees is not particularly limited as long as it is generated in a conventional Japanese sake production process, and there is no particular limitation on the production method, the type of sake, or the like.

[0049] Specifically, sake lees is the solid content remaining after pressing fermented material (moromi) such as raw material rice and koji. In the present production method, sake lees obtained by a conventional pressing step, sake lees in the form of fine powder obtained by further subjecting said sake lees to processing such as pulverization treatment, or a mixture of these can be used.

[0050] The type of rice used as raw material is not particularly limited, and examples include non-glutinous rice, glutinous rice, and indica rice. Sake lees are obtained in the pressing process using these raw materials. The sake lees obtained in the pressing process may be further crushed.

[0051] The sake lees used in the liquid culture medium of this manufacturing method may be any commercially available product. For example, "Junmai Sake Lees" from Hakutsuru Sake Brewing Co., Ltd. and "Takinoizumi" from Yamada Sake Brewing & Food Co., Ltd. are available.

[0052] The total content of sake lees is not particularly limited and can be set as appropriate. For example, the total content of sake lees is preferably 0.1 w / v% or more, more preferably 0.5 w / v% or more, even more preferably 1 w / v% or more, and particularly preferably 2 w / v% or more, relative to the total amount of liquid culture medium. On the other hand, although not limited to the following, it is preferably 15 w / v% or less, more preferably 13 w / v% or less, even more preferably 10 w / v% or less, and particularly preferably 8 w / v% or less.

[0053] <Beer grounds> Beer grounds are a by-product of the beer manufacturing process. Beer grounds can be any by-product of the normal beer manufacturing process, and there are no particular limitations on the manufacturing method or type of malt used.

[0054] Beer lees are the residue discharged during the beer manufacturing process. Specifically, they are the residue obtained by filtering and removing the wort after saccharifying malted barley. In this manufacturing method, beer lees obtained in the normal beer manufacturing process, beer lees that have been further processed by crushing or other methods to become fine powder, or mixtures thereof can be used.

[0055] The type of barley used as raw material is not particularly limited; for example, six-row barley and two-row barley can be used. In the manufacturing process using these raw materials, brewer's lees are obtained. The obtained brewer's lees may be further crushed.

[0056] The average particle size of the brewer's lees used in this manufacturing method is not particularly limited, but is preferably 500 μm or less, more preferably less than 400 μm, and even more preferably less than 300 μm. The lower limit is not particularly limited, but is, for example, 10 μm or more.

[0057] The brewer's lees used in the liquid culture medium of this manufacturing method may be any commercially available product. Examples include "brewer's lees" manufactured by Shimizu Flour Milling Co., Ltd. and "brewer's lees" manufactured by Aoyama Shoten Co., Ltd.

[0058] The total content of beer lees is not particularly limited and can be set as appropriate. For example, the total content of beer lees is preferably 0.26 w / v% or more, more preferably 0.3 w / v% or more, even more preferably 0.4 w / v% or more, and particularly preferably 0.6 w / v% or more, relative to the total amount of liquid culture medium. On the other hand, although not limited to the following, it is preferably 3 w / v% or less, more preferably 2.75 w / v% or less, even more preferably 2.5 w / v% or less, and particularly preferably 2 w / v% or less.

[0059] <Soybean Flour> Soybean flour is a powder obtained by grinding the seeds (cotyledons) of soybeans, which belong to the genus Glycine in the legume family. Specifically, it is a powder obtained by grinding the residue remaining after removing the outer skin from soybeans, or after further removing the endosperm and germ attached thereto.

[0060] The type of soybean used as raw material is not particularly limited, and examples include yellow soybeans, white soybeans, green soybeans, black soybeans, and saddle beans. Using these raw materials, soybean flour is obtained through a dehulling process and a grinding process. For the dehulling process, conventionally known methods, such as using a dehulling machine and a wind separator, are used to dehull the soybeans. For the grinding process, conventionally known methods, such as dry grinding, wet grinding, and freeze grinding, are used as appropriate to grind the soybeans. A more specific example of a soybean flour manufacturing method is, but is not limited to, a method that includes a dehulling process, a heating process, a drying process, and a grinding process. The heating process may be performed before the dehulling process or after the grinding process. Furthermore, the soybean flour used in this manufacturing method may be defatted soybean flour obtained through a defatting process.

[0061] The average particle size of soy flour is not particularly limited, but is preferably 100 μm or less, more preferably 70 μm or less, and even more preferably less than 50 μm. The lower limit is not particularly limited, but is, for example, 5 μm or more. In this invention, the average particle size refers to the volume cumulative particle size D50 (median diameter) at a cumulative volume of 50% when measured dry using a laser diffraction scattering particle size distribution analyzer.

[0062] The soy flour used in the liquid culture medium of this manufacturing method may be any commercially available product. For example, Marukome's "Soybean Nutritional Soy Flour" and Kosei Foods' "Soybean Flour with the Bounty of the Field" are examples of products that can be used.

[0063] The total soy flour content in the liquid culture medium of this manufacturing method is preferably 0.26 w / v% or more, more preferably 0.4 w / v% or more, even more preferably 0.5 w / v% or more, and particularly preferably 0.7 w / v% or more, relative to the total amount of liquid culture medium. On the other hand, it is preferably 2.4 w / v% or less, more preferably 2.3 w / v% or less, even more preferably 2.2 w / v% or less, and particularly preferably 1.5 w / v% or less.

[0064] [Cultivation Conditions] The cultivation conditions typically involve culturing for 7 to 60 days at an initial pH of 2 to 8 and a temperature of 15 to 35°C. When performing aeration and stirring culture, although there may be slight variations depending on the shape of the culture tank, it is appropriate to carry out the culture with an aeration rate of 0.01 to 2.5 vvm (aeration capacity per unit volume per minute) and a stirring speed in the range of 10 to 800 rpm. For example, 400 L of a solution with the above-described component composition is placed in a 500 L fermentation tank, sterilized as usual, and after the temperature of the culture solution has fallen to 25°C or below, 8 L of pre-cultured inoculum prepared by the method described later is inoculated. Culturing at 25°C and an aeration rate of 0.5 vvm for 30 days can yield a culture of Lion's Mane mycelium.

[0065] Furthermore, since erinacin A is a secondary metabolite produced by Lion's Mane mushroom, and is usually hardly produced before the mid-logarithmic growth phase, it is preferable to cultivate the mushroom at least until the late logarithmic growth phase when considering the productivity of erinacin A. More preferably, the cultivation should extend beyond the late logarithmic growth phase to the stationary phase (quiescent phase) and even more preferably beyond the stationary phase (quiescent phase) to the death phase. If cultivation is stopped before reaching the late logarithmic growth phase, although the weight of the Lion's Mane mycelium (cell weight) increases, the production of erinacin A, a secondary metabolite, is small, and it is not possible to obtain a large amount of erinacin A.

[0066] The "logarithmic growth phase" refers to the stage in which cells divide and proliferate at regular intervals, and the logarithm of the number of cells relative to time becomes a straight line rising to the right. This phase can be divided into the "early phase," where the rate of cell proliferation begins to increase from the induction phase; the "mid-phase," where cells proliferate exponentially and exhibit the aforementioned linear proliferation; and the "late phase," before the rate of proliferation begins to decline and the stationary phase is reached. The "late logarithmic growth phase" refers to the stage in which the rate of cell proliferation begins to decline, and more precisely, before the actual proliferation falls below the linear proliferation shown in the growth curve and the phase transitions to the stationary phase.

[0067] The incubation period varies depending on conditions such as incubation temperature, pH, medium composition, and degree of aeration (stirring), but can be appropriately adjusted within a range of 7 to 60 days, for example, from the late logarithmic growth phase to the death phase. From the viewpoint of significantly achieving the effects of the present invention, it can be appropriately adjusted within a range of 10 to 30 days, for example, from the stationary phase to the death phase.

[0068] The culture conditions in this manufacturing method are not particularly limited, except for the culture time conditions mentioned above, and are set according to known methods. For example, the pH of the liquid medium can be appropriately adjusted within the range of pH 2 to 8. From the viewpoint of significantly achieving the effects of the present invention, it is preferable to appropriately adjust the pH within the range of pH 4.5 to 7.

[0069] Furthermore, the culture temperature can be appropriately adjusted within the range of 15 to 35°C, and it is preferable to adjust it appropriately within the range of 20 to 30°C, for example.

[0070] [Cultivation Method] The cultivation method in this manufacturing method is not limited, but examples include a cultivation method that includes a pre-pre-culturing step, a pre-culturing step, and a main cultivation step. Specifically, for example, a cultivation method that includes step I (pre-pre-culturing step) of inoculating Lion's Mane mycelium onto an agar plate and culturing it, step II (pre-culturing step) of inoculating the Lion's Mane mycelium cultivated in step I into a liquid medium such as in a flask and culturing it, and step III (main cultivation step) of inoculating the Lion's Mane mycelium cultivated in step II into a liquid medium in a culture tank and culturing it. Furthermore, the cultivation method may also include step IV of removing the supernatant (water) of the liquid medium containing the Lion's Mane mycelium cultivated in step III to obtain a Lion's Mane mycelium culture.

[0071] More specifically, the mycelium of Lion's Mane mushroom is inoculated onto an agar plate and cultured for approximately 7 to 14 days at an appropriate temperature of 15 to 32°C. Then, the mycelium is inoculated into a culture medium in a flask and cultured with shaking for approximately 3 to 5 days at a temperature of 20 to 30°C, pH 4.5 to 6.5, and a shaking speed of 100 to 250 rpm until the early logarithmic growth stage. Subsequently, the culture from the flask is inoculated into a culture medium in a culture tank and cultured at a temperature of approximately 22 to 32°C and a density of 0.8 to 1.2 kg / cm³. 2 Under tank pressure and pH 4.5 to 5.5 conditions, a predetermined gas is introduced at a gas flow rate of 0.5 to 1 vvm, and the culture is carried out for 7 to 30 days at a stirring speed of 10 to 150 rpm to obtain a culture solution containing cultured mycelium and supernatant. From the culture solution thus obtained, the supernatant can be removed, for example, using a filter press, to obtain a culture of Lion's Mane mycelium.

[0072] <<Enzyme Treatment Process>> The enzyme treatment process in this manufacturing method is a process of performing enzyme treatment on a culture of Lion's Mane mushroom mycelium containing erinacin A. In detail, the enzyme treatment process is a process of treating the cell wall of the cultured Lion's Mane mushroom mycelium with enzymes, and is a process in which some or all of the components that make up the cell wall, such as chitin and glucan, are broken down by the enzyme.

[0073] The enzymes used in the enzymatic treatment process are not particularly limited as long as they have the effect of degrading the cell wall of the Lion's Mane mushroom mycelium, but chitinases and β-glucanases are preferred. Specifically, chitinases and chitobiases are particularly preferred as enzymes to be used in the enzymatic treatment process. Chitinases and chitobiases can be used individually or in combination. For example, Denatzyme CBB-P1 / R (manufactured by Nagase Vita Co., Ltd.) can be used as a chitinase, but purified chitinases and chitobiases are difficult to obtain, so generally enzyme preparations such as cellulases, hemicellulases, and pectinases that have chitinase activity and chitobiase activity can be used. Examples include hemicellulase Amano 90 (manufactured by Amano Enzyme Co., Ltd.), cellulosin TP25 (manufactured by HBI Co., Ltd.), soluble pectinase T (manufactured by HBI Co., Ltd.), tunicase SP-EN (manufactured by Amano Enzyme Co., Ltd.), cellulase T "Amano" 4 (manufactured by Amano Enzyme Co., Ltd.), and cellulosin HC100 (manufactured by HBI Co., Ltd.).

[0074] The enzyme used in the enzymatic treatment process is preferably an enzyme having at least one of chitinase activity or chitobiase activity, from the viewpoint of improving the bioavailability of erinacin A derived from the mycelium of Lion's Mane mushroom. Specifically, the chitinase activity of the enzyme is preferably 700 U / g or more, more preferably 800 U / g or more, even more preferably 1,000 U / g or more, particularly preferably 1,100 U / g or more, and most preferably 1,200 U / g or more. Furthermore, there is no particular upper limit to the chitinase activity of the enzyme, but it is usually 10,000 U / g or less. Furthermore, the enzyme used in the enzyme treatment process preferably has a chitobias activity of 60,000 U / g or more, more preferably 70,000 U / g or more, even more preferably 80,000 U / g or more, particularly preferably 100,000 U / g or more, and most preferably 120,000 U / g or more, from the viewpoint of improving the bioavailability of erinacin A derived from Lion's Mane mycelium. The upper limit of the chitobias activity of the enzyme is not particularly limited, but is usually 1,000,000 U / g or less.

[0075] In one example of the embodiment of this manufacturing method, although not limited to the following, it is preferable that the chitinase activity of the enzyme used in the enzyme treatment step is 800 U / g or more and the chitobiase activity is 70,000 U / g or more, more preferably that the chitinase activity of the enzyme used in the enzyme treatment step is 1,000 U / g or more and the chitobiase activity is 80,000 U / g or more, and even more preferably that the chitinase activity of the enzyme used in the enzyme treatment step is 1,100 U / g or more and the chitobiase activity is 100,000 U / g or more.

[0076] The chitinase activity of the enzyme is measured by defining 1 U as the amount of enzyme that releases 1 μmol of N-acetylglucosamine per minute, and is specifically measured by the method described in the test examples below. The chitobias activity of the enzyme is measured by defining 1 U as the amount of enzyme that releases 1 μmol of p-nitrophenol per minute, and is specifically measured by the method described in the test examples below.

[0077] The amount of enzyme added in the enzyme treatment process should be appropriately set according to the type of enzyme and the amount of cultured mycelium of Lion's Mane mushroom that is the target of the enzyme treatment. For example, the amount of enzyme added is about 0.001 to 1 part by mass per 100 parts by mass of solid matter (containing cultured mycelium) obtained by filtering the culture solution obtained after the liquid culture process, and preferably 0.01 to 0.1 parts by mass. It is also preferable that the chitinase activity is 0.6 U / mL or more and the chitobiase activity is 50 U / mL or more relative to the enzyme treatment solution.

[0078] The temperature, time, and pH conditions in the enzyme treatment process are set appropriately according to the type of enzyme, etc. The temperature is usually around 20 to 60°C, with 30 to 50°C being preferred. The time is usually around 0.5 to 5 hours, with 1 to 3 hours being preferred. The pH is usually around 3 to 9, with 4 to 8 being preferred, taking into consideration the optimal pH.

[0079] Furthermore, the enzyme treatment step may be performed on the culture medium obtained in the liquid culture step, or on the Hericium erinaceus mycelium culture separated and recovered from the culture medium obtained in the liquid culture step. Specifically, for example, the enzyme treatment may be performed on the residue (containing cultured mycelium) obtained by filtering the culture medium obtained in the liquid culture step.

[0080] <<Heat Treatment Process>> This manufacturing method improves the bioavailability of erinacin A derived from Lion's Mane mushroom mycelium by performing a heat treatment process after the enzyme treatment process described above. As mentioned above, this manufacturing method is characterized by the order of enzyme treatment and heat treatment, and by including a step of heat treatment after enzyme treatment, the bioavailability of erinacin A derived from Lion's Mane mushroom mycelium can be improved. Conversely, if enzyme treatment is performed after heat treatment, the excellent effect of this manufacturing method cannot be obtained.

[0081] The specific method of heat treatment is not particularly limited and may be either wet or dry heat treatment. Examples include wet heat treatments such as steam heating (including high-pressure steam heating), superheated steam treatment, boiling, and steaming, and dry heat treatments such as direct flame heating, hot air treatment, electric heating, infrared heating, electromagnetic heating, and high-frequency heating. Specifically, for example, one method is to add water to the mycelial culture of Lion's Mane mushroom after enzyme treatment and then perform heat treatment using an autoclave or heat block.

[0082] The heat treatment temperature is usually 80°C or higher, preferably 90°C or higher, and more preferably 100°C or higher. Furthermore, the heat treatment temperature conditions are preferably 80 to 150°C, even more preferably 85 to 140°C, and particularly preferably 90 to 130°C.

[0083] The heat treatment time can be set appropriately according to the heating temperature, but for example, it is 0.5 hours or more, preferably 1 to 10 hours, and more preferably 3 to 7 hours.

[0084] <<Drying Process>> In this manufacturing method, it is preferable to perform a drying process before the grinding process described later, in order to facilitate the grinding process, finely grind the material, and turn it into a powder. The drying process may be performed before the heating process, and the drying process is not a mandatory step and may be omitted.

[0085] As for the drying process, known methods can be used as appropriate, such as natural drying, heat drying, spray drying, and freeze-drying. For freeze-drying, for example, a known freezer is used to perform a freezing process in an atmosphere set to -80°C or below, followed by freeze-drying by vacuum drying. The vacuum level is, for example, in the range of 0 to 20 Pa.

[0086] <<Grinding Process>> In this manufacturing method, from the viewpoint of further improving the bioavailability of erinacin A derived from Lion's Mane mushroom mycelium, it is preferable to perform the grinding process at at least one of the following stages: before or after the enzyme treatment process, or after the heat treatment process. That is, it is preferable to perform the grinding process at at least one of the following stages: before the enzyme treatment process, after the enzyme treatment process, or after the heat treatment process. Specifically, for example, it is preferable to perform the grinding process in this order on the Lion's Mane mushroom mycelium processed product after the heat treatment process. Specifically, it is preferable to perform enzyme treatment and heat treatment in this order on the Lion's Mane mushroom mycelium culture separated and recovered from the liquid medium, and then perform the grinding process to obtain the Lion's Mane mushroom mycelium processed product.

[0087] As for the grinding process, known methods can be used as appropriate, such as hammer mill grinding, cutter mill grinding, pin mill grinding, jet mill grinding, ball mill grinding, or grinding using a mortar and pestle or homogenizer pestle.

[0088] The particle size of the Hericium erinaceus mycelium culture and / or processed Hericium erinaceus mycelium after pulverization is, for example, 250 μm or less, and is measured using a sieve.

[0089] In one embodiment of the present invention, from the viewpoint of further improving the bioavailability of erinacin A derived from Lion's Mane mycelium, a manufacturing method is preferred in which the enzyme treatment step, heat treatment step, and pulverization step are carried out in this order. Similarly, from the viewpoint of further improving the bioavailability of erinacin A derived from Lion's Mane mycelium, a manufacturing method is preferred in which the enzyme treatment step, heat treatment step, freeze-drying step, and pulverization step are carried out in this order. In addition, this manufacturing method may further include a sterilization step, but it is preferable that the sterilization step be incorporated into the heat treatment step.

[0090] <<Composition containing cultured lion's mane mushroom mycelium>> The composition containing cultured lion's mane mushroom mycelium preferably has cultured mycelium as its main component. Typically, the proportion of cultured mycelium to the total amount of the composition is 80% by mass or more, preferably 90% by mass or more, and more preferably 95% by mass or more. As described above, in one embodiment of the present invention, for example, after a liquid culture step using a liquid culture medium containing solids, the cultured mycelium separated and recovered from the liquid culture medium by known separation means such as filtration may contain residual manufacturing raw materials such as solids from the culture medium raw materials. However, it is preferable that the proportion of residues in the lion's mane mushroom cultured mycelium composition containing cultured mycelium and residues of manufacturing raw materials be as low as possible.

[0091] Furthermore, since the processed lion's mane mushroom mycelium obtained by this manufacturing method exhibits significantly improved in vivo absorption of erinasin A derived from lion's mane mushroom mycelium, it is preferable to use it as a processed lion's mane mushroom mycelium for promoting erinasin A absorption in embodiments of the present invention.

[0092] <<Applications>> The processed lion's mane mushroom mycelium obtained by this manufacturing method can be suitably used as a raw material for various foods, supplements, quasi-drugs, and pharmaceuticals. For example, it can be suitably used as a food material consisting of dried and pulverized material obtained by drying and pulverizing the processed lion's mane mushroom mycelium obtained by this manufacturing method, or as a food composition containing the said food material. The food composition may optionally contain known raw materials, such as excipients, additives, binders, thickeners, emulsifiers, colorants, flavorings, and seasonings.

[0093] [Method to improve the bioavailability of erinasin A] As an example of an embodiment of the present invention, a method to improve the bioavailability of erinasin A contained in processed lion's mane mushroom mycelium is performed in this order on a liquid-cultured lion's mane mushroom mycelium culture by enzymatic treatment and heat treatment. By performing each step and treatment described in the above manufacturing method, the bioavailability of erinasin A contained in processed lion's mane mushroom mycelium can be improved.

[0094] [Hetericium erinaceus mycelium processed product] As an example of an embodiment of the present invention, a Hetericium erinaceus mycelium processed product is obtained by performing enzyme treatment and heat treatment in this order on a liquid-cultured Hetericium erinaceus mycelium culture. By performing each step and treatment described in the above manufacturing method, a Hetericium erinaceus mycelium processed product with excellent bioavailability of erinasin A can be obtained. Another example of an embodiment of the present invention is a Hetericium erinaceus mycelium processed product for promoting erinasin A absorption, obtained by performing enzyme treatment and heat treatment in this order on a liquid-cultured Hetericium erinaceus mycelium culture.

[0095] The present invention will be described in more detail below with reference to examples and comparative examples, but the present invention is not limited to the following examples unless it exceeds the gist of the invention.

[0096] <<Test Example 1: Extractability Test of Elinasine A>> Test Example 1 is a test demonstrating the improvement in extraction efficiency of elinasine A by performing a heat treatment step after an enzyme treatment step in the manufacturing process.

[0097] [Test Species] We used strain Y3 of Hericium erinaceus, which was provided by the Nagano Prefectural Forestry Research Center.

[0098] The pre-culture step, pre-culture step, and main culture step were carried out sequentially according to the method shown below.

[0099] [Pre-culturing process] Potato dextrose agar medium (manufactured by Nissui Pharmaceutical Co., Ltd.), autoclaved at 121°C for 20 minutes, was dispensed in 10-15 mL portions into a sterile petri dish, cooled and solidified to prepare agar medium (1). Using a cork borer (7 mm), agar medium containing subcultured Lion's Mane mycelium (strain Y3, a strain transferred from the Nagano Prefectural Forestry Research Center) was punched out, inoculated onto the agar medium (1) prepared above, and cultured at 25°C for approximately 7-14 days.

[0100] [Pre-culture step, Main culture step] A 500 mL baffled Erlenmeyer flask with a pH of approximately 6.1, as shown in Table 1 below, was filled (20% by weight of the flask volume), and autoclaved at 121°C for 20 minutes to prepare liquid culture medium (2). Three pieces of agar medium (1) obtained in the pre-culture step were punched out with a cork borer (7 mm), and inoculated into the liquid culture medium (2) (pre-culture step). The solution cultured for 7 days was used as the pre-culture solution, and 2% of the pre-culture solution was inoculated into a 500 mL baffled Erlenmeyer flask filled with the culture medium shown in Table 1 below. Rotary shaking culture was performed for 14 days at 25°C, 125 rpm, in the dark using a constant temperature shaking incubator (TAITEC, BR-3000LF) (Main culture step). The wheat bran added to the liquid culture medium was manufactured by Yuutech Co., Ltd., and the sake lees used were Hakutsuru Junmai sake lees manufactured by Hakutsuru Sake Brewing Co., Ltd.

[0101]

[0102] [Preparation of Control] The control is an example in which only the heat treatment process is performed. Specifically, the residue (cultured mycelium) obtained by vacuum filtration of the main culture medium was hydrated, and after heat treatment at 100°C for 3 hours in a heat block, a solid was obtained by vacuum filtration.

[0103] [Preparation of Comparative Examples 1-1 and 1-2] Comparative Examples 1-1 and 1-2 show examples in which an enzyme treatment step is performed after the heat treatment step. Specifically, the residue obtained by vacuum filtration of the culture medium was added to water, and after heat treatment at 100°C for 3 hours in a heat block, an enzyme treatment (37°C for 1 hour) was performed (enzyme treatment step). Next, the filtrate was removed by vacuum filtration to obtain a solid product (Lion's Mane mushroom mycelium processed product). In the enzyme treatment step, the enzyme (E1) or (E2) described below was used, and 4.0 mg of the enzyme was added to 2.5 g of the residue obtained by vacuum filtration of the culture medium (value after subtracting the weight of solids mixed in the liquid medium (sake lees and wheat bran)), and the solution was adjusted to 5 mL with 20 mM acetate buffer to prepare the enzyme reaction solution. The enzyme concentration in the enzyme reaction solution was 0.8 mg / ml (20 mM acetate buffer). • Enzyme (E1) "Hemicellulase Amano 90" (manufactured by Amano Enzyme Co., Ltd., derived from Aspergillus niger) • Enzyme (E2) "Cellulosin TP25" (manufactured by HBI Co., Ltd., derived from Trichoderma Reesei)

[0104] [Preparation of Examples 1-1 and 1-2] Examples 1-1 and 1-2 show examples in which an enzyme treatment step is performed first, followed by a heat treatment step. Specifically, the residue obtained by vacuum filtration of the culture medium was subjected to enzyme treatment (37°C, 1 hour) (enzyme treatment step), and then heat treatment was performed in a heat block at 100°C for 3 hours (heat treatment step). Next, the filtrate was removed by vacuum filtration to obtain a solid (Lion's Mane Mushroom mycelium processed product according to the present invention). In the enzyme treatment step, the enzyme (E1) or (E2) described above was used, and 4.0 mg of the enzyme was added to 2.5 g of the residue obtained by vacuum filtration of the culture medium (value after subtracting the weight of solids blended in the liquid medium (sake lees and wheat bran)), and the solution was adjusted to 5 mL with 20 mM acetate buffer to prepare the enzyme reaction solution. The enzyme concentration in the enzyme reaction solution was also 0.8 mg / ml (20 mM acetate buffer) as described above.

[0105] [Measurement of Elinacin A content] Each solid obtained above was ground using a Biomassher SP (manufactured by Nippi Corporation), liquid-liquid extraction was performed with water and hexane, the hexane layer was discarded, and liquid-liquid extraction was performed with water and ethyl acetate. The ethyl acetate layer (containing Elinacin A) was recovered and dried by centrifugal concentration. It was then dissolved in 1 mL of methanol and used as an HPLC analysis sample. Elinacin A standard was prepared according to Non-Patent Document 13. HPLC analysis was performed under the following conditions to quantify the amount of Elinacin A contained per 1 g of wet cell weight. The results are shown in Table 2 and Figure 1. Note that the wet cell weight is the value obtained by subtracting the weight of the solids (sake lees and wheat bran) added to the liquid culture medium. [HPLC Conditions] • Column: Capcellpak C18 AQ Φ4.6 mm × 250 mm (Osaka Soda Co., Ltd.) • Solvent: 80% methanol • Detection: UV (λ=340 nm) • Flow rate: 0.5 mL / min • Column temp.: 30°C • Inject volume: 10 μL

[0106]

[0107] As shown in Table 2 and Figure 1, the amount of erinacin A contained in the processed lion's mane mushroom mycelium was significantly increased when the enzyme treatment was performed first, followed by the heat treatment (Examples 1-1 and 1-2), compared to when only the heat treatment process was performed (control) or when the enzyme treatment process was performed after the heat treatment process (Comparative Examples 1-1 and 1-2).

[0108] In detail, comparing the case where only the heat treatment step is performed (control) with the case where the enzyme treatment step is performed after the heat treatment step (Comparative Examples 1-1 and 1-2), the increase in the amount of erinasin A extracted from the processed lion's mane mushroom mycelium was slight (for example, an increase of about 4.4% in Comparative Example 1-1). However, when the enzyme treatment step was performed first, followed by the heat treatment step (Examples 1-1 and 1-2), the erinasin A content in the processed lion's mane mushroom mycelium increased significantly (for example, an increase of about 31% in Example 1-1). Thus, it was shown that the manufacturing method, in which the enzyme treatment step is performed first, followed by the heat treatment step, significantly improves the extraction efficiency of erinasin A derived from lion's mane mushroom mycelium.

[0109] <<Test Example 2: In vivo absorption test (measurement of blood erinacin A concentration)>> Test Example 2 is a test that demonstrates the improvement in the in vivo absorption of erinacin A by a manufacturing method that involves a heat treatment step after an enzyme treatment step.

[0110] The main culture process was carried out according to the method shown below. The pre-culture and pre-culture processes were performed in the same manner as in Test Example 1.

[0111] [Main Culture Process] 2% of the pre-culture solution was inoculated into a 3L jar fermenter (manufactured by Marubishi Bioengin Co., Ltd.) filled with the culture medium shown in Table 1, and cultured with rotational shaking at 25°C, 110-125 rpm, in the dark for 14 days (main culture process). The wheat bran added to the liquid culture medium was wheat bran manufactured by Yutec Co., Ltd., and the sake lees used was Hakutsuru Junmai sake lees manufactured by Hakutsuru Sake Brewing Co., Ltd.

[0112] [Preparation of Control] The control is an example in which only freeze-drying and grinding processes are performed, without heat treatment or enzyme treatment. Specifically, the residue obtained by vacuum filtration of the culture solution was freeze-dried and then ground in a mill (Panasonic MX-X108) (conditions: 15 seconds x 3 times). After that, it was passed through a 250 μm mesh to obtain a powdered processed product of Lion's Mane mushroom mycelium. For the freeze-drying process, the product was frozen in a freezer set to -80°C, and the frozen product was vacuum-dried under conditions of 6 Pa.

[0113] [Preparation of Comparative Example 2-1] Comparative Example 2-1 shows an example in which a heat treatment process is performed, followed by a freeze-drying process and a grinding process. Specifically, the residue obtained by vacuum filtration of the culture solution from Test Example 1 was mixed with water, and the mixture was heat-treated at 100°C for 3 hours in a heat block. The residue obtained by vacuum filtration was freeze-dried and then ground in a mill (Panasonic MX-X108) (conditions: 15 seconds x 3 times). After that, it was passed through a 250 μm mesh to obtain a powdered Hericium erinaceus mycelium product. For the freeze-drying process, the mixture was frozen in a freezer set to -80°C, and the frozen product was vacuum-dried under conditions of 6 Pa.

[0114] [Preparation of Example 2-1] Example 2-1 shows an example in which an enzyme treatment step is performed first, followed by a heat treatment step, and then a freeze-drying step and a grinding step are performed in that order. Specifically, the residue obtained by vacuum filtration of the culture solution from Test Example 1 was subjected to enzyme treatment (37°C, 1 hour) (enzyme treatment step), the culture solution after enzyme treatment was heat-treated at 100°C for 3 hours in a heat block (heat treatment step), and the residue obtained by vacuum filtration and removal of the filtrate was freeze-dried and then ground in a mill (Panasonic MX-X108) (conditions: 15 seconds x 3 times). After that, it was passed through a mesh with a mesh opening of 250 μm to obtain a powdered Hericium erinaceus mycelium processed product according to the present invention. In the freeze-drying step, the product was frozen in a freezer set to -80°C, and the frozen product was vacuum-dried under conditions of 6 Pa. Furthermore, the enzyme used in the enzyme treatment process was the aforementioned enzyme (E1), and the enzyme concentration was 0.8 mg / mL (20 mM acetate buffer).

[0115] [Measurement of blood erinacin A concentration] The concentration of erinacin A in the blood of animals that ingested powdered Lion's Mane mushroom mycelium was measured by the following test.

[0116] [1] About the animals used in the study: • Animals used (Crlj:WI rat, male, genotype: WT, Jackson Laboratory Japan). • Age at start of study: 7 weeks old. • Feed: CRF-1 (15 kGy gamma ray irradiation, manufactured by Oriental Yeast Co., Ltd.) • Bedding: White flakes (manufactured by Jackson Laboratory Japan). • Drinking water: Ad libitum.

[0117] [2] Name and composition of the test area

[0118] [3] Weight measurement and group division After a one-week acclimatization period, weight measurements were taken at 8 weeks of age, and the groups were formed (6 animals per group) so that the average weight of each group was equal. [4] Preparation and administration method of the test substance (Lion's Mane mushroom mycelium processed product) At 8 weeks of age, each test substance (Example 2-1, Comparative Example 2-1, or control) was prepared by the following method and administered. ・Test substance: Powdered Lion's Mane mushroom mycelium processed product ・Preparation method: 8.0 g of the test substance was added to 100 mL of 0.5% methylcellulose, a suspension was prepared using a mortar and pestle, and the concentration was adjusted to 0.08 g / mL. • Route: Oral administration • Dosage: 40 mL / kg BW (3.2 g / kg BW of processed lion's mane mushroom mycelium) • Number of administrations: Single dose [5] Blood collection After administration of the test substance, blood was collected from the jugular vein with heparin as an anticoagulant at the specified times (0 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours). The collected blood was centrifuged (4°C, 10,000 rpm, 5 minutes) and 100 μL of plasma was collected. The plasma was frozen and stored at -80°C until measurement.

[0119] [6] Measurement of blood elinacin A concentration In accordance with Non-Patent Document 10, elinacin A was extracted from plasma with ethyl acetate, the ethyl acetate layer was collected and dried, and the sample was prepared. The blood elinacin A concentration was then analyzed using LC-MS / MS. The results are shown in Table 4 and Figure 2. <Equipment Used> ・ACQUITY UPLC H-Class PLUS (Waters) ・Xevo TQ-S micro (Waters) ・N2 Supplier Model T30F (System Instruments) <Measurement Conditions> ・Column: InertSustain Phenyl, particle size 5 μm, Φ2.1 × 150 mm (GL Sciences) ・Solvent: 0-8 min: Water:Methanol = 40:60 8.1-12 min: 100% methanol 12.1-15 min: Water:Methanol = 40:60 ・Flow rate: 0.4 mL / min ・Injection volume: 2 μL <Internal Standard> ・2,4,5-Trimethylbenzoic acid (Tokyo Chemical Industries, Ltd.)

[0120]

[0121] As shown in Table 4 and Figure 2, the amount of erinasin A absorbed into the blood increased when the enzyme treatment step was performed, followed by the heat treatment step, and then the freeze-drying and grinding steps (Example 2-1), compared to the case where only the freeze-drying and grinding steps were performed without the heat treatment or enzyme treatment steps (control), or when the freeze-drying and grinding steps were performed after the heat treatment step (Comparative Example 2-1).

[0122] Specifically, in the control group, i.e., the group administered the processed lion's mane mushroom mycelium obtained through the freeze-drying and grinding processes, no erinasin A was detected in the blood. This indicates that simply freeze-drying or grinding the lion's mane mushroom mycelium culture results in very little erinasin A being absorbed into the bloodstream. Furthermore, in the group of Comparative Example 2-1, i.e., the group administered the processed lion's mane mushroom mycelium obtained through the heat treatment process followed by the freeze-drying and grinding processes, erinasin A was absorbed into the bloodstream, but the amount was less than in the group of Example 2-1. In other words, in the group administered the processed lion's mane mushroom mycelium obtained through the enzyme treatment process followed by the heat treatment process, freeze-drying process, and grinding process, the amount of erinasin A absorbed into the bloodstream increased by approximately 2.67 times compared to the group of Comparative Example 2-1, demonstrating an improvement in the amount of erinasin A absorbed into the bloodstream. Furthermore, in both the Comparative Example 2-1 group and the Example 2-1 group, the blood erinasin A concentration reached its maximum 4 hours after administration. Therefore, it was found that processing the mycelium of Lion's Mane mushroom obtained by a manufacturing method involving first performing an enzyme treatment step followed by a heat treatment enhances the absorption of erinasin A into the body.

[0123] <<Test Example 3: Intracellular Absorption Test (Artificial Digestive Solution Test)>> Test Example 3 was conducted in accordance with the following references. This test demonstrates the improvement in extraction efficiency of erinacin A by using a manufacturing method that involves a heat treatment step after an enzyme treatment step. <References> Minekus, M., Alminger, M., Alvito, P., Ballance, S., Bohn, T., Bourlieu, C., Carriere, F., Boutrou, R., Corredig, M., Dupont, D., Dufour, C., Egger, L., Golding, M., Karakaya, S., Kirkhus, B., Le Feunteun, S., Lesmes, U., Macierzanka, A., Mackie, A., Marze, S., McClements, DJ., Menard, O., Recio, I., Santos, CN., Singh, RP., Vegarud, GE., Wickham, MSJ., Weitschies, W. and Brodkorb, A. 2014. A standardized static in vitro digestion method suitable for food‐an international consensus. Food Func. 5: 1113-1124.

[0124] [Preparation of Control] The control represents an example in which no heat treatment or enzyme treatment is performed at all. Specifically, the residue obtained by vacuum filtration of the culture medium from Test Example 1 was mixed with artificial digestate (oral phase) and reacted (37°C, 5 minutes), then artificial digestate (gastric phase) was mixed and reacted (37°C, 2 hours), then artificial digestate (intestinal phase) was mixed and reacted (37°C, 2 hours), and finally solid matter was removed by vacuum filtration to obtain the filtrate.

[0125] [Preparation of Comparative Example 3-1] Comparative Example 3-1 shows an example in which only the heat treatment step is performed and the enzyme treatment step is not performed. Specifically, the residue obtained by vacuum filtration of the culture solution from Test Example 1 was mixed with water, and the mixture was heat-treated at 100°C for 3 hours in a heat block, and then vacuum filtration was performed to obtain a solid (Lion's Mane mushroom mycelium processed product). Artificial digestant (oral phase) was added to the solid and reacted (37°C, 5 minutes), then artificial digestant (gastric phase) was added and reacted (37°C, 2 hours), and then artificial digestant (intestinal phase) was added and reacted (37°C, 2 hours), and the solid was removed by vacuum filtration again to obtain a filtrate.

[0126] [Preparation of Comparative Example 3-2] Comparative Example 3-2 shows an example in which a heat treatment process and a grinding process are performed, but an enzyme treatment process is not performed. Specifically, the residue obtained by vacuum filtration of the culture solution of Test Example 1 was mixed with water, and the mixture was heat-treated at 100°C for 3 hours in a heat block. The residue obtained by vacuum filtration was freeze-dried and then ground in a mill (Panasonic MX-X108) (conditions: 15 seconds x 3 times). After that, it was passed through a 250 μm mesh to obtain a powdered Hericium erinaceus mycelium product. Artificial digestant (oral phase) was added to the powdered Hericium erinaceus mycelium product and reacted (37°C, 5 minutes), then artificial digestant (gastric phase) was added and reacted (37°C, 2 hours), and then artificial digestant (intestinal phase) was added and reacted (37°C, 2 hours). Solid matter was removed by vacuum filtration to obtain a filtrate.

[0127] [Preparation of Example 3-1] Example 3-1 shows an example in which a heat treatment step is performed after the enzyme treatment step. Specifically, the residue obtained by vacuum filtration of the culture solution from Test Example 1 was subjected to enzyme treatment (37°C, 1 hour) (enzyme treatment step), then heat treatment was performed in a heat block at 100°C for 3 hours (heat treatment step), and a solid (Lion's Mane mushroom mycelium processed product according to the present invention) was obtained by vacuum filtration. Artificial digestant (oral phase) was added to the solid and reacted (37°C, 5 minutes), then artificial digestant (gastric phase) was added and reacted (37°C, 2 hours), and then artificial digestant (intestinal phase) was added and reacted (37°C, 2 hours), and the solid was removed by vacuum filtration again to obtain the filtrate. The enzyme used was the enzyme (E1) described above, and the enzyme concentration in the enzyme reaction solution was 0.8 mg / mL (20 mM acetate buffer).

[0128] [Preparation of Example 3-2] Example 3-2 shows an example in which an enzyme treatment step is performed first, followed by a heat treatment step, and then a grinding treatment step. Specifically, the residue obtained by vacuum filtration of the culture solution from Test Example 1 was subjected to enzyme treatment (37°C, 1 hour) (enzyme treatment step), then heat treatment was performed in a heat block at 100°C for 3 hours (heat treatment step), and solid material was obtained by vacuum filtration. The solid material was crushed in a mill (Panasonic MX-X108) (15 seconds x 3 times), passed through a 250 μm mesh to obtain a powdered Hericium erinaceus mycelium product according to the present invention. Artificial digestate (oral phase) was added to the powdered Hericium erinaceus mycelium product according to the present invention and reacted (37°C, 5 minutes), then artificial digestate (gastric phase) was added and reacted (37°C, 2 hours), and then artificial digestate (intestinal phase) was added and reacted (37°C, 2 hours), and the solid material was removed by vacuum filtration again and the filtrate was collected. The enzyme used was the aforementioned enzyme (E1), and the enzyme concentration was 0.8 mg / mL (20 mM acetate buffer).

[0129] [Measurement of Erinacin A Content] By quantifying the elinacin A contained in the filtrate using the method described in Test Example 1, the amount of elinacin A contained in 1 g of wet bacterial cells (extracted by artificial digestion solution) was quantified. The results are shown in Table 5 and Figure 3.

[0130]

[0131] As shown in Table 5 and Figure 3, compared to cases where only the heat treatment step is performed (Comparative Example 3-1) or where the grinding step is performed after the heat treatment step (Comparative Example 3-2), the case where the enzyme treatment step is performed first and then the heat treatment step is performed (Examples 3-1 and 3-2) showed a significant increase in the amount of erinasin A present in the filtrate due to the artificial digestate. This indicates that, according to the manufacturing method in which the enzyme treatment step is performed first and then the heat treatment step is performed, the processed lion's mane mushroom mycelium is more easily broken down by the digestate, releasing erinasin A contained in the mycelium outside the mycelium, and significantly improving the bioavailability of erinasin A. Furthermore, a comparison of Example 3-1 and Example 3-2 showed that the bioavailability of erinasin A is further improved according to the manufacturing method in which the grinding step is performed after the enzyme treatment step and the heat treatment step.

[0132] <<Test Example 4: Examination of Enzyme Types>> Test Example 4 is a test to examine suitable enzymes for use in the enzyme processing process. In addition to the enzymes (E1) and (E2) mentioned above, the following enzymes (E3) to (E6) were prepared, and their chitinase activity and chitobiase activity were measured according to the method described below. The results are shown in Table 6, Figure 4, and Figure 5. ・Enzyme (E3) "Soluble Pectinase T" (manufactured by HBI Co., Ltd., derived from Aspergillus niger) ・Enzyme (E4) "Tunicase SP-EN" (manufactured by Amano Enzyme Co., Ltd.) ・Enzyme (E5) "Cellulase T "Amano" 4" (manufactured by Amano Enzyme Co., Ltd., derived from Trichoderma genus) ・Enzyme (E6) "Cellulosin HC100" (manufactured by HBI Co., Ltd., derived from Aspergillus niger)

[0133] [Chitinase Activity Measurement] The chitinase activity of each enzyme is measured using the amount of enzyme that releases 1 μmol of N-acetylglucosamine per minute as 1 U. Take 1 mL of reagent B shown below into a test tube and pre-incubate at 37°C for 5 to 10 minutes. Add 0.2 mL of reagent C shown below, and carry out the reaction at 37°C for 1 hour with gentle shaking. After deactivation by heating (boiling for 5 minutes), centrifuge the reaction solution (3,000 rpm, 10 minutes) and measure the amount of N-acetylglucosamine in the supernatant by the Reissig method. The amount of N-acetylglucosamine released by the enzymatic reaction was calculated from the standard curve. The results are shown in Table 6 and Figure 4. (Reagent A: Acetate buffer) Dissolve 0.82 g of sodium acetate in approximately 90 mL of distilled water. Add acetic acid little by little to this solution to adjust the pH to 6.0. Fill up to 100 mL to make 0.1 M acetate buffer. (Reagent B: Substrate solution) Suspend chitin powder in solution A (Reagent A) to a concentration of 0.5% to prepare the substrate solution. (Reagent C: Enzyme solution) Dissolve the enzyme in distilled water to a concentration of 0.8 mg / mL.

[0134] [Chitobiase Activity Measurement] The chitobiase activity of each enzyme is measured, with 1 U representing the amount of enzyme that releases 1 μmol of p-nitrophenol per minute. 0.75 mL of reagent A and 0.2 mL of reagent B are placed in a test tube and pre-incubated at 37°C for 5-10 minutes. 50 μL of reagent C is added, and the mixture is quickly mixed thoroughly with a test tube mixer to start the reaction. After gently shaking for 10 minutes, 2.0 mL of reagent D is added, and after quick mixing, the absorbance at 405 nm is measured. The amount of p-nitrophenol released by the enzymatic reaction was calculated from the standard curve. The results are shown in Table 6 and Figure 5. (Reagent A: McLvain buffer) 0.2 M disodium hydrogen phosphate solution is added to 0.1 M citrate to adjust the pH to 6.0. (Reagent B: Substrate solution) 8.56 mg of p-nitrophenol-N-acetyl-β-D-glucosaminid is dissolved in 5.0 mL of reagent A. (Reagent C: Enzyme solution) Dissolve in distilled water to an enzyme concentration of 0.8 mg / mL. (Reagent D: Reaction stop solution) Dissolve 2.65 g of anhydrous sodium carbonate in distilled water to make 100 mL.

[0135]

[0136] As shown in Table 6, Figures 4 and 5, chitinase activity and chitobiase activity were confirmed for all enzymes (E1) to (E6). However, enzymes (E1), (E2), and (E3) showed high activity, with enzymes (E1) and (E2) showing particularly high activity. Therefore, it was found that enzymes (E1), (E2), and (E3) are preferred as enzymes to be used in the enzyme treatment step of this manufacturing method, and among them, enzymes (E1) and (E2) are particularly preferred.

[0137] While the above embodiments illustrate specific forms of the present invention, these embodiments are merely illustrative and should not be interpreted restrictively. Various modifications that are obvious to those skilled in the art are intended to fall within the scope of the present invention.

[0138] The present invention's method for producing processed lion's mane mushroom mycelium significantly improves the bioavailability of erinacin A, making it suitable for use as a raw material in various foods, supplements, quasi-drugs, and pharmaceuticals.

Claims

1. A method for producing processed lion's mane mushroom mycelium, comprising performing enzyme treatment and heat treatment in that order on a liquid-cultured lion's mane mushroom mycelium culture.

2. The method for producing a processed mycelium of Lion's Mane mushroom according to claim 1, wherein the enzyme treatment is an enzyme treatment using an enzyme having at least one of chitinase activity or chitobiase activity.

3. The method for producing a processed lion's mane mushroom mycelium according to claim 1 or 2, wherein the heat treatment is a heat treatment at 100°C or higher.

4. A method for producing a processed mycelium of Lion's Mane mushroom according to any one of claims 1 to 3, wherein the chitinase activity of the enzyme used in the enzyme treatment is 700 U / g or more, and / or the chitobiase activity of the enzyme is 60,000 U / g or more.

5. A method for producing a processed mycelium of Lion's Mane mushroom according to any one of claims 1 to 4, comprising performing a grinding treatment at at least one of the following stages: before or after the enzyme treatment, and after the heat treatment.

6. A method for improving the bioavailability of erinacin A contained in processed lion's mane mushroom mycelium by performing enzyme treatment and heat treatment in this order on liquid-cultured lion's mane mushroom mycelium culture.

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