Method for producing filamentous fungus

Abstract

To provide a new method for producing a filamentous fungus, capable of easily separating fungal cells from a culture medium and ensuring high safety when utilizing the fungal cells as food.SOLUTION: The method for producing a filamentous fungus comprises a step of inoculating and culturing the filamentous fungus on a gel or film disposed in contact with a culture medium, wherein the gel or film contains one or more thickening polysaccharides selected from the group consisting of agar, gelatin, carrageenan, xanthan gum, locust bean gum, and curdlan.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing a filamentous fungus. [Background technology]

[0002] In recent years, technology to process microorganisms, including their entire bodies, into meat substitutes has been attracting attention as one solution to the problem of global food shortages. In particular, filamentous fungi are formed from hyphae, and by collecting the hyphae, they form fibrous masses with a texture similar to meat, making them a promising raw material for meat substitutes.

[0003] Edible filamentous fungi are usually cultivated in solid or liquid media, and then harvested and processed. However, when cultivated in solid or liquid media, it is not easy to separate the fungal bodies from the medium. Considering that the fungal bodies are edible, contamination of the medium must be avoided.

[0004] Therefore, the use of a membrane (polymers such as polypropylene, polytetrafluoroethylene, polycarbonate, and polyamide, glass fiber materials, porous ceramic materials, cloth, etc.) has been proposed as a method for recovering bacterial cells from the culture medium (Patent Document 1). The bacterial cells are separated from the liquid culture medium by a membrane that allows only nutrients to pass through, and can be easily recovered from the liquid culture medium by applying pressure to peel the bacterial cells from the membrane surface.

[0005] A method for recovering bacterial cells from a solid medium using a porous membrane has also been proposed (Patent Document 2). The porous membrane may be a semipermeable membrane sheet such as cellophane, collodion membrane, bladder membrane, eggshell membrane, or acetylcellulose membrane, or a membrane filter for filtration sterilization.

[0006] However, in order to use filamentous fungi for meat substitutes, production efficiency must be increased and safety for use as food must be ensured, and further improvements are being sought in terms of practical application. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Special Publication No. 2022-521993 [Patent Document 2] Japanese Patent Application Publication No. 2019-122346 Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a novel method for producing filamentous fungi, which allows for easy separation of the fungal cells from the medium and is highly safe when the fungal cells are used as food. [Means for solving the problem]

[0009] The present inventors discovered that by seeding and culturing bacterial cells on an edible gel or film containing a specific thickening polysaccharide, the bacterial cells can be easily separated from the medium and safety for food use can be ensured, leading to the completion of the present invention.

[0010] That is, the present invention includes the following inventions. (1) A method for producing a filamentous fungus, comprising a step of seeding and culturing a filamentous fungus on a gel or film placed in contact with a culture medium, wherein the gel or film contains one or more thickening polysaccharides selected from the group consisting of agar, gelatin, carrageenan, xanthan gum, locust bean gum, and curdlan. (2) The method for producing a filamentous fungus according to item 1, further comprising the step of recovering the filamentous fungus cultured on the gel or film together with the gel or film. (3) The method for producing a filamentous fungus according to item 1 or 2, further comprising the step of recovering the filamentous fungus together with the gel or film, dissolving the gel or film at a high temperature (80°C or higher), and isolating the filamentous fungus. (4) The method for producing a filamentous fungus according to item 3, wherein the gel or film is dissolved in hot water (80°C or higher). (5) A method for producing a filamentous fungus according to any one of items 1 to 4, further comprising the step of removing the filamentous fungus cultured on the gel or film together with the gel or film, and replacing the medium. (6) A filamentous fungus culture composition comprising a gel or film and a filamentous fungus cultured on the gel or film, wherein the gel or film contains one or more thickening polysaccharides selected from the group consisting of agar, gelatin, carrageenan, xanthan gum, locust bean gum, and curdlan. (7) A culture medium for a filamentous fungus, comprising a culture medium layer and a thickening polysaccharide layer disposed in contact with the culture medium layer, wherein the thickening polysaccharide layer contains one or more thickening polysaccharides selected from the group consisting of agar, gelatin, carrageenan, xanthan gum, locust bean gum, and curdlan, and a filamentous fungus is seeded and cultured on the thickening polysaccharide layer. [Effects of the Invention]

[0011] According to the present invention, the fungus cells can be easily separated from the medium, and the cultured filamentous fungus can be recovered from the medium together with the gel or film. Furthermore, since the specific thickening polysaccharide used in the gel or film of the present invention is edible, there is little risk of it being mixed with the bacterial cells during recovery.

[0012] Furthermore, by using a gel or film that melts at high temperatures, bacterial cells can be easily separated simply by treating them at high temperatures, and the high temperature kills the bacterial cells (including spores), so they can be used safely in food factories without contaminating production lines, etc. with bacteria.

[0013] Furthermore, according to the present invention, the cells can be removed together with the gel or film during the cultivation and the medium can be replaced, which has the advantage of increasing the yield of cells.

[0014] Furthermore, by adjusting the concentration and thickness of the components contained in the gel or film, it is possible to prevent bacterial cells from remaining in the culture medium, making it easier to reuse the culture medium.It is also thought that when recovering bacterial cells from the culture medium, metabolites of the bacterial cells remaining on the gel or film can also be easily recovered. DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention will be described in detail below. In the present invention, the "filamentous fungus" is not particularly limited as long as it is an edible filamentous fungus, and is preferably, for example, a filamentous fungus belonging to the genus Aspergillus or Fusarium. Examples of filamentous fungi belonging to the genus Aspergillus include Aspergillus oryzae, Aspergillus sojae, Aspergillus luchuensis, Aspergillus kawachii, and Aspergillus tamarii. Examples of filamentous fungi belonging to the genus Fusarium include Fusarium venenatum.

[0016] In the present invention, the term "gel or film" or "thickening polysaccharide layer" is not particularly limited as long as it contains one or more edible thickening polysaccharides selected from the group consisting of agar, gelatin, carrageenan, xanthan gum, locust bean gum, and curdlan, maintains its shape without dissolving at the culture temperature, allows the components of the culture medium to pass through it and allows the filamentous fungus to be cultured thereon, and allows the fungus to be recovered after the culture. Here, a "gel" refers to a semi-solid or solid state in which colloidal particles are dispersed in a liquid or gas, and has a thickness of, for example, 0.025 cm to 10 cm. A "film" refers to a polymer component or the like molded into a thin film, and has a thickness of, for example, 0.001 to 0.025 cm. A "thickening polysaccharide layer" is, for example, a gel or film containing a thickening polysaccharide. The one or more thickening polysaccharides are preferably agar, gelatin, carrageenan, or curdlan from the viewpoint of the degree of residue in the medium at the time of recovery or separation from the medium, and are preferably agar, gelatin, carrageenan, xanthan gum, or locust bean gum that dissolves in water at 80°C or higher from the viewpoint of separation of the gel or film from the fungal cells.

[0017] In the present invention, the "gel or film" or "thickening polysaccharide layer" is not particularly limited and can be prepared by known methods. It can be obtained by dissolving one or more thickening polysaccharides selected from agar, gelatin, carrageenan, xanthan gum, locust bean gum, and curdlan in water at a concentration sufficient for gelling (solidifying) or film formation, followed by gelling or film formation, and may contain other ingredients (other edible thickening polysaccharides, starch, emulsifiers, etc.). Commercially available products may also be used; an example of a film is Clair High Temperature (manufactured by Ina Foods Co., Ltd.).

[0018] In the present invention, the "gel or film" or "thickening polysaccharide layer" preferably melts at high temperatures (preferably 80° C. or higher). Furthermore, the thickness of the "gel or film" or "thickening polysaccharide layer" and the concentration of the components contained therein can be adjusted as appropriate, and a thickness and concentration that prevents bacterial cells from remaining in the medium after collection of the "gel or film" or "thickening polysaccharide layer" is preferred. In the case of a gel, a thickness of 0.3 cm to 1.0 cm is preferred, and in the case of a film, a thickness of 0.003 cm to 0.01 cm is preferred. Furthermore, the concentration of the one or more thickening polysaccharides in the gel is preferably 1% by mass to 10% by mass.

[0019] In the present invention, the method for culturing filamentous fungi is not particularly limited, and can be carried out by any known method, except that the filamentous fungi are seeded and cultured on a "gel or film" or "thickening polysaccharide layer" arranged in contact with a medium or medium layer, more specifically on the surface not in contact with the medium or medium layer (i.e., the surface in contact with air). Here, the "gel or film" or "thickening polysaccharide layer" can be present in a form that covers the surface of the medium or medium layer, and in the case of a liquid medium, a portion of it may be submerged in the liquid, but preferably it covers (floats on) the surface of the medium or medium layer.

[0020] In the present invention, the "medium" or "medium layer" refers to a liquid or solid medium, and any medium capable of growing filamentous fungi can be used. Examples of liquid media include synthetic media supplemented with carbon sources, nitrogen sources, inorganic salts, vitamins, minerals, and amino acids, as well as commercially available media such as YDP medium, PDA medium, and SCD medium. Examples of carbon sources include monosaccharides, disaccharides, polysaccharides, starch, and blackstrap molasses. More specifically, glucose, maltose, and sucrose can be used. Examples of nitrogen sources include ammonia, ammonium sulfate, ammonium phosphate, ammonium carbonate, ammonium acetate, peptone, yeast extract, casein hydrolysate, bran, and meat extract. Examples of inorganic salts include potassium salts, magnesium salts, sodium salts, phosphate salts, manganese salts, iron salts, zinc salts, and copper salts. Liquid media may contain solid components. Solid media may also be prepared by gelling liquid media with agar, gelatin, or the like. The pH of the medium is adjusted to 5.0 to 8.0. The culture temperature is set to 20 to 40°C, preferably 25 to 35°C, and the culture time is 1 to 10 days, preferably 3 to 7 days, more preferably 3 to 4 days.

[0021] After cultivation, the bacterial cells can be easily recovered by, but not limited to, separating the gel or film from the medium, or by scraping the bacterial cells from the gel or film, etc. Furthermore, when a gel or film that dissolves at high temperatures is used, the gel or film can be dissolved (either by itself or in the liquid) by, for example, autoclaving or exposing it to hot water, and the bacterial cells can be easily separated by solid-liquid separation such as filtration or centrifugation.

[0022] In the present invention, the "filamentous fungus culture composition" is not particularly limited as long as it comprises a gel or film and a filamentous fungus cultured thereon, and includes, for example, a gel or film on which a filamentous fungus is layered over the entire surface or a portion thereof. The filamentous fungus culture composition can be made edible by washing, shaping, heating, freezing, seasoning, etc., using common means (methods, conditions, etc.).

[0023] In the present invention, the "filamentous fungal culture medium" is not particularly limited as long as it comprises a medium layer and a thickening polysaccharide layer arranged in contact with the medium layer and is capable of culturing filamentous fungi, and includes, for example, a two-layer structure formed by a solid medium layer and a thickening polysaccharide layer covering the solid medium layer, and a liquid medium layer and a thickening polysaccharide layer covering the liquid medium layer or having a portion thereof submerged in liquid. [Example]

[0024] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples.

[0025] Experimental Method (1) Preparation of spore suspension Approximately 20 ml of suspension solution (composition below) was added to approximately 10 g of rice koji and suspended by vortexing. After suspension, the solution was filtered using a cell strainer (mesh size: 70 μm, material: nylon), and the filtrate was used as the spore solution. The spore solution contained 1.0 × 10 5 After adjusting the concentration to cells / ml, 1 ml of the solution was dispensed into microtubes and stored frozen at -80°C. When using the spore liquid, it was taken out of the freezer, thawed at room temperature, and tapped to ensure a sufficiently uniform concentration before use.

[0026] <Composition of suspension solution> Sodium chloride: 0.9g Tween® 20: 0.05g The above substances were dissolved in 100 ml of water and sterilized by autoclaving.

[0027] (2) Culture method - Liquid medium After preparing the medium (mixing the following components 1 to 4), approximately 20 ml of the liquid was dispensed into petri dishes to prepare liquid media. Then, various gels were placed in the liquid media or the surface of the liquid media was covered with a film, and the spore solution was then sown on top of the film. The spore solution was dripped onto approximately 9 points on the surface of the liquid media without spreading it over the surface. The culture was carried out for 4 days in an incubator set at 30°C (gel) or 28°C (film).

[0028] <Liquid medium composition> Composition 1: Ammonium sulfate: 20g Disodium hydrogen phosphate: 1.36g Potassium sulfate: 2.7g Yeast extract, Meat Powder N (Asahi Group Foods): 2g The above material was dissolved in 880 ml of water and sterilized by autoclaving. Composition 2: Glucose: 50g The above substances were dissolved in 100 ml of water and sterilized by autoclaving. Composition 3: Calcium acetate: 0.2g The above substances were dissolved in 10 ml of water and sterilized by autoclaving. Composition 4: Magnesium sulfate: 0.6g The above substances were dissolved in 10 ml of water and sterilized by autoclaving.

[0029] (3) Culture method - solid medium After preparing the medium (mixing the following compositions 1 to 4), approximately 20 ml of the medium was dispensed into petri dishes and cooled at room temperature to prepare plate media. Various gels or films were then placed on the plate media, and the spore solution was then sown on top of them. The spore solution was sown by dropping it at approximately 9 points, without spreading it over the medium. The culture was carried out in an incubator set at 30°C (gel) or 28°C (film) for 4 or 6 days.

[0030] <Solid medium composition> Composition 1: Ammonium sulfate: 20g Disodium hydrogen phosphate: 1.36g Potassium sulfate: 2.7g Yeast extract, Meat Powder N (Asahi Group Foods): 2g Agarose XP (coagulant): 5g The above material was dissolved in 880 ml of water and sterilized by autoclaving. Composition 2: Glucose: 50g The above substances were dissolved in 100 ml of water and sterilized by autoclaving. Composition 3: Calcium acetate: 0.2g The above substances were dissolved in 10 ml of water and sterilized by autoclaving. Composition 4: Magnesium sulfate: 0.6g The above substances were dissolved in 10 ml of water and sterilized by autoclaving.

[0031] (4) Preparation of gels and films The gel was prepared by dissolving the following components in water and gelling them. Xanthan gum + locust bean gum (1:1 by mass ratio): 2% by mass concentration ·Agar: 2% concentration by mass K-carrageenan: 2-4% by mass concentration Gelatin: 10% by weight Curdlan: 5% by mass concentration Sodium alginate: 2% by weight The films used were agar film ("Clair High Temperature", manufactured by Ina Foods Co., Ltd.), wafer film, and polyvinyl alcohol (PVA) film.

[0032] Example 1: Evaluation of culture in liquid medium Various gels (each component is placed in a base area of ​​9cm 2 The cells were cultured in liquid medium for 4 days using the film and 10 ml of the gel was poured into a petri dish (gelling) and the film was then evaluated as follows. Evaluation items:

[0033] <Status at culture temperature> It was confirmed whether the gel remained solid and whether the film retained its shape at the incubation temperature. <Residual degree in culture medium or separation from culture medium> It was confirmed whether the gel could be recovered without remaining in the medium, and whether the film would dissolve in the medium and become impossible to separate. <Permeability to medium components> It was confirmed that the components in the medium could pass through the gel or film and be cultured. <Bacteria cell impermeability> When the gel or film was collected after the culture, it was confirmed whether or not bacteria were present in the medium (it is better if they were not permeated). <Solubility at high temperatures> When the gel or film recovered after cultivation was exposed to hot water (80°C), it was confirmed whether the gel or film would dissolve and only the bacterial cells could be recovered. The evaluation results for the gel are shown in Table 1, and the evaluation results for the film are shown in Table 2.

[0034] [Table 1]

[0035] [Table 2]

[0036] As shown by the data in Tables 1 and 2, the gels and films of the present invention were able to maintain their shape without dissolving during cultivation at the cultivation temperature, allowing the components in the medium to permeate, enabling the cultivation of bacterial cells. Furthermore, the gels and films of the present invention could be recovered and separated after cultivation, and all except curdlan could be dissolved at high temperatures (curdlan could be separated by scraping the bacterial cells from the gel). Furthermore, no penetration of bacterial cells into the medium was observed when recovering the gels and films.

[0037] Example 2: Evaluation of culture on solid medium Various gels (K-carrageenan has a base area of ​​63.5 cm 210 ml of gelatin is placed in a petri dish with a base area of ​​9 cm 2 The culture medium was cultured for 6 days (gelatin and film for 4 days) on solid medium, and the following evaluations were carried out.

[0038] Evaluation items: <Status at culture temperature> It was confirmed whether the gel remained solid and whether the film retained its shape at the incubation temperature. <Removability from culture medium> It was confirmed that the gel could be peeled off without collapsing when collected after culture, and that the film would dissolve during culture and become impossible to peel off. <Permeability to medium components> It was confirmed that the components in the medium could pass through the gel or film and be cultured. <Bacteria cell impermeability> When the gel or film was collected after the culture, it was confirmed whether or not bacteria were present in the medium (it is better if they were not permeated). <Solubility at high temperatures> When the gel or film recovered after cultivation was exposed to hot water (80°C), it was confirmed whether the gel or film would dissolve and only the bacterial cells could be recovered. The evaluation results for the gel are shown in Table 3, and the evaluation results for the film are shown in Table 4.

[0039] [Table 3]

[0040] [Table 4]

[0041] As shown in the data in Tables 3 and 4, the gels and films of the present invention were able to maintain their shape without dissolving during culture at the culture temperature, allowing the components in the medium to permeate, enabling the cultivation of bacterial cells. Furthermore, the gels and films of the present invention were able to be peeled off after culture and were able to dissolve at high temperatures.

[0042] Regarding K-carrageenan, the penetration of the bacterial cells into the medium was observed, but this could be improved by adjusting the thickness and concentration of the ingredients.

[0043] Example 3: Evaluation of gel concentration and thickness K-carrageenan was used to evaluate gel consistency and thickness. Each concentration and amount of K-carrageenan (base area 63.5 cm 2 The mixture was cultured in a solid medium for 6 days, and the conditions were divided by concentration and thickness, and the evaluation was carried out in the same manner as in Example 2. The results are shown in Table 5.

[0044] [Table 5]

[0045] As shown in Table 5, the impermeability of bacterial cells is related not only to the gel thickness but also to the concentration of its components, and bacterial impermeability can be achieved by appropriately adjusting both the concentration and thickness. Adjusting these factors is also thought to improve separation and peelability from the medium.

Claims

1. A method for producing a filamentous fungus, comprising a step of seeding and culturing a filamentous fungus on a gel or film placed in contact with a culture medium, wherein the gel or film contains one or more thickening polysaccharides selected from the group consisting of agar, gelatin, carrageenan, xanthan gum, locust bean gum, and curdlan.

2. The method for producing a filamentous fungus according to claim 1, further comprising a step of recovering the filamentous fungus cultured on the gel or film together with the gel or film.

3. 3. The method for producing a filamentous fungus according to claim 2, further comprising the step of recovering the filamentous fungus together with the gel or film, dissolving the gel or film at a high temperature (80°C or higher), and isolating the filamentous fungus.

4. 3. The method for producing a filamentous fungus according to claim 1, further comprising the step of removing the filamentous fungus cultured on the gel or film together with the gel or film, and replacing the culture medium.

5. A filamentous fungus culture composition comprising a gel or film and a filamentous fungus cultured on the gel or film, wherein the gel or film contains one or more thickening polysaccharides selected from the group consisting of agar, gelatin, carrageenan, xanthan gum, locust bean gum, and curdlan.

6. A culture medium for a filamentous fungus, comprising a culture medium layer and a thickening polysaccharide layer disposed in contact with the culture medium layer, wherein the thickening polysaccharide layer contains one or more thickening polysaccharides selected from the group consisting of agar, gelatin, carrageenan, xanthan gum, locust bean gum, and curdlan, and a filamentous fungus is seeded and cultured on the thickening polysaccharide layer.

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