Novel grifola frondosa mushroom strain and method for culturing same
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2026-02-05
- Publication Date
- 2026-08-13
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Abstract
Description
Novel maitake strain and its cultivation method
[0001] The present invention relates to a novel maitake strain and its cultivation method. The present invention also relates to a composition containing such a maitake strain.
[0002] Maitake (Grifola frondosa) belongs to the wood-rotting fungus of the order Polyporales, family Grifolaceae, genus Grifola. The characteristics of the maitake fruit body are that, while many edible mushrooms produced by artificial cultivation such as enoki mushrooms and buna shimeji have a clearly distinguishable stalk and cap (so-called mushroom type), in the case of maitake, the stalk and the cap part cannot be clearly distinguished, and light black-brown, dark brown to brown caps overlap to form a large fruit body (so-called monkey saddle type) (Non-Patent Document 1 and Non-Patent Document 4).
[0003] The fruit body of maitake is useful as an edible mushroom, and an artificial cultivation method has been established, and cultivation is carried out in large-scale factories. In factory cultivation, if strains with high productivity such as high yield and strains that are easy to handle such as those with fruit bodies that are not easily damaged are used, the production efficiency will be high and it will be more advantageous than others.
[0004] Since the fruit body of maitake performs various physiological and chemical reactions by its own life activities even while being stored after harvest, changes such as browning and wilting of the fruit body occur, affecting freshness and quality (Non-Patent Document 2). Changes due to decreased freshness soften and make the fruit body brittle, deteriorating the appearance and texture. Also, such changes will deteriorate the texture (Non-Patent Document 3), which is said to be an important element of deliciousness for mushrooms. Therefore, strains that maintain firmness and are not easily brittle even after harvest are desired as strains that can suppress quality deterioration.
[0005] In addition, maitake is not only edible, but also expected to have health effects, and many studies have been conducted. As functions of maitake, immunostimulatory action, antioxidant activity, antiviral activity, etc. have been reported (for example, Patent Document 1 and Non-Patent Document 4), and health foods using extracts from maitake have also been produced.
[0006] Japanese Patent Application Laid-Open No. 2020-2052
[0007] New Classification Mushroom Illustrated Guide, Hokuryukan, p. 356, 2021 Specialty Products Information Mushrooms etc., Vol. 20, No. 8, pp. 58-60, 1999; J. Home Econ. Jpn., vol. 47, No. 8, pp. 765-768, 1996; Biotechnology, Vol. 92, No. 10, pp. 572-575, 2014.
[0008] As mentioned earlier, the production costs of maitake mushrooms cultivated artificially in factories are rising due to soaring costs for culture medium materials and fuel. Therefore, there is a demand for highly productive strains, such as those with a high yield of fruiting bodies per unit of culture medium. In addition, to enhance the commercial value, there is a demand for strains that do not easily detach from the fruiting bodies during transportation and processing, and strains that do not deteriorate or become brittle easily during storage.
[0009] To solve the above problems, the inventors crossbred various types of maitake mushrooms and selected strains based on fruiting body yield, shape, and color, and were able to obtain strains with these promising characteristics. Further detailed examination of the characteristics of these strains revealed, unexpectedly, that they had a thicker mycelium, were harder, and were less prone to brittleness after storage than previous strains. This invention was completed based on the above findings.
[0010] The present invention includes, for example, the following embodiments: [1] A Maitake strain selected from (A), (B), and (C) below: (A) A Maitake strain having accession number NITE BP-04194 or its offspring; (B) A Maitake strain derived from a Maitake strain having accession number NITE BP-04194, characterized in that it has a mycelial thickness equal to or greater than that of a Maitake strain having accession number NITE BP-04194 and / or equal to or greater robustness; (C) A Maitake strain that does not form a band in confrontation culture with a Maitake strain having accession number NITE BP-04194, and is characterized in that it has a mycelial thickness equal to or greater than that of a Maitake strain having accession number NITE BP-04194 and / or equal to or greater robustness. [2] The Maitake strain according to [1], wherein the thickness of the fungus is equal to or greater than the above, and is expressed by the indicator that (a) the thickness of the fungus of the fruiting body is an average of 2.2 mm or more, preferably an average of 2.35 mm or more. [3] The Maitake strain according to [1], wherein the strength is equal to or greater than the above, and is expressed by at least one indicator selected from the group consisting of (b) the average force required to crush the fruiting body to 80% of its height is 1.3 times or more, preferably 1.5 times or more, that of the standard strain, and (c) the degree of detachment of the fruiting body when the fruiting body is dropped from a height of 2 m is 5% or more lower than that of the standard strain, and the degree of detachment remains 10% or more lower even after 14 days of refrigerated storage.
[0011] [4] A method for cultivating maitake mushrooms, comprising inoculating a culture medium with the spawn of the maitake strain described in [1] and producing fruiting bodies or mycelium. [5] The method according to [4], wherein the culture medium is prepared by mixing 2 to 5 parts by dry weight of hardwood sawdust with 1 part by dry weight of a mixture of one or more plant-derived nutrients selected from wheat bran, corn bran, hominy feed, okara, rice bran, beet pulp, cotton hull, and corn cob, and adjusting the moisture content to 50 to 75%. [6] The method according to [4], comprising culturing the spawn or mycelium at room temperature of 20 to 30°C and humidity of 50 to 75% RH to form fruiting body primordia, and then growing the fruiting bodies in a room at room temperature of 15 to 21°C and humidity of 85 to 100% RH.
[0012] [7] A composition comprising the Maitake mushroom strain described in [1], a part thereof, and / or processed products thereof. [8] The composition described in [7], which is a food or beverage, a supplement, a pharmaceutical or cosmetic.
[0013] The new maitake mushroom strain according to the present invention produces fruiting bodies with thick mycelium and high yield. Furthermore, these fruiting bodies are less prone to mycelium detachment, do not deteriorate easily during storage, and are not easily brittle. As a result, this strain has higher commercial value than conventional maitake mushrooms and offers industrial advantages that lead to reduced production costs.
[0014] These are photographs showing the side-culture results of Yukiguni Mai No. 15 and various strains. These are photographs showing the mycelium when Yukiguni Mai No. 15 and the mycelium of other strains used in the test were cultured. These are photographs of the fruiting bodies of Yukiguni Mai No. 15 and its parent plant. These are photographs of the fruiting bodies of Yukiguni Mai No. 15 and a commercially available strain used for comparison. This is a graph showing the force applied when the fruiting body of Example 5 was compressed. This is a graph showing the detachment rate when the fruiting body of Example 6 was dropped.
[0015] This specification encompasses the disclosures of Japanese Patent Application No. 2025-019487, filed on 7 February 2025, which forms the basis of the priority of this application. The present invention will now be described in detail.
[0016] This invention relates to a novel strain of maitake mushroom, a method for cultivating the same, and its uses. The novel maitake strain (hereinafter referred to as "Yukiguni Mai No. 15") was obtained by crossing primary mycelium obtained from Yukiguni Mai No. 9 and the company's proprietary strain C6510. The fruiting body of Yukiguni Mai No. 15 is characterized by its thick, slightly dark brown color and firm, compact texture.
[0017] Furthermore, this new strain, Yukiguni Mai No. 15, has been sent by the applicant for international deposit to the Patent Microorganism Depository Center of the National Institute of Technology and Evaluation (NITE), an international depositary authority under the provisions of the Budapest Convention for the Deposit of Patent Microorganisms (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818 Japan), and was accepted on October 31, 2024 (Depositary Number NITE BP-04194).
[0018] In one embodiment, the present invention provides a Maitake mushroom strain selected from the following (A), (B), and (C): (A) a Maitake mushroom strain (Yukiguni Mai No. 15) having accession number NITE BP-04194 or its offspring; (B) a Maitake mushroom strain derived from a Maitake mushroom strain having accession number NITE BP-04194, characterized by having a mycelial thickness equal to or greater than that of the Maitake mushroom strain having accession number NITE BP-04194 and / or equal to or greater robustness; (C) a Maitake mushroom strain that does not form a band in confrontation culture with a Maitake mushroom strain having accession number NITE BP-04194, and is characterized by having a mycelial thickness equal to or greater than that of the Maitake mushroom strain having accession number NITE BP-04194 and / or equal to or greater robustness. In this specification, these Maitake strains are collectively referred to as "the Maitake strains of the present invention."
[0019] The mycological properties of Yukiguni Mai No. 15 are as follows: (1) Taxonomic characteristics: Basidiomycete, genus Griffola, maitake mushroom (Griffola frondosa). When confrontation culture, a method for determining genetic differences on agar plates, was performed, it showed bands with the parent strain Yukiguni Mai No. 9, the company's proprietary strain C6510, and commercially available standard strains Mori M51 and Yukiguni Mai No. 10, indicating that it is genetically different from these maitake strains. Therefore, this strain was determined to be a new strain (Example 1).
[0020] (2) Physiological characteristics (Example 3): When cultured on potato dextrose agar at 25°C for 14 days, the physiological characteristics compared to the parent strain Yukiguni Mai No. 9 were that the mycelial density was slightly lower, the aerial mycelial development was slightly greater, the peripheral area of the mycelium was more homogeneous, and the thickness of the mycelium was slightly lower. Similarly, compared to the parent strain C6510, the mycelial density was slightly lower, the aerial mycelial development was slightly greater, and the thickness of the mycelium was thinner. Furthermore, compared to the conventional strain Mori M51, the thickness of the mycelium was slightly lower, the shape of the peripheral area of the mycelium was more homogeneous, and the shape of the mycelium surface was smooth. In addition, compared to the commercially available strain Yukiguni Mai No. 10, the mycelial density was slightly lower, the thickness of the mycelium was thinner, and the shape of the mycelium surface was smooth. The optimal temperature for mycelial growth is around 26.5°C.
[0021] (3) Cultivation characteristics (Examples 4 and 5): Compared to conventional strains, Yukiguni Mai No. 15 of the present invention is characterized by its high fruiting body yield and thicker mycelium. This was determined by comparing the weight of the fruiting bodies and the thickness of the mycelium obtained when cultivation tests were conducted under the same conditions as Mori M51, a commercially available standard strain. The specific methods and results are described in the examples.
[0022] Other characteristics include the fact that the fruiting bodies of Yukiguni Mai No. 15 are rounder in shape and have a neat fan shape compared to the parent strain Yukiguni Mai No. 9, and yield higher fruiting bodies. Also, compared to the other parent strain C6510, the mycelium is less wavy and darker in color. These characteristics allow it to be distinguished from the parent strain. Furthermore, compared to the fruiting bodies of commercially available strains, the color of the mycelium is the same as or slightly darker than the standard Mori M51, and darker than the commercially available Yukiguni Mai No. 10, and the thickness of the mycelium is thicker than not only Mori M51 but also Yukiguni Mai No. 10 (see Example 4).
[0023] The Yukiguni Mai No. 15 variety of this invention is characterized by its robust fruiting body, which makes it difficult for the mycelium to detach from the fruiting body. Maitake mycelium can detach from the main body during harvesting, sorting, and even during transportation after packaging, which can lead to adverse effects such as reduced yield and deterioration of appearance. By making it difficult for the mycelium to detach from the fruiting body of Yukiguni Mai No. 15, it can contribute to improved yield and prevention of deterioration of appearance.
[0024] The Yukiguni Mai No. 15 variety of the present invention also has the characteristic of having a fruiting body that does not deteriorate easily over time. Maitake mushrooms can become brittle over time, causing the mycelium to break and resulting in a deterioration of appearance. In addition, they can become softer over time, resulting in a withered appearance and a decrease in commercial value. On the other hand, the fruiting body obtained from Yukiguni Mai No. 15 is less prone to becoming brittle over time, maintains its hardness, and is less prone to deterioration overall, thus extending its shelf life. Specific evaluation methods are described in the examples.
[0025] Regarding the strength of the fruiting body, the average force required to crush a fruiting body obtained from bottle cultivation to 80% of its height is more than 1.5 times higher for Yukiguni Mai No. 15 than for Mori M51 or Yukiguni Mai No. 10, indicating its superior strength (see Example 5). Furthermore, when a Yukiguni Mai No. 15 fruiting body is dropped from a height, the degree of mycelial detachment does not change significantly even after 14 days of refrigerated storage, remaining below 70% of that of Mori M51 or Yukiguni Mai No. 10, indicating that it does not become brittle even after storage (see Example 6).
[0026] The thick mycelium and firm fruiting body of Yukiguni Mai No. 15 contribute to a satisfyingly firm texture, offering a different texture from previous varieties such as Mori M51.
[0027] The offspring and derived strains of Yukiguni Mai No. 15 are also included in the present invention, insofar as they possess characteristics equivalent to or better than those described above. Offspring refer to strains obtained using Yukiguni Mai No. 15 (accession number NITE BP-04194) as a parent, and may possess characteristics equivalent to or better than those of the parent Yukiguni Mai No. 15. Derived strains refer to strains obtained by spontaneously mutating Yukiguni Mai No. 15 during culture, by artificially inducing mutations using ultraviolet light, chemicals, or radiation, or by artificially modifying genes through genetic recombination or genome editing, and which are different from Yukiguni Mai No. 15 but possess characteristics equivalent to or better than those described above. In the present invention, equivalent or better characteristics refer to having a thickness of equal or greater than that of the fungus and / or equal or greater robustness. Such offspring and derived strains can be understood by those skilled in the art, and it is also understood that they possess characteristics equivalent to or better than those described above. Whether a particular strain is an offspring or derived strain possessing characteristics equivalent to those described above can be determined based on the contents of this specification and the common technical knowledge in the art. For example, the descendants of Yukiguni Mai No. 15 can be determined by comparing the genome sequence of Yukiguni Mai No. 15, which is deposited under accession number NITE BP-04194, with DNA markers, which are characteristic sequences of that genome.
[0028] For example, the derived strain of Yukiguni Mai 15 can be determined by comparing the genome sequence of Yukiguni Mai 15, which is deposited under accession number NITE BP-04194, with a DNA marker, which is a characteristic sequence of that sequence. The derived strain is a bacterial strain having a genome containing a base sequence that has 90% or more, preferably 95% or more, more preferably 98% or more, even more preferably 99% or more, and particularly preferably 99.5% or more identity with the genome sequence of Yukiguni Mai 15. In this specification, "identity" refers to the identity of a base sequence or amino acid sequence determined by techniques known in the art. As a method of sequence alignment, alignment programs known in the art can be used. Examples include the CLUSTAL W program, FASTA program, and BLAST program. Using such a publicly available program, the genome sequence of Yukiguni Mai 15 can be aligned with the genome sequence of a certain strain, and the identity of the two sequences can be calculated. For example, if the two sequences have more than 90% identity, the strain can be determined to be an induced strain of Yukiguni Mai 15.
[0029] When using DNA markers, identification can also be achieved, for example, by performing PCR (Polymerase Chain Reaction) to amplify the region containing the DNA marker, followed by gel electrophoresis, and checking for matching amplification patterns and the size of the amplified products. The determination of offspring and derivatives of Yukiguni Mai No. 15 is performed by statistical processing such as constructing a phylogenetic tree using the maximum likelihood method of genome sequencing and electrophoretic patterns, or by visual comparison, to ensure consistency as offspring. As DNA markers, markers such as RAPD (random amplified polymeric DNA), AFLP (Amplified Fragment Length Polymerism), SSR (Simple Sequence Repeat), and SNP (Single Nucleotide Polymerism), which are used in known DNA variety identification methods, can be used. For example, when examining a large number of DNA markers using a genome sequencing method, if more than 90% of the examined DNA markers match those of Yukigunimai No. 15, the strain can be determined to be an induced strain of Yukigunimai No. 15.
[0030] Contrast culture refers to culturing the mycelium of two strains of fungi facing each other on the same culture medium. It is known that when the two strains are different varieties, a distinct band is formed on the culture medium. Therefore, a Maitake strain that does not form a band in contrast culture with Yukiguni Mai No. 15 can be considered to be the same strain as Yukiguni Mai No. 15 or substantially the same strain (a strain that has slight genetic differences from Yukiguni Mai No. 15 but cannot be considered a different variety), and is thought to possess characteristics equivalent to those described above. The presence or absence of band formation in contrast culture can be determined by methods known in the art, specifically by the method described in the examples.
[0031] Whether a particular strain of maitake mushroom possesses characteristics equivalent to or better than those described above can be determined based on the contents of this specification and the common technical knowledge in the art.
[0032] In one embodiment, characteristics equivalent to or better than Yukiguni Mai No. 15 can be evaluated by equivalent or better mycelial thickness and / or equivalent or better rigidity, and specifically, this is represented by at least one indicator selected from the group consisting of: (a) the average thickness of the mycelial layer of the fruiting body being 2.2 mm or more; (b) the average force required to crush the fruiting body to 80% of its height being 1.3 times or more that of the standard strain; and (c) the degree of detachment of the fruiting body portion when the fruiting body is dropped from a height of 2 m being 5% or more lower than that of the standard strain, and the degree of detachment remaining 10% or more lower even after 14 days of refrigerated storage.
[0033] The thickness of the bacteria can be evaluated, for example, by measuring the thickness of a portion 10 mm inward from the tip of the bacteria. This thickness can be used as an indicator if it averages 2.2 mm or more, for example, an average of 2.2 mm to 2.9 mm, preferably an average of 2.35 mm or more, and more preferably an average of 2.4 mm or more.
[0034] The force required to crush the fruiting body to 80% of its height can be calculated, for example, by using a compression jig to compress the fruiting body to 80% of its height and measuring the maximum force applied at that time (maximum test force, N). The standard strain is Mori M51, which is cultivated by many producers, and an indicator can be used that the average force required to crush the fruiting body to 80% of its height is 1.3 times or more, for example, 1.3 to 2.5 times, preferably 1.5 times or more, compared to the standard strain.
[0035] The degree of detachment of the fruiting body when dropped from a height of 2m can be calculated, for example, by first measuring the weight of the fruiting body (weight before drop), dropping the fruiting body from a height of 2m with the stem facing upwards and the mycelium facing downwards, and taking the weight of the heaviest mass as the weight after drop. The detachment weight (the difference between the weight before drop and the weight after drop) is then calculated as the ratio of the weight before drop to the weight after drop (a smaller value indicates less detachment, i.e., less brittleness). Compared to a standard strain, an indicator of a good strain is one in which the degree of detachment of the fruiting body when dropped from a height of 2m is 5% or more lower, for example, 5-60% lower, and the degree of detachment after 14 days of refrigerated storage is 10% or more lower, for example, 10-35% lower.
[0036] The Maitake strain of the present invention satisfies at least one of the indicators (a) to (c) above. Preferably, it satisfies two or three of the indicators (a) to (c) above.
[0037] The method for cultivating maitake mushrooms according to the present invention can be carried out by conventional methods used for cultivating maitake mushrooms (for example, the "Maitake" section in the 2002 edition of the Mushroom Yearbook and Plants World) or by modifying those methods according to the circumstances of the cultivator. Specifically, it includes inoculating a culture medium with the spawn of the maitake strain of the present invention and producing fruiting bodies or mycelium.
[0038] There are two methods for cultivating maitake mushrooms: controlled cultivation and natural cultivation. Controlled cultivation involves managing sterilized and inoculated mushroom beds in an artificial environment to produce fruiting bodies, while natural cultivation involves using mushroom beds or logs in a natural environment to produce fruiting bodies.
[0039] Cultivation under controlled temperatures is carried out using bags or bottles made of polypropylene, polyethylene, etc., as cultivation containers, but bag cultivation is the most common. In a specific example of bag cultivation, sawdust (from broadleaf trees) and nutrient additives are mixed in a volume ratio of, for example, about 8:1, water is added and kneaded thoroughly, and the culture medium is packed into the cultivation container. After sterilization, the following steps are taken: inoculation with spawn, cultivation, growth, and harvesting. The period from inoculation to harvest is about 55 to 90 days.
[0040] The cultivation medium mainly consists of sawdust and nutrient additives. The sawdust is preferably from broad-leaved trees, including species of the Fagaceae family such as Quercus crispula, Quercus serrata, Quercus acutissima, and Fagus crenata, and species of the Betulaceae family, and an appropriate particle size can be selected. The nutrient additives are mainly plant-derived nutrients and include one or more selected from, for example, wheat bran, corn bran, hominy feed, okara, rice bran, beet pulp, cotton hull, and corn cob. The nutrient additives can be appropriately determined depending on the cultivation method adopted and the type of sawdust used. The mixing ratio of sawdust to nutrient additives should be, for example, approximately 3:1 by dry weight. After thoroughly mixing the sawdust and additives, water is added to preferably achieve a moisture content of 50-75%, more preferably 60-70%.
[0041] In one embodiment, the medium used is adjusted to a water content of 50 to 75% by mixing 2 to 5 parts by dry weight of broad-leaved tree sawdust with 1 part by dry weight of a mixture of one or more plant-derived nutrient sources selected from bran, corn bran, hominy feed, okara, rice bran, beet pulp, cotton hull, and corn cob.
[0042] For the cultivation container, a bag made of polypropylene, polyethylene, etc. with a ventilation filter or a bottle equipped with a lid with such a filter is used.
[0043] In order to propagate only the target strain in the medium, it is necessary to kill the fungi that originally exist. The sterilization methods include atmospheric pressure sterilization using steam at 98 to 100 °C and pressure sterilization using a pressure vessel at 100 °C or higher. To kill bacteria that do not die at 100 °C or lower, pressure sterilization is preferred. After sterilization, the medium is cooled to 30 °C or lower in preparation for inoculation.
[0044] The inoculation of the strain into the medium is carried out in a room as clean as possible. When using a bag for the cultivation container, after inoculation, the bag may be folded to secure a space for forming a primordium (described later) at the upper part of the bag. This space is, for example, about 5 cm in height and about 1 / 4 of the upper surface of the medium centered on the filter part in area. Cultivation is possible even without this space. When choosing a bottle for the cultivation container, a lid with a filter may be placed on the bottle.
[0045] During cultivation, mainly the temperature, humidity, ventilation, and light are adjusted and managed. Generally, for shiitake, since the suitable temperature for mycelial growth and the suitable temperature for fruiting body formation are approximate, usually, cultivation is carried out in the procedure of forming fruiting body primordia in the culture room first and then transferring them to the growth room for growth. As the cultivation of shiitake mycelium progresses and the mycelium spreads, the respiratory action becomes more active, and accordingly, the temperature inside the mushroom bed also rises, becoming 2 to 5 °C higher than the indoor temperature around the 14th to 20th day of cultivation. Therefore, the indoor temperature is set lower than the suitable temperature for mycelial growth. The temperature of the culture room is preferably 20 to 30 °C, more preferably 22 to 28 °C. Normal primordium formation occurs in the mushroom bed where nutrients have been accumulated for a certain period.
[0046] The humidity condition in the cultivation room is preferably about 50 to 75% RH. When the carbon dioxide concentration in the cultivation room increases, ventilation is carried out to introduce fresh air. In addition, it is advisable that the carbon dioxide concentration does not exceed 2,500 ppm. The presence of light is not necessary for mycelial growth but is necessary for the formation of fruit bodies. Therefore, light may be applied after the start of cultivation, but preferably, light is not applied during the mycelial elongation period (up to about 30 to 35 days of cultivation), and then cultivation is carried out while irradiating light.
[0047] When cultivated under appropriate conditions, mycelia gather in the space above the culture medium surface from around the 35th day after inoculating the inoculum and rise in a mat-like shape. When light is irradiated at this time, water droplets are generated on the surface, and further, a part of the mycelial mat becomes hard and dense, bulges in a lump, and folds occur on its surface, and the location where the water droplets are generated sinks. The site where such morphological changes occur is called a fruit body primordium. After the formation of this fruit body primordium, a development operation is performed. For the development operation, the culture medium may be directly transferred to the development room, but if necessary, before the development operation, in an environment of around 20°C, humidity of 60 to 95% RH, and illuminance of about 300 to 1500 lux, further induction of primordium formation may be carried out.
[0048] After moving to the development room, ensure the space for the growth of the fruit body. As an example of the method, when the cultivation container is a bag, cut a cross about 4 to 5 cm with a knife near the part where the primordium has grown the largest, or cut off a part of the bag to open the bag. In the case of a bottle, just remove the lid.
[0049] The environment of the development room is also an important factor for the traits and yield of the fruit body, so mainly manage conditions such as temperature, humidity, ventilation, and light. The growth temperature of shiitake has a wide range of 15 to 21°C, but the optimum growth temperature of Yukiguni Mai 15th is 19 to 21°C, so it is preferable to manage based on this temperature as a guide. The humidity in the development room is preferably managed at 90 to 100% RH. Also, similar to the cultivation room, the development room should be managed to introduce fresh air so that the carbon dioxide concentration in the room is 1,000 ppm or less. The light condition is preferably such that the illuminance is maintained at 500 lux or more.
[0050] After being moved to the growth chamber, the mushrooms will grow to harvestable size in about 20 days, or in about 10 days if primordia induction was performed before growth. Harvesting should be done when pores begin to form on the underside of the mushroom. Preferably, harvesting should be done when the pores have grown from about 3 mm from the edge of the mushroom to covering the entire underside. In this way, the maitake mushroom strain of the present invention can be cultivated.
[0051] In one embodiment, the method for cultivating maitake mushrooms according to the present invention includes culturing the spawn or mycelium of the maitake strain of the present invention at a room temperature of 20 to 30°C and a humidity of 50 to 75% RH to form fruiting body primordia, and then growing the fruiting bodies in a room at a room temperature of 15 to 21°C and a humidity of 85 to 100% RH.
[0052] The present invention provides a composition comprising the Maitake mushroom strain (fruiting body and / or mycelium), a part thereof, and / or processed products thereof. Processed products include, but are not limited to, dried products, crushed products, extracts, fermented products, heat-treated products, and the like.
[0053] A dried product can be prepared by drying the fruiting body and / or mycelium (or a part thereof) of the Maitake strain of the present invention using known drying methods, such as heat drying, vacuum drying, and air drying, preferably by heat drying and vacuum drying.
[0054] The fruiting bodies and / or mycelium (or a part thereof) of the Maitake strain of the present invention can be crushed, pulverized, or ground to prepare crushed products and extracts. For example, such crushing can be carried out by physical crushing (stirring, filter filtration, etc.), enzymatic dissolution treatment, chemical treatment, etc.
[0055] An extract can be obtained by extracting the fruiting body and / or mycelium (or a part thereof) of the Maitake strain of the present invention using a suitable aqueous solvent or organic solvent. The extraction method is not particularly limited as long as it uses an aqueous solvent or organic solvent as the extraction solvent, but known methods such as immersion, stirring, or reflux in aqueous or organic solvent (e.g., water, methanol, ethanol, etc.) can be mentioned.
[0056] A fermented product can be prepared by fermenting the fruiting body and / or mycelium (or a part thereof) of the Maitake strain of the present invention with lactic acid bacteria. For example, lactic acid bacteria can be inoculated into a suspension of the fruiting body and / or mycelium (or a part thereof) of the Maitake strain of the present invention, and fermentation can be carried out under lactic acid bacteria fermentation conditions known in the art. The resulting fermented product may be used as is, or it may be subjected to other treatments such as filtration, sterilization, dilution, or concentration.
[0057] Furthermore, the fruiting body and / or mycelium (or a part thereof) of the Maitake strain of the present invention can be prepared as a heat-treated product by heat treatment. To prepare the heat-treated product, the fruiting body and / or mycelium (or a part thereof) of the Maitake strain of the present invention is subjected to high-temperature treatment (for example, 80 to 150°C) for a certain period of time, for example, about 10 minutes to 1 hour (for example, about 10 to 20 minutes).
[0058] The above-described processes may be performed individually or in combination as appropriate. In the present invention, such processed products can also be used in the composition of the present invention.
[0059] In addition to the above, the composition of the present invention may also contain additives, other known drugs, nutritional supplements, etc., either individually or in combination with others.
[0060] The form of the composition of the present invention is not particularly limited. For example, if it is a pharmaceutical composition, it may be in the form of oral preparations such as tablets, capsules, granules, powders, syrups, dry syrups, liquids, suspensions, and inhalants, enteral preparations such as suppositories, intravenous drips, and injections. Of these, oral preparations are preferred. Such compositions in dosage forms can be manufactured by adding commonly used excipients, disintegrants, binders, wetting agents, stabilizers, buffers, lubricants, preservatives, surfactants, sweeteners, flavoring agents, fragrances, acidulants, and colorants to the above-mentioned components according to the dosage form and following conventional methods.
[0061] Furthermore, the compositions of the present invention are highly safe and easy to ingest and use continuously over long periods. Therefore, the compositions of the present invention can also be used in food and beverages (including animal feed), supplements, and cosmetics. Specific examples of food and beverages include liquid foods such as enteral nutrition formulas, and health and nutritional supplement foods and beverages in the form of formulations such as tablets, candies, chewable tablets, powders, capsules, granules, and drinks. Food and beverages and supplements can be manufactured according to conventional methods by incorporating other food materials, various nutrients, various vitamins, minerals, dietary fiber, and various additives (e.g., flavoring agents, sweeteners, acidulants such as organic acids, stabilizers, and flavors) used in the manufacture of the food and beverages, in addition to the above-mentioned active ingredients. Cosmetics can also be manufactured according to conventional methods by incorporating excipients and additives used in cosmetics, and can be produced according to conventional methods.
[0062] In the composition of the present invention, the amount of the fruiting body and / or mycelium of the Maitake strain of the present invention, a part thereof, and / or the processed product thereof can be appropriately determined by a person skilled in the art.
[0063] The subjects to whom the composition of the present invention is administered or ingested or used are, but are not limited to, mammals (e.g., primates such as humans and chimpanzees, laboratory animals such as rats and mice, domestic animals such as pigs, cattle, horses, sheep and goats, and pet animals such as dogs and cats, preferably humans or mice, most preferably humans), birds (poultry such as chickens, quail, turkeys, ducks and geese, pet birds such as parakeets, parrots and zebra finches), and fish (farmed fish such as tuna, sea bream, yellowtail and amberjack, ornamental fish such as koi and goldfish), preferably mammals. The amount, interval, and duration of use of the composition of the present invention can be appropriately determined by a person skilled in the art, depending on the purpose of use, taking into consideration the sex, weight, and age of the subject.
[0064] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these specific examples. Furthermore, the development of Yukiguni Mai No. 15 and each example described below were carried out at the Research and Development Headquarters of Yukiguni Factory Co., Ltd. (formerly the Research and Development Office of Yukiguni Maitake Co., Ltd.).
[0065] [Example 1: Creation and Selection of Novel Strains] After isolating spores from the fruiting bodies of Maitake strains Yukiguni Mai No. 9 and C6510, which were owned by the applicant, dozens of primary mycelial lines derived from these spores were cross-pollinated to create hybrid strains. The cross-pollination was performed by bringing the primary mycelial lines of each of the aforementioned strains into contact with each other on the same potato dextrose agar medium at 25°C through opposite culture. The resulting hybrid strains were subjected to cultivation tests in 500cc bottles filled with a culture medium mixed with broadleaf tree sawdust and nutrients, in a room with controlled temperature and humidity. More than a dozen hybrid strains that produced high yields and fruiting bodies with good mycelium and shape were selected. These hybrid strains were further cultivated in bags using the same culture medium and Maitake cultivation bags to select strains with excellent characteristics such as high fruiting body yield, dark mycelium color, and thick mycelium. This strain was designated as a new strain (hereinafter referred to as "Yukiguni Mai No. 15"), and its various properties were confirmed to be stable before cultivation was completed.
[0066] The mycelial growth of Yukiguni Mai No. 15 was carried out by inoculating a portion of the mycelium onto potato dextrose agar and culturing it at 25°C.
[0067] [Example 2: Genetic Characteristics] It is well known that when the mycelia of different mushrooms are cultured side-by-side on agar plates, zoning occurs between the two colonies. Therefore, Yukiguni Mai 15, its parent strains Yukiguni Mai 9 and C6510, the commercially available standard strain Mori M51, and the commercially available strain Yukiguni Mai 10 were cultured side-by-side on potato dextrose agar plates at 25°C for 28 days, and the presence or absence of zoning was observed. A 9 cm diameter petri dish was used to prepare the culture medium. The results are shown in Table 1 and Figure 1. In Table 1, + indicates that zoning was present between colonies, and - indicates that it was not.
[0068]
[0069] Yukiguni Mai No. 15 exhibited band formation in all the tested strains, indicating that it is genetically distinct from these strains.
[0070] [Example 3: Physiological Characteristics] The mycelial density, the development of aerial mycelia, the shape of the margins of the mycelium, the thickness of the mycelium, the presence or absence of coloration on the surface of the mycelium, and the shape of the surface of the mycelium were observed when cultured on potato dextrose agar at 25°C for 14 days (Figure 2 and Table 2).
[0071] As a result, Yukiguni Mai No. 15 differed from its parent strain Yukiguni Mai No. 9 in that its mycelial density was slightly lower, the development of aerial mycelia was slightly greater, the peripheral area of the mycelium was more homogeneous, and the thickness of the mycelium was slightly lower. It also differed from the other parent strain C6510 in that its mycelial density was slightly lower, the development of aerial mycelia was slightly greater, and the thickness of the mycelium was thinner.
[0072] Furthermore, compared to Mori M51, Yukiguni Mai No. 15 had a thinner mycelial colony, a more homogeneous shape at the periphery of the mycelial colony, and a smoother surface. Compared to Yukiguni Mai No. 10, it tended to have a thinner mycelial density and colony, a more homogeneous shape at the periphery of the mycelial colony, and a smoother surface.
[0073] The optimal growth temperature was determined by first pre-culturing the mycelium on potato dextrose agar at 23±1°C for 4 days to standardize hyphal regeneration (to a diameter of approximately 10 mm), and then culturing at 22°C, 24°C, 26°C, 28°C, 30°C, and 32°C for 10 days. The mycelial growth distance was measured, and a graph was created using Microsoft Excel with culture temperature on the x-axis and mycelial growth rate on the y-axis. A quadratic approximation curve was then created, and the optimal growth temperature was determined from its maximum value. As a result, it was confirmed that the optimal growth temperature for Yukiguni Mai No. 15 is around 26.5°C, which is not significantly different from that of Mori M51 and Yukiguni Mai No. 10. The results are summarized in Table 2.
[0074]
[0075] The evaluation criteria in the table were set as follows: Mycelial density When the petri dish is placed on the palm of the hand and viewed from above the mycelium, the palm is: Coarse: Clearly visible Slightly coarse: Intermediate between coarse and medium Medium: Outline is visible Slightly dense: Intermediate between medium and dense Dense: Not visible Aerial mycelium When the mycelium is viewed from the side of the petri dish, the height of the mycelium standing up is: Low: Less than 5% between the mycelium and the petri dish lid Slightly low: 5% or more but less than 10% between the mycelium and the petri dish lid Medium: 10% or more but less than 20% between the mycelium and the petri dish lid Slightly high: 20% or more but less than 30% between the mycelium and the petri dish lid High: 30% or more between the mycelium and the petri dish lid Mycelial thickness When the mycelium is viewed from the side of the petri dish, the thickness of the mycelium is: Thin: Less than 10% of the height of the central inoculated area Slightly thin: 10% or more but less than 35% of the height of the central inoculated area Medium: 35% or more but less than 65% of the height of the central inoculated area Slightly thick: 65% or more but less than 90% of the height of the central inoculated area Thick: 90% or more of the height of the central inoculated area The determination was made by evaluating three of the five inoculated petri dishes (four for Yukiguni Mai No. 9 and C6510), excluding the largest and smallest colonies, and averaging the results. When four dishes were used, those that were particularly larger or smaller than the other three were excluded.
[0076] Furthermore, the growth rate at different temperatures was determined by pre-culturing on potato dextrose agar at 23±1°C for 4 days to standardize mycelial regeneration (to a diameter of approximately 10 mm), and then pre-culturing at each temperature range of 10°C, 15°C, 20°C, 25°C, and 30°C, and measuring the mycelial growth rate for 10 days. The results are shown in Table 3. From the experimental results, it was confirmed that Yukiguni Mai No. 15 had a faster mycelial growth rate at all temperatures compared to Mori M51 and Yukiguni Mai No. 10.
[0077]
[0078] [Example 4: Cultivation Characteristics] The cultivation characteristics test was conducted using a cultivation medium prepared by mixing beech wood-based hardwood sawdust and hominy feed in a dry weight ratio of 75:25, and adjusting the moisture content to approximately 61%. A polypropylene cultivation bag with a ventilation filter (square, 8500cc, opening diameter approximately 200 x 120 mm, height approximately 440 mm) was used as the cultivation container. The cultivation amount was 2500 ± 50 g per bag, which was compacted to a height of approximately 15 cm, two inoculation holes of approximately 15 mm in diameter were made towards the bottom, and the bags were sterilized by autoclaving. After sterilization and cooling, approximately 20-25 ml of sawdust spawn was inoculated into each bag. The sawdust spawn used was prepared by filling 500cc polypropylene bottles with the same broadleaf tree sawdust medium as the cultivation medium described above, autoclaving them, and then inoculating them with mycelium from a Maitake mushroom strain cultured on potato dextrose agar. The bottles were then cultured at 22±1°C. After inoculation, the top of the bag was folded in so that the filter surface was facing outwards. The bottles were cultured at a temperature of 22±1°C, a humidity of approximately 70-75% RH, and an illumination of 200-500 lux. Culture was completed when primordia formed. The mushroom beds with formed primordia were moved to a growth chamber at a temperature of 17±1°C, a humidity of 90-95% RH, and an illumination of 500-1500 lux to promote primordia formation. Once the primordia had grown to near the filter, the filter portion was removed to promote growth into fruiting bodies. When the fruiting bodies had grown sufficiently and the pores on the underside of the fungus had formed to about 2 mm from the edge, the fruiting bodies were harvested. After removing the culture medium attached to the base of the peduncle of the fruiting body, the yield of the fruiting bodies was measured.
[0079] The period from inoculation with spawn to primordia formation, the period to the peak of development, and fruiting body yield were all investigated, and the average value was calculated. In this cultivation experiment, Yukiguni Mai No. 9 and C6510 were conducted with 14 bags per test plot, and the others with 21 bags per test plot. However, two bags of Yukiguni Mai No. 10 were discarded due to cultivation failure midway through the experiment.
[0080] As part of the morphological characteristics investigation of the fruiting bodies, three standard fruiting body strains were selected from each replicate, and the traits shown in Tables 4 and 5 were observed and recorded for a total of nine strains. The color of the fungal surface was identified using the Royal Horticultural Society (RHS) color chart. The diameter of the fruiting body strain was measured by taking the major and minor axes of the crosshairs that intersect at the center of each fruiting body, and calculating the average value. For the size of the fungus, 10 specimens with standard formation were selected from each fruiting body strain, and the average value was calculated for 30 specimens from each strain.
[0081] The thickness of the fungal specimens was measured using calipers at a point 10 mm inward from the tip. Similar to measuring the size of the fungal specimens, measurements were taken on 30 specimens of each strain, and the average value was calculated. The criterion for judgment was that an average thickness of 2.2 mm or more on the 30 specimens was considered acceptable (meeting the desired standard). A thickness of 2.35 mm or more is preferable, and 2.40 mm or more is even more desirable.
[0082] The firmness of the fungal tissue was measured using a fruit hardness meter KM-1 (Fujiwara Seisakusho) and the included metal triangular pyramidal tip (base 12 mm, height 10 mm) to determine the pressure applied when the meter was pressed against the fungus. Measurements were taken on 30 fungal samples of each strain, similar to the size of the fungus, and the average value was used for evaluation. The evaluation criteria were as follows: Soft: pressure less than 0.05 kg Medium: pressure 0.05 kg or more and less than 0.10 kg Hard: pressure 0.10 kg or more
[0083] Tables 4 and 5 show the cultivation characteristics obtained from the above, and Figures 3 and 4 show the shapes of typical fruiting bodies. As a result, compared to its parent strain, Yukiguni Mai No. 9, Yukiguni Mai No. 15 had rounder fruiting body shapes, the mycelium was neatly fan-shaped, there was less discoloration on the surface of the culture substrate, and the yield was higher. Furthermore, compared to the other parent strain, C6510, the mycelium was less wavy, the color was dark greyish yellowish brown, and the yield was higher. Based on these points, the fruiting bodies of Yukiguni Mai No. 15 and its parent strain can be distinguished (Table 4 and Figure 3).
[0084] Furthermore, compared to the commercially available strain Mori M51, Yukiguni Mai No. 15 had a smaller fruiting body diameter, and the fungus was thicker and firmer. It also showed increased yield, and the time to primordia formation and the time to peak harvest were shorter. For yield, a 5% or more increase compared to Mori M51 was considered acceptable. Yukiguni Mai No. 15 showed an approximately 18% increase in yield compared to Mori M51, thus meeting the criteria.
[0085] Compared to the commercially available Yukiguni Mai No. 10, Yukiguni Mai No. 15 had thicker, harder fungal growths and a darker brown color. Furthermore, its yield increased, and the time to the peak harvest period was shorter. These points allow for the distinction of Yukiguni Mai No. 15 fruiting bodies from commonly available strains (Table 5 and Figure 4).
[0086]
[0087]
[0088] [Example 5: Measurement of Fruiting Body Strength of Yukiguni Mai No. 15] The fruiting bodies of Yukiguni Mai No. 15, Mori M51, and Yukiguni Mai No. 10 were examined using a small benchtop testing machine.
[0089] The fruiting bodies for the experiment were cultivated as follows: A 500cc polypropylene bottle was used as the cultivation container. A mixture of broadleaf tree sawdust, hominy feed, and okara (soybean pulp) was prepared as the culture medium, with a moisture content of 65%, and approximately 370g of this mixture was filled into each bottle. A vent was made in the center. A lid with a filter was placed over the bottle, and after steam sterilization and cooling, various Maitake mycelium strains were inoculated. These were then cultured for 40 days under conditions of 25-27°C and 50-60% RH to allow the mycelium to spread throughout the medium. After culturing, sprouting was carried out for 11 days in a room at 19°C and 95-100% RH. After that, the lids of the bottles were removed, and the fruiting bodies were grown in a room under the same conditions as sprouting. When the fruiting bodies had grown sufficiently and the pores on the underside of the mycelium had formed to about 2mm from the edge, the fruiting bodies were harvested. For the subsequent strength measurement tests, harvested fruiting bodies that had been stored in a refrigerator for one day and those that had been stored for 14 days were used.
[0090] The hardness of the fruiting bodies was measured using a small benchtop testing machine (EZ Test EZ-LX, Shimadzu Corporation). The fixtures used were disc-shaped compression fixtures (118 mm in diameter) on both the upper and lower plates. For the test, the entire fruiting body was used, placed on the lower pressure plate with the mycelial part facing down, and compressed at a speed of 1 mm / second with the upper pressure plate. Compression was continued until the fruiting body height was reduced to 80%, and the maximum force applied at that time (maximum test force, N) was used as an indicator of strength. A strength value of 1.3 times or more compared to Mori M51 was considered acceptable. Furthermore, over time, if the hardness on the 14th day of storage remained at 60% or more compared to the hardness on the 1st day, it was considered that the fruiting body would not deteriorate easily even after storage. The number of samples used was 6 strains of all fungal strains on the 1st day of storage, and on the 14th day of storage, 4 strains each of Yukiguni Mai 15 and Yukiguni Mai 10, and 3 strains of Mori M51 were used.
[0091] As shown in Figure 5, the force required to compress the fruiting body to 80% of its height was 1.7 and 1.6 times higher for Yukigunimai No. 15 than for Mori M51 and Yukigunimai No. 10, respectively, on the first day of storage. This indicates that Yukigunimai No. 15 is a stronger strain than the other two, and it was judged to be acceptable. Furthermore, due to this difference in strength, it was determined that Yukigunimai No. 15 has a distinctive texture.
[0092] Furthermore, its strength did not decrease easily even after storage. While the force required for Mori M51 and Yukiguni Mai 10 on the 14th day of storage decreased to 43% and 49% respectively compared to the first day, the force required for Yukiguni Mai 15 remained at 69%. Moreover, the force required for Yukiguni Mai 15 on the 14th day (355N) was equal to or greater than that of Mori M51 and Yukiguni Mai 10 on the first day of storage (312 and 329N respectively), indicating that it maintains the hardness of other strains even after storage. In other words, Yukiguni Mai 15 was judged to have properties that make it resistant to deterioration even after storage.
[0093] [Example 6: Measurement of brittleness and resistance to mycelial detachment of Yukiguni Mai No. 15] The fruiting bodies of Yukiguni Mai No. 15, Mori M51, and Yukiguni Mai No. 10 were dropped from the air, and their brittleness and resistance to mycelial detachment were examined by how much the plants broke apart.
[0094] The fruiting bodies used in the experiment were obtained by cultivating them in the same manner as in Example 4.
[0095] The drop test was conducted by measuring the weight of the fruiting body, then dropping it from a height of 2 meters (the distance from the bottom of the fruiting body to the stainless steel plate) onto a 5 mm thick stainless steel plate with the stem facing upwards and the mycelium facing downwards. The weight of the heaviest clump (the one that remained connected and did not detach) after the drop was defined as the post-drop weight, and the difference between this and the pre-drop weight was defined as the weight of the detached portion. The ratio of the weight of the detached portion to the pre-drop weight was defined as the detachment rate and used as an indicator of brittleness (a smaller value indicates less brittleness).
[0096] The tests were conducted on two types of fruiting bodies, one on day 1 and the other on day 14 of storage in a refrigerator, to investigate the effect of storage on brittleness. If the shedding rate was 5% or more lower than that of Mori M51 on day 1, the brittleness and resistance to mycelial detachment were judged to be satisfactory. Similarly, if the same test was conducted on day 14 of storage, and the shedding rate was 10% or more lower than that of Mori M51, the brittleness, resistance to mycelial detachment, and resistance to deterioration during storage were all judged to be satisfactory. Three strains were used on day 1 of storage, and six strains were used on day 14 (four Mori M51 strains were used due to severe deterioration), and the average values were calculated.
[0097] Figure 6 shows the results. On the first day of storage, the shedding rate of Yukigunimai No. 15 (18%) was significantly lower than that of Mori M51 (30%), but higher than that of Yukigunimai No. 10 (8%). However, on the 14th day, while the shedding rates of the other two strains (Yukigunimai No. 10: 27%, Mori M51: 45%) increased, the shedding rate of Yukigunimai No. 15 (15%) did not change significantly from the first day and became lower than that of the other two strains. Therefore, it was found that Yukigunimai No. 15 is less fragile and less prone to mycelial shedding, and this characteristic is maintained even during storage. Incidentally, Yukigunimai No. 10, immediately after harvest, has a flexible characteristic, so its shedding rate on the first day was low.
[0098] As described above, Yukiguni Mai No. 15 passed the test in terms of the fragility of the fruiting body and the resistance to the detachment of the fungus on the first day. Furthermore, its properties remained stable over time, and its resistance to deterioration was also deemed satisfactory.
[0099] Accession number NITE BP-04194 (Y15M, accessed October 31, 2024)
Claims
1. Maitake mushroom strains selected from (A), (B), and (C) below: (A) Maitake mushroom strain having accession number NITE BP-04194 or its offspring; (B) Maitake mushroom strains derived from Maitake mushroom strain having accession number NITE BP-04194, characterized by having a mycelial thickness equal to or greater than that of Maitake mushroom strain having accession number NITE BP-04194 and / or equal to or greater robustness; and (C) Maitake mushroom strains that do not form a band in confrontation culture with Maitake mushroom strain having accession number NITE BP-04194, and are characterized by having a mycelial thickness equal to or greater than that of Maitake mushroom strain having accession number NITE BP-04194 and / or equal to or greater robustness.
2. The Maitake strain according to claim 1, wherein the thickness of the fungus equivalent to or greater than the above is expressed by the indicator that (a) the thickness of the fungus in the fruiting body is 2.2 mm or more on average.
3. The Maitake strain according to claim 1, wherein the equivalent or superior toughness is represented by at least one indicator selected from the group consisting of (b) the average force required to crush the fruiting body to 80% of its height being 1.3 times or more that of the standard strain, and (c) the degree of detachment of the fruiting body portion when the fruiting body is dropped from a height of 2 m being 5% or more lower than that of the standard strain, and the degree of detachment remaining 10% or more lower even after 14 days of refrigerated storage.
4. A method for cultivating maitake mushrooms, comprising inoculating a culture medium with the spawn of the maitake strain described in claim 1, and producing fruiting bodies or mycelium.
5. The method according to claim 4, wherein the culture medium is prepared by mixing 2 to 5 parts by dry weight of hardwood sawdust with 1 part by dry weight of a mixture of one or more plant-derived nutrients selected from wheat bran, corn bran, hominy feed, okara, rice bran, beet pulp, cotton hull, and corn cob, and adjusting the moisture content to 50 to 75%.
6. The method according to claim 4, comprising culturing the spawn or mycelium under conditions of room temperature of 20 to 30°C and humidity of 50 to 75% RH to form fruiting body primordia, and then growing the fruiting body in a room at room temperature of 15 to 21°C and humidity of 85 to 100% RH.
7. A composition comprising the Maitake mushroom strain described in claim 1, a part thereof, and / or processed products thereof.
8. The composition according to claim 7, which is a food or beverage, a supplement, a pharmaceutical, or a cosmetic.