Method for producing 14-dehydroergosterol

Abstract

A method for producing 14-dehydroergosterol is provided. According to the present invention, there is provided a method for producing 14-dehydroergosterol (14-DHE), which comprises a step of culturing a microorganism belonging to the genus Aspergillus to produce 14-DHE, wherein the initial spore concentration in a medium of the microorganism is 1.0 x 10 4 The present invention also provides a method for producing 14-DHE, which comprises culturing a microorganism belonging to the genus Aspergillus to produce 14-DHE, wherein the initial spore concentration in the medium of the microorganism is 1.0 × 10 spores / mL or more and the medium comprises at least malt extract and an enzymatic digest of a protein. 4 The present invention provides a method for producing 14-DHE, wherein the concentration of 14-DHE is 10 g / L or more, the medium comprises at least malt extract and sugars, and the concentration of the sugars in the medium is 10 g / L or more.

Description

[Technical Field]

[0001] The present invention relates to a method for producing 14-dehydroergosterol. In a method for producing 14-dehydroergosterol by culture of a plant, The present invention relates to a method for enhancing the production of sterols. [Background technology]

[0002] In recent years, 14-dehydroergosterol (hereinafter simply referred to as "14-DHE") has become It has been attracting attention as a substance with physiological activity related to immunosuppression and improvement of lipid metabolism. (Patent Document 1) Regarding the production of 14-DHE, methods using microbial fermentation such as koji mold have been proposed. However, these conventional methods do not provide sufficient amounts of To obtain 14-DHE, a culture period of about 6 days is required. A method for producing DHE was desired. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Laid-Open No. 2016-204342 [Patent Document 2] Japanese Patent Application Laid-Open No. 2013-138617 [Patent Document 3] Japanese Patent Application Laid-Open No. 2015-123013 Summary of the Invention [Problem to be solved by the invention]

[0004] An object of the present invention is to provide a novel method for producing 14-DHE. A novel method for enhancing 14-DHE production in a microbial culture method The present invention aims to provide a method for [Means for solving the problem]

[0005] The present inventors have found that, in the culture of a microorganism belonging to the genus Aspergillus, By setting specific conditions for the initial spore concentration and medium components, 14 The present invention is based on this finding. It is something that happens.

[0006] According to the present invention, the following inventions are provided. [1] A method for producing 14-dehydroergosterol (14-DHE), comprising the steps of: The method comprises culturing a microorganism belonging to the genus Gillus to produce 14-DHE, The initial spore concentration in the biological medium is 1.0 × 10 4 More than 100 cells / mL and the medium is small Both of the ingredients comprise a malt extract and an enzymatic protein digest. [2] The method according to [1] above, wherein the protein enzymatic digest is peptone. [3] The method according to [2] above, wherein the concentration of peptone in the medium is 4.5 g / L or more. [4] A method for producing 14-dehydroergosterol (14-DHE), comprising the steps of: The method comprises culturing a microorganism belonging to the genus Gillus to produce 14-DHE, The initial spore concentration in the biological medium is 1.0 × 10 4 More than 100 cells / mL and the medium is small Both contain malt extract and sugars, and the concentration of the sugars in the medium is 10 g / The method is greater than or equal to L. [5] The method according to [4] above, wherein the sugar comprises maltose and / or dextrose. . [6] The method according to any one of [1] to [5] above, wherein the culture period is 3 to 5 days. [7] The total concentration of malt extract, yeast extract, protein enzyme digest, and sugars in the medium is 1 The method according to any one of [1] to [6] above, wherein the concentration is 8 g / L or more. [8] The microorganism according to any one of [1] to [7] above, wherein the microorganism is Aspergillus kawachii. method. [9] The method according to any one of [1] to [8] above, further comprising a pre-culture step.

[10] Method for producing 14-dehydroergosterol (14-DHE) by microbial culture A method for enhancing the production of 14-DHE in a microorganism belonging to the genus Aspergillus, to produce 14-DHE, The concentration is 1.0×10 4 The medium contains at least malt extract and tamarind. The method comprises the step of:

[11] Method for producing 14-dehydroergosterol (14-DHE) by microbial culture A method for enhancing the production of 14-DHE in a microorganism belonging to the genus Aspergillus, to produce 14-DHE, The concentration is 1.0×10 4 / mL or more, and the medium contains at least malt extract and sugar. and the concentration of the sugar in the medium is 10 g / L or more. Law.

[0007] The manufacturing method of the above [1] and the manufacturing method of the above [4] are collectively referred to in this specification as "the present invention It is sometimes called the "manufacturing method."

[0008] According to the method of the present invention, it is possible to obtain a production equivalent to or greater than that of the conventional method in a shorter culture period than the conventional method. This allows for the efficient production of 14-DHE, which is a physiologically active substance. It is advantageous in that HE can be produced. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 shows the wet cell weight of Aspergillus kawachii obtained by culturing under conditions 1 to 6 shown in Example 1. [Figure 2] FIG. 2 shows the amount of 14-DHE produced per unit weight of wet fungal cells of Aspergillus kawachii (14-DHE production amount / unit weight of wet fungal cells) under conditions 1 to 6 shown in Example 1. [Figure 3] FIG. 3 shows the amount of 14-DHE produced per culture batch (total amount of 14-DHE produced) under conditions 1 to 6 shown in Example 1. [Figure 4] Figure 4 shows the amount of 14-DHE produced per unit weight of wet mycelia (14-DHE production / unit weight of wet mycelia) of Aspergillus kawachii when the culture period was 4 to 6 days under conditions 4 and 6 shown in Example 1, as conditions 4' and 6', respectively. Specific Description of the Invention

[0010] The 14-DHE obtained by the production method of the present invention is a compound represented by the following structural formula: It has the following physicochemical properties:

[0011] (structural formula) [ka]

[0012] (Physicochemical properties) (1) Molecular weight: 394.63 (2) Molecular formula: CHO (observed by high-resolution APCI-Orbitrap method: m / z 395.33057 (M+H) + , Theoretical value: 395.33139) (3) Solubility in solvents: Insoluble in water, slightly soluble in ethanol, easily soluble in chloroform (4) Ultraviolet absorption spectrum (MeCN): 391 nm (5) 1 H-NMR(CD3OD): 6.15 (1H, m), 5.75 (1H, m), 5.65 (1H, dd, J = 2.2, 5.9Hz), 5.27 (1H, dd, J = 7.0, 15.1 Hz), 5.21 (1H, dd, J = 7.9, 15.1 Hz), 3.64 (1H, m), 2.51 (1H, ddd, J = 2.2, 5.1, 10.6 Hz), 2.30 (1H, m), 2.20 (1H, m), 2.20 (1H, dd , J = 3.2, 7.8 Hz), 2.06 (1H, m), 2.05 (1H, m), 1.94 (1H, m), 1.90 (1H, m),1.87 (2H, m), 1.87 (1H, ddd, J = 3.2, 7.0, 7.3 Hz), 1.71 (1H, m), 1.59 (1H, m),1.57 ( 1H, m), 1.45 (1H, m), 1.45 (1H, ddd, J = 3.2, 6.3, 6.5 Hz), 1.30 (1H, m),1.05 (3 H, d, J = 6.8 Hz ), 0.93 (3H, d, J = 7.3 Hz), 0.92 (3H, s,), 0.89 (3H, s), 0.85 (3H, d, J = 6.5 Hz), 0.83 (3H, d, J = 6.3 Hz). (6) 13 C-NMR(CD3OD):149.2 (s), 143.0 (s), 135.4 (s), 132.2 (s), 132.0 (s),120. 5 (s), 120.4 (s), 117.4 (s), 70.4 (s), 58.1 (s), 46.3 (s), 45.4 (s), 42.8 (s), 4 1.0 (s), 39.0 (s), 38.9 (s), 37.8 (s), 37.0 (s), 36.0 (s), 33.1 (s), 32.0 (s), 2 1.1 (s), 19.9 (s), 19.7 (s), 19.6 (s), 17.6 (s), 16.8 (s), 14.5 (s).

[0013] The microorganisms belonging to the genus Aspergillus used in the present invention are capable of producing 14-DHE. There is no particular limitation as long as it can be used, but for example, black koji mold (Aspergillus awamori, A Spergillus niger), yellow koji mold (Aspergillus oryzae), soy sauce koji mold (Aspergillus Aspergillus sojae, and white koji mold (Aspergillus kawachii). Spergillus kawachii and Aspergillus awamori are preferred, and Aspergillus Kawachi (including subspecies) is preferred because it produces a high amount of 14-DHE. It can be obtained from Akita Konno Co., Ltd., Higuchi Moyashi Co., Ltd., Nihon Jozo Kogyo Co., Ltd., etc.

[0014] According to a first aspect of the production method of the present invention, a microorganism belonging to the genus Aspergillus is cultured to produce a fermented food product. A method for producing 14-DHE, comprising the step of producing 14-DHE, The initial spore concentration in the biological medium is 1.0 × 10 4 More than 100 cells / mL and the medium is small The present invention provides a method for producing a malt extract containing a malt extract and a protein enzyme digest. will be done.

[0015] In the first embodiment of the production method of the present invention, a microorganism belonging to the genus Aspergillus is cultured. The initial number of spores of the microorganism added to the medium at the beginning, i.e., the number of spores of the microorganism in the medium The lower limit of the initial spore concentration (above or above) is 5.0 × 10 per mL of medium.3 pieces, 7 .5×10 3 pieces, 1.0×10 4 pieces, 1.25×10 4 pieces, 1.5×10 4 pieces, 1.75 x10 4 pieces, 2.0×10 4 The upper limit (or less than or equal to) 1.0 x 10 per mL of medium 5 pieces, 2.0×10 4 pieces, 3.0×10 4 pieces, 4.0×1 0 4 pieces, 5.0×10 4 The lower and upper limits of these can be set to Any combination can be used. The above initial spore concentration ranges are, for example, 5.0×10 3 pieces~5.0×10 4 pieces, 7.5×10 3 pieces~4.0×10 4 To be an individual The initial spore concentration of the microorganism belonging to the genus Aspergillus added to the medium can be increased. By setting the above, the number of microorganisms in the 14-DHE-producing phase can be sufficiently confirmed. Microorganisms in the 14-DHE-producing phase that can be protected and does not lead to bacteriolysis can be sufficiently present.

[0016] In the first embodiment of the production method of the present invention, the medium contains at least malt extract and protein. Includes enzymatic digests.

[0017] Malt extract is made by germinating wheat, especially barley seeds, and then roasting them in some cases, and then mixing them with water. It is an extract obtained by extracting with a solvent, and contains maltose, glucose, fructose, etc. It contains a lot of reducing sugars, as well as peptides, amino acids, purines, vitamins, etc. Malt extract is available in various forms, including solid powder and paste. As is known, the form of the malt extract used in the present invention is not particularly limited, and From the viewpoint of durability, powdered materials can be preferably used.

[0018] The minimum concentration (solids concentration) of malt extract in the medium is 9 g / L. , 10g / L, 15g / L, 18g / L, 21g / L, 22g / L, and The upper limit (less than or equal to) can be 30g / L, 35g / L, or 40g / L. These lower and upper limits can be arbitrarily combined, and the above malt The range of the concentration of the extract in the medium is, for example, 10 to 40 g / L, 15 to 35 g / L, 18 to The concentration of malt extract in the medium can be set to 30 g / L, or 21 to 30 g / L. By setting the temperature in this way, it is possible to provide the nutrients necessary for the sufficient growth and development of microorganisms. At the same time, it is possible to prevent excess nutrients from remaining in the medium after the expected culture period has elapsed. By doing so, many microorganisms are able to produce 14-DHE through secondary metabolism (14-DHE is thought to be produced through secondary metabolism of microorganisms). It is possible to make the powder phase. In addition to solid ingredients such as malt extract, malt extract in a water-containing form such as paste can also be used. In that case, it is sufficient to convert the concentration into solid concentration and then use the required amount. Needless to say (the same goes for other ingredients below).

[0019] The enzymatic digest of proteins includes peptone. The origin of peptone is not particularly limited. Even if it is derived from meat, yeast, milk casein, soy, or blood cell components, The origin and type of the enzyme are not particularly limited. For example, pepsin, papain, bread Examples include creatine, trypsin, and bacterial proteinase.

[0020] The lower limit (above or beyond) of the concentration (solid concentration) of the protein enzymatic digest in the medium is: The upper limit can be set to 2g / L, 3g / L, 4g / L, or 5g / L. The lower limit of these and the lower limit of the saturation ... The upper limit values can be arbitrarily combined, and the concentration of the enzyme digest of the protein in the medium The concentration range can be, for example, 2 to 11 g / L, 3 to 9 g / L, or 3 g / L. By setting the concentration of the protein enzyme digest in the medium as above, sufficient growth of the microorganisms can be achieved. It can supply nutrients (especially nitrogen sources with adequate small molecules) necessary for growth and development. At the same time, the nutrients in the medium (especially nitrogen sources with moderately small molecules) should be ) is not left in excess, so that many microorganisms are in the secondary metabolism phase. 14-DHE is thought to be produced by secondary metabolism in microorganisms. can be.

[0021] In the first embodiment of the production method of the present invention, the medium contains no components other than those described above (for example, yeast enzymes). Although it may contain other ingredients (such as cereals and sugars), it is recommended that these ingredients be substantially free of the ingredients. Even if the medium does not substantially contain sugars, sugars derived from malt extract can be used. It is acceptable for bacteria to be included in the culture medium.

[0022] According to a second aspect of the production method of the present invention, a microorganism belonging to the genus Aspergillus is cultured to produce a fermented food product. A method for producing 14-DHE, comprising the step of producing 14-DHE, The initial spore concentration in the biological medium is 1.5 × 10 4 More than 100 cells / mL and the medium is small Furthermore, a production method is provided which is characterized in that the product contains malt extract.

[0023] In a second embodiment of the production method of the present invention, in a medium of a microorganism belonging to the genus Aspergillus The lower limit of the initial spore concentration (above or above) is 5.0 × 10 per mL of medium. 3 pieces, 7 .5×10 3 pieces, 1.0×10 4 pieces, 1.25×10 4 pieces, 1.5×10 4 pieces, 1.75 x10 4 pieces, 2.0×10 4 pieces, 2.25×10 4 pieces, 2.5×10 4 It can be done individually The upper limit (below or below) is 1.0 x 10 5 pieces, 2.0×10 4 pieces, 3.0 x10 4 pieces, 4.0×10 4 pieces, 5.0×10 4 The lower limit of these The upper and lower limits can be arbitrarily combined, and the range of the initial spore concentration is, for example, For example, 5.0 x 10 4 pieces~5.0×10 4 pieces, 7.5×10 3 pieces~4.0×10 4 Individual The initial spore concentration in the medium of the microorganism belonging to the genus Aspergillus added to the medium can be By setting the concentration as above, the number of microorganisms in the 14-DHE-producing phase can be It is possible to secure sufficient 14-DHE production without causing bacteriolysis. Microorganisms can be present in sufficient quantities.

[0024] In a second embodiment of the production method of the present invention, the medium contains at least malt extract. The lower limit of the extract concentration (solid concentration) in the medium (above or beyond) is 10 g / L, 1 The upper limit can be set to 1g / L, 12.0g / L, 13g / L, or 14g / L. Below or below) are 15g / L, 20g / L, 25g / L, 30g / L, and 40g / L. These lower and upper limits can be combined in any desired manner. The range of the concentration of the malt extract in the medium is, for example, 9 to 30 g / L, 10 to 25 g / L, The concentration of malt extract in the medium can be 10-20g / L, 10-15g / L. By setting the temperature as above, the nutrients necessary for the sufficient growth and development of microorganisms can be obtained. It is possible to supply nutrients to the medium without excess remaining after the expected cultivation period has elapsed. By preventing this, many microorganisms are in the secondary metabolic phase. This can be done.

[0025] In a second embodiment of the production method of the present invention, the medium contains yeast extract and sugars in addition to the above components. It may include.

[0026] The lower limit of the concentration (solids concentration) of yeast extract in the medium (above or beyond) is 1.2 g / L, 1.8g / L, 2.4g / L, and 3.0g / L, and the upper limit (hereinafter The standard deviation (or less) can be 2.2g / L, 2.8g / L, or 3.4g / L. These lower and upper limits can be arbitrarily combined, and the amount of the sugar in the medium can be adjusted. The concentration range can be, for example, 1.2 to 3.4 g / L or 1.8 to 2.8 g / L. By setting the concentration of yeast extract in the medium as described above, the growth and It can provide nutrients (especially sugars, vitamins, and minerals) necessary for growth and development. By preventing excess nutrients from remaining in the medium after the expected culture period has elapsed, It is possible to ensure that the microorganisms are in the secondary metabolic phase.

[0027] Sugars include, for example, glucose, fructose, maltose, lactose, and dextrose. The sugars in the medium are preferably maltose and dextrose. The limits (above or above) are 7.8g / L, 10g / L, 13g / L, and 15.6g / L. The upper limit (or less than or equal to) is 16g / L, 18g / L, 20g These lower and upper limits can be arbitrarily set. The concentration range of the above sugars in the medium is, for example, 10 to 25 g / L, 1 It can be 3 to 20 g / L, or 13 to 18 g / L.

[0028] In the production method of the present invention, malt extract, yeast extract, protein enzyme digest, and sugars The total concentration (solid concentration) of each component in the medium should be 18g / L or more (preferably 20g / L In addition, malt extract, yeast extract, protein The total concentration of enzyme digests and sugars is calculated by adding the concentrations of each component added in the medium. It is possible.

[0029] In the production method of the present invention, the culture period can be 3 to 5 days, and From the viewpoint of maximizing the production amount, the incubation period is preferably 3.5 to 4.5 days, more preferably 4. days or less or about 4 days.

[0030] In the production method of the present invention, the lower limit of the culture temperature (above or beyond) is 20°C, 25°C, The upper limit (less than or equal to) can be 40°C or 35°C. These lower and upper limits can be combined arbitrarily, and the culture temperature The range is, for example, 20 to 40°C, 20 to 35°C, 25 to 35°C, 30 to 35°C, most preferably The temperature may be set to about 30°C.

[0031] In the production method of the present invention, the microbial culture can be carried out in liquid culture. Using a fermenter capable of controlling conditions, for example, stirring culture, aeration culture, shaking culture, etc. The cultivation is carried out under aerobic conditions. The agitation speed and aeration volume vary depending on the type of microbial cells and the It is selected appropriately depending on the culture conditions.

[0032] In the case of shaking culture, the shaking conditions are not particularly limited, but the shaking direction is one-dimensional (forward and backward). It can be in two dimensions (rolling vibration) or two-dimensional (rotating vibration), or it can be in three dimensions. For example, when using a 5L jar fermenter as a culture vessel, When shaking, the stirring speed can be 100 to 1000 rpm, preferably 200 to 800 rpm. When culturing under aeration conditions, the aeration rate is For example, the concentration can be set to 0.4 to 0.6 vvm. By setting appropriate shaking conditions, air is supplied to the entire culture medium, and the growth of microorganisms is prevented. This prevents imbalance of nutrients and growth-inhibiting substances in the medium, and improves the efficiency of 14-DHE. This ensures efficient production.

[0033] The production method of the present invention may further include a pre-culture step prior to the aforementioned culture step. (In this specification, the culture process already described is called "main culture" to distinguish it from "preculture"). Pre-culture is a method for culturing microorganisms by increasing the initial number of spores to a desired number in advance. This is done from the perspective of adjusting to the growth phase, and can be done, for example, as follows: This can be done.

[0034] In the production method of the present invention, the culture period of the pre-culture is not particularly limited, but may be, for example, 8 to 14 It can be 4 hours, 10-80 hours, 15-40 hours, 15-30 hours, and the most preferred In the production method of the present invention, the incubation time is preferably 24 hours or more. The culture temperature, shaking conditions, and aeration amount can be the same as those for the main culture. Here, pre-culture is carried out on a relatively small scale in flasks, etc., but a shaking incubator is used. In this case, the shaking mode can be selected according to the shape of the culture vessel. For example, When using a Sakaguchi flask as a culture vessel, vibration should be unidirectional (reciprocating). When using an Erlenmeyer flask, the amplitude is two-dimensional (rotational shaking). For example, the amplitude (spm) or rotation speed (rpm) when the volume of the culture vessel is 500 mL ) is 10 to 500, 50 to 300, 70 to 200, and more preferably about 120, The most preferable flow rate is about 120 spm when using a Sakaguchi flask.

[0035] In the production method of the present invention, after the main culture step, a fermentation product containing the bacterial cells obtained by the main culture is obtained. The method may further include a step of extracting the extract using a suitable solvent by a conventional method. The process for extracting the fermentation product includes a step of disrupting the cells by ultrasonic treatment and a soaking step described below. It can be a combination.

[0036] The organic solvents used for solvent extraction are ethanol, methanol, n-propanol, and ethanol. Examples include isopropanol, methyl acetate, ethyl acetate, hexane, and heptane. Of these, ethanol, methanol, ethyl acetate, and hexane are preferred. Two or more types of organic solvents may be mixed and used, or two or more types of organic solvents may be used in two or more stages. Alternatively, instead of or in combination with such organic solvents, an extraction step may be performed. In combination, edible oils and fats can be used as the extraction solvent. Although it may be an animal fat such as soybean oil, it is preferably a vegetable fat, for example soybean oil. , rapeseed oil, olive oil, palm oil, sesame oil, corn oil, safflower oil, cottonseed oil, peanut oil Examples include corn oil, grapeseed oil, perilla oil, camellia oil, and flaxseed oil.

[0037] The step of extracting the fermentation product may be, for example, a step of extracting the fermentation product containing the bacterial cells from ethanol. and a step of immersing the cells in a soaking solution containing water and organic solvent. The extraction process can be carried out by adding a solvent and / or edible oils and fats. In the extraction process using the culture medium, the soaking process is essential. The ethanol in the soaking liquid containing ethanol and water is used to soak the fermented product containing the bacteria obtained by cultivation. The concentration of the alcohol is 20 (w / w)% or more, especially 30 (w / w)% or more, and especially 40 (w / w)% or more. )% or more, and 95(w / w)% or less, particularly 90(w / w)% or less. The immersion liquid is preferably a mixture of water and ethanol, and more preferably 80 (w / w) % or less. The soaking liquid containing ethanol and water may be an aqueous ethanol solution containing the fermented product. It weakens the cell membrane or cell wall of the bacteria contained within, allowing the 14-DHE stored inside the bacteria to be extracted. Immersion should be for at least 1 hour, especially for at least 6 hours, and especially It is preferable to carry out the treatment for a period of 12 hours or more, and more preferably 24 hours or more.

[0038] The fermented product is soaked in a soaking solution containing ethanol and water, and then soaked in an organic solvent and / or food. The amount of edible oil is 10 mL or less per 1 g of culture product. In particular, when used in an amount of 8 mL or less, especially 6 mL or less, 14% of the koji fermentation extract obtained This is preferable because it increases the concentration of -DHE. There is no need to use ultrasonic treatment to destroy the bacteria. By soaking, the cell membrane or cell wall of the fungus is sufficiently weakened, and it is possible to simply add edible oils and fats. By simply stirring the mixture, the 14-DHE stored in the cells can be extracted more efficiently. The extraction process may be repeated multiple times as necessary to extract 14-DHE.

[0039] According to the present invention, in the method for producing 14-DHE by microbial culture, the production of 14-DHE is A method for enhancing the viability of a microorganism belonging to the genus Aspergillus, which comprises culturing the microorganism in a liquid culture to produce 14-DH E, wherein the initial spore concentration in the culture medium of the microorganism is 1.0 x 10 4 The concentration is ≥ 1000 / mL, and the medium contains at least malt extract and enzyme digest of protein. The enhancement method of the present invention comprises the steps of: and the protein enzymatic digest is prepared according to the description of the first embodiment of the production method of the present invention. otherwise, the method can be carried out in accordance with the description of the manufacturing method of the present invention. can.

[0040] The present invention also provides a method for producing 14-DHE by microbial culture. A method for enhancing the production of a compound comprising: culturing a microorganism belonging to the genus Aspergillus in a liquid culture medium for 14- and producing DHE, wherein the microorganism has an initial spore concentration of 1.0×1 0 4 The concentration of the microorganisms is at least 1 / mL, and the medium contains at least malt extract and sugars. The enhancement method of the present invention is characterized in that the initial spore concentration is The manufacturing method of the present invention can be carried out according to the description of the second embodiment, and the rest of the manufacturing method of the present invention can be carried out according to the description of the second embodiment. This can be carried out according to the description of the manufacturing method described above. [Example]

[0041] The present invention will be described in more detail based on the following examples, but the present invention is not limited to these examples. It's not something like that.

[0042] Example 1: Examination of culture conditions for fermentative production of 14-DHE (1) In Example 1, the culture conditions for fermentation production of 14-DHE were as follows: The study focused on the concentration (number of spores added) and medium composition.

[0043] (1) Method A. Pre-culture Aspergillus kawachii ( Aspergillus kawachii ) The NBRC4308 strain (distributed by the National Institute of Technology and Evaluation) was used. The medium contains Malt extract (Bacto Malt extract). ract 218630) 6.0g / L, maltose (Fujifilm Wako Pure Chemical Industries) 1.8 g / L, dextrose (Fujifilm Wako Pure Chemical Industries) 6.0 g / L, yeast extract Y Add 150 mL of culture medium containing 1.2 g / L of yeast extract (Difco). The medium was prepared in a 500 mL Sakaguchi flask. The spore concentration was adjusted to 1.0 x10 5 The NBRC4308 strain was inoculated at a density of 100 cells / mL and incubated at 30°C and 120 spm. The cells were cultured for 24 hours.

[0044] B. Main culture The main culture medium was prepared in a 5 L Jafermenter (Marubishi Bio) with the composition and amount shown in Table 1. The pre-culture solution (a) was prepared in 3L of a container (Oenji Co., Ltd.). After that, the mixture was cultured at 30°C, 400 rpm, and 0.5 VVM for 96 hours (4 days) (condition 1). The culture was carried out for 144 hours (6 days) (conditions 2 to 6). FCO) was used, and malt extract, yeast extract, maltose, and dextrose were used as pre-culture medium. The peptone used was the same as that used for the fermentation. It is a protein enzymatic digest.

[0045] [Table 1]

[0046] C. Extraction and quantification of 14-DHE To collect the cultured bacteria in step (a), the culture liquid (liquid koji) is absorbed using filter paper. The mixture was filtered by suction, and the cells were washed with water. After that, the mixture was filtered by suction again to remove as much water as possible. 6 mL of ethanol was added to 2 g of the collected wet cells, and the mixture was left to stand overnight. Then, 3 mL of water and 6 mL of hexane were added. After vigorously stirring for 2 minutes, the mixture was sonicated for 30 minutes. The mixture was centrifuged at 100 rpm and the supernatant (hexane layer) was collected. Then, add 6 mL of hexane to the residue (suspended aqueous layer) other than the hexane layer, stir, and sonicate. The hexane layer was collected and dried. This was repeated twice for 2 g of cells. 2 mL of ethanol was added to the resulting hexane dry matter, and 2- After diluting 40 times with propanol, 20 μL was applied to liquid chromatography (HPLC). The HPLC conditions are shown in Table 2. Under these conditions, 14-DHE was detected within 12.5 minutes. The amount of 14-DHE produced was measured using a 14-DHE standard (produced by fermentation and purification). The values were calculated using the calibration curve method.

[0047] [Table 2]

[0048] (2) Results The results are shown in Figures 1 to 3. Under the culture conditions equivalent to those of the conventional method (condition 1), malt When cultured for 6 days in a medium containing extract at a certain concentration or higher, the wet bacterial weight is approximately 90g, the amount of 14-DHE produced per wet cell unit weight was approximately 680μg, and the total 14-DHE produced was The yield was approximately 60 mg / 3 L.

[0049] A. Initial spore concentration When the initial spore concentration (the number of spores added in the main culture) was increased to 8 times that of condition 1, the culture was continued for 4 days. Even under the condition (Condition 2), the wet bacterial weight was equal to or greater than that of the 6-day culture (Condition 1) (Fig. 1). The amount of 14-DHE produced per unit weight (Fig. 2) and the total amount of 14-DHE produced (Fig. 3) were It was confirmed that this could be achieved.

[0050] B. Medium composition i) Medium Components <Malt extract + peptone> The initial spore concentration was increased by 8 times, and the medium composition was changed to two components: malt extract and peptone. The total concentration of the medium components was changed to about twice that of Condition 1 and Condition 2 (both 18 g / L). (Condition 4: total concentration of medium components 30 g / L) (Condition 4) Compared with condition 1) and condition 2, the wet cell weight and 14- The amount of DHE increased (Figs. 1 and 2), and the total amount of 14-DHE was approximately three times that of the 6-day culture (Condition 1). It was confirmed that this was the case (Figure 3).

[0051] <Malt extract + yeast extract> On the other hand, when yeast extract was added instead of peptone as a medium component (condition 5), The wet cell weight increased significantly, but the amount of 14-DHE and total 14-D per wet cell weight also decreased. It was confirmed that the amount of HE decreased.

[0052] ii) Medium concentration Condition 1 (cultured for 6 days) and Condition 2 differ in that the total concentration of the medium components was doubled without changing the medium components. When the total amount of medium components was 30 g / L (conditions 3 and 6), the number of spores added increased by 8 times. When the condition 3 was met, only the wet cell weight increased significantly, and the amount of 14-DHE per unit wet cell weight increased. The amount of 14-DHE and the total amount of 14-DHE were clearly reduced. When the concentration was increased by 12 times (condition 6), the wet cell weight and the wet cell weight increased by 14-fold in 4 days. The amount of DHE was approximately 1.5 times higher than that of the 6-day culture (condition 1), and the total amount of 14-DHE was It was confirmed that this was approximately three times that of condition 1 (cultured for 6 days) (Figure 3).

[0053] From these results, when the initial spore concentration was increased by 8 times with the same medium composition, the number of spores increased by 4 days after the main culture. At this point, almost no 14-DHE was produced, but when the initial spore concentration was further increased (e.g. For example, when the concentration is increased by 12 times, the transition from the growth phase to the secondary metabolism phase of the fungus occurs. This may have accelerated the production of 14-DHE, a secondary metabolite of the fungus. can be done.

[0054] Example 2: Examination of culture conditions for fermentative production of 14-DHE (2) From the results of Example 1, among the culture conditions for fermentation production of 14-DHE, the initial spore concentration and the medium By changing the culture conditions of the composition, the culture period can be changed from 4 days to 6 days (Example 1). It was confirmed that the amount of 14-DHE produced was equal to or greater than that of condition 1) (especially under condition 1). Therefore, in Example 2, the initial spore concentration and culture conditions were the same as those in Conditions 4 and 6 of Example 1. Under the same culture conditions, how much 14-DHE production was affected when the culture period was extended beyond 4 days? We examined what kind of impact this would have.

[0055] (1) Method The conditions were the same as Conditions 4 and 6 in Example 1, except that the culture period was 5 and 6 days. went.

[0056] (2) Results The results are shown in Figure 4. Specifically, when the medium composition was Condition 4 (●) in Example 1, The amount of 14-DHE produced per cell was almost unchanged between 4 and 5 days of culture. On the sixth day, the amount of bacteria also decreased, indicating that bacteria were lysed. On the other hand, when the medium composition was condition 6 (▲) in Example 1, the culture period was 5 days. The amount of 14-DHE produced per unit weight of bacterial cells decreased during the 6-day incubation period compared to the 4-day incubation period (condition 6 in Figure 4). ).

[0057] Example 3: Examination of culture conditions for fermentative production of 14-DHE (3) In Condition 4 of Example 1, when meat-derived peptone was added as a medium component, 14-DH This result indicates that the use of enzyme digested protein as a nitrogen source increases the amount of E produced. , which contributes to the increased production of 14-DHE by microorganisms belonging to the genus Aspergillus. Therefore, in Example 3, the production of 14-DHE when the origin (type) of peptone was changed was investigated. The effect on the yield was examined.

[0058] (1) Method Other than using yeast-derived peptone (Oriental Yeast Co., Ltd.) as the peptone, The test was carried out under the same conditions as in Condition 4 of Example 1.

[0059] (2) Results When yeast-derived peptone was used, the production volume per batch was 100% of that in peptone-free medium (Example 1). This was approximately 1.75 times higher than that of condition 1) (data not shown). It was confirmed that the amount of 14-DHE produced increased regardless of the type (origin) of peptone used as the digest. It was recognized.

Claims

1. A method for producing 14-dehydroergosterol (14-DHE), comprising the step of culturing Aspergillus kawachii to produce 14-DHE, wherein the initial spore concentration of the Aspergillus kawachii in a medium is 1.0 x 10 ４ the cell density is from 2.0 x 10 4 cells / mL to 2.0 x 10 4 cells / mL, the medium comprises at least malt extract and a protein enzymatic digest, the concentration of the malt extract in the medium is from 9 g / L to 40 g / L, the concentration of the protein enzymatic digest in the medium is from 2 g / L to 11 g / L, and the culture period is 3 to 5 days.

2. 2. The method of claim 1, wherein the protein enzymatic digest is peptone.

3. The method according to claim 2, wherein the concentration of peptone in the medium is 4.5 g / L or more and 11 g / L or less.

4. A method for producing 14-dehydroergosterol (14-DHE), comprising the step of culturing Aspergillus kawachii to produce 14-DHE, wherein the initial spore concentration of the Aspergillus kawachii in a medium is 1.0 x 10 ４ the cell density is from 0.01 to 0.2 × 10 cells / mL, the culture medium contains at least malt extract and sugars, the concentration of the malt extract in the culture medium is from 9 g / L to 40 g / L, the concentration of the sugars in the culture medium is from 10 g / L to 25 g / L, and the culture period is 3 to 5 days.

5. 5. The method of claim 4, wherein the sugars include maltose and / or dextrose.

6. The method according to any one of claims 1 to 5, wherein the total concentration of the malt extract, yeast extract, protein enzymatic digest, and sugars in the medium is 18 g / L or more and 30 g / L or less.

7. The method according to any one of claims 1 to 6, further comprising a pre-culture step.

8. A method for enhancing production of 14-dehydroergosterol (14-DHE) in a method for producing 14-DHE by microbial culture, comprising the step of culturing Aspergillus kawachii to produce 14-DHE, wherein the initial spore concentration of the Aspergillus kawachii in a medium is 1.0 x 10 ４ the cell density is from 2.0 x 10 4 cells / mL to 2.0 x 10 4 cells / mL, the medium comprises at least malt extract and a protein enzymatic digest, the concentration of the malt extract in the medium is from 9 g / L to 40 g / L, the concentration of the protein enzymatic digest in the medium is from 2 g / L to 11 g / L, and the culture period is 3 to 5 days.

9. A method for enhancing production of 14-dehydroergosterol (14-DHE) in a method for producing 14-DHE by microbial culture, comprising the step of culturing Aspergillus kawachii to produce 14-DHE, wherein the initial spore concentration of the Aspergillus kawachii in a medium is 1.0 x 10 ４ the cell density is from 0.01 to 0.2 × 10 cells / mL, the culture medium contains at least malt extract and sugars, the concentration of the malt extract in the culture medium is from 9 g / L to 40 g / L, the concentration of the sugars in the culture medium is from 10 g / L to 25 g / L, and the culture period is 3 to 5 days.

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