Method for producing fragrance-carrying filamentous fungi

Abstract

To provide a filamentous fungus in which flavor is supported on fungal cells when processing the filamentous fungus into food, in order to solve the problem that when the flavor is added to a culture medium in the production of a flavoring-supported filamentous fungus, the filamentous fungus is killed by the effect of the flavor so that the filamentous fungus cannot be proliferated.SOLUTION: In the production of a flavor-supported filamentous fungus, it has been found that by culturing a filamentous fungus in a medium containing flavor and oil, it is possible to prepare fungal cells supporting the flavor. Based on this knowledge, a method for producing a flavor-supported filamentous fungus, including a step of culturing a filamentous fungus in a medium containing flavor and oil is provided.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a technique for supporting a fragrance on the cells of filamentous fungi.

Background Art

[0002] As one of the solutions to the global food problem, a technique for processing microorganisms into alternative meat and the like as whole cells has been attracting attention. In particular, filamentous fungi are formed from hyphae, and by collecting the hyphae, a fibrous mass with a texture similar to meat can be obtained, so it is regarded as a promising raw material for alternative meat. Among filamentous fungi, Aspergillus oryzae, which is the national fungus of Japan, has been used for the production of fermented foods such as miso, soy sauce, and sake since ancient times, and its safety as a food is guaranteed. Therefore, it is also attracting attention as an alternative protein, and technologies for mass-producing koji as a protein source have been studied (Patent Document 1). In addition to Aspergillus oryzae, filamentous fungi of the genus Fusarium have attracted attention as a raw material for alternative meat because of their texture similar to meat, and are labeled as mycoprotein and are already sold in some countries.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] When processing filamentous fungi as food, excellent texture could be achieved by collecting and solidifying the hyphae into a fibrous mass. However, in order to add flavor, it was necessary to separately add a fragrance or the like. However, since the added fragrance adheres only to the surface of the cells, it is likely to volatilize and it is difficult to maintain the scent, which is a problem. In addition, when a fragrance is added to the medium, the filamentous fungi may die due to the influence of the fragrance, and growth may become difficult.

Means for Solving the Problems

[0005] The inventors, in the production of food products derived from filamentous fungi, conceived the idea of ​​incorporating fragrances into the fungal cells themselves. They discovered that by culturing filamentous fungi in a culture medium containing fragrances and oils, it is possible to support fragrances in the fungal cells without reducing growth efficiency, leading to the present invention. Therefore, the present invention relates to the following: [1] A method for producing fragrance-carrying filamentous fungi, The process includes cultivating filamentous fungi in a culture medium containing fragrances and oils, The manufacturing method wherein the filamentous fungus is a filamentous fungus belonging to the genus Aspergillus or Fusarium. [2] The manufacturing method according to item 1, wherein the fragrance has an n-octanol / water partition coefficient of 2 to 6. [3] The manufacturing method according to item 1 or 2, wherein the concentration of the fragrance in the culture medium is 0.01% by mass to 0.1% by mass. [4] The manufacturing method according to any one of items 1 to 3, wherein the concentration of oil and fat in the culture medium is 0.1% by mass to 20% by mass. [5] A method for producing fragrance according to any one of items 1 to 4, wherein the fragrance is selected from the group consisting of aliphatic higher alcohols, aliphatic higher aldehydes, aromatic aldehydes, indoles or derivatives thereof, ketones, fatty acids, lactones, terpene hydrocarbons, phenol ethers, and esters. [6] The manufacturing method according to any one of items 1 to 5, wherein the oil is selected from the group consisting of olive oil, rapeseed oil, almond oil, sesame oil, camellia oil, perilla oil, linseed oil, grapeseed oil, coconut oil, rice oil, palm oil, safflower oil, corn oil, shiso oil, and sunflower oil. [Effects of the Invention]

[0006] The present invention makes it possible to produce filamentous fungi on which fragrance is supported. [Modes for carrying out the invention]

[0007] The present invention relates to a method for producing fragrance-carrying filamentous fungi. More specifically, the method for producing fragrance-carrying filamentous fungi includes the step of culturing filamentous fungi in a culture medium containing fragrance and oils and fats. In yet another embodiment, the present invention may also relate to fragrance-carrying filamentous fungi produced by the production method according to the present invention, and to food products produced using live and / or dead cells of fragrance-carrying filamentous fungi.

[0008] In this invention, filamentous fungi refer to fungi that form hyphae. From the viewpoint of supporting fragrances, filamentous fungi belonging to the genera Aspergillus or Fusarium are particularly preferred. From the viewpoint of using the hyphae for food, mycotoxin-non-producing filamentous fungi are preferred.

[0009] Examples of filamentous fungi belonging to the genus Aspergillus include Aspergillus oryzae, Aspergillus sojae, Aspergillus luchuensis, Aspergillus kawachii, and Aspergillus tamarii. As for strains used as koji mold, in addition to strains available from public institutions, such as the Aspergillus oryzae RIB strain, strains contained in koji sold for food can be used. Some filamentous fungi of the genus Aspergillus are known to produce mycotoxins such as aflatoxin, ochratoxin, and patulin, and strains that do not produce these mycotoxins are particularly preferred. An example of a filamentous fungus belonging to the genus Fusarium is Fusarium venenatum. Some filamentous fungi of the genus Fusarium are known to produce mycotoxins such as deoxynivalenol and its acetylated derivatives, nivalenol, T2-toxin, HT-2-toxin, and fusarenone-X, which share a common structure called the trichothecene skeleton. Strains that do not produce these mycotoxins are particularly preferred.

[0010] Immobilizing a fragrance onto filamentous fungi means culturing the filamentous fungi in the presence of a fragrance to allow the fragrance to be incorporated into the fungal cells. From the viewpoint of fragrance immobilization, it is preferable to include a step of culturing the filamentous fungi in a culture medium containing the fragrance and oils. The fragrance can be any fragrance as long as it can be immobilized, but from the viewpoint of promoting immobilization onto filamentous fungi, a fragrance with an n-octanol / water partition coefficient of 2 to 6 may be used. From the viewpoint of better immobilization, an n-octanol / water partition coefficient of 3 or higher is preferable. From the viewpoint of better immobilization, an n-octanol / water partition coefficient of 5 or lower is preferable. The concentration of the fragrance in the culture medium can be appropriately selected according to the type and strength of the fragrance to be immobilized, but as an example, a concentration of 0.01% to 0.1% by mass can be used. The n-octanol / water partition coefficient refers to the concentration ratio (Pow) of the chemical substance in the two solvent phases containing n-octanol and water when the chemical substance is added to the two solvent phases and an equilibrium state is reached. Pow is a physicochemical index that represents the solubility of a chemical substance in lipids. A higher Pow indicates greater solubility in lipids, while a lower Pow indicates greater solubility in water.

[0011] Any compound may be used as the supported fragrance, but as examples, compounds such as aliphatic higher alcohols, aliphatic higher aldehydes, aromatic aldehydes, indoles or their derivatives, ketones, fatty acids, lactones, terpene hydrocarbons, phenol ethers, and esters can be used as fragrance components. In addition to the above compounds, compounds listed in the "Survey on the Actual Use of Food Fragrance Compounds in Japan" (FY2000 Ministry of Health, Labour and Welfare Research Report, Japan Fragrance Manufacturers Association, March 2001) can be used as fragrances. The fragrance may consist of one type of fragrance, or multiple types, such as two, three, four, five, or more types of fragrances, may be supported in combination. Representative compounds of each classification are listed below, but the present invention is not intended to be limited to these compounds:

[0012] Examples of aliphatic higher alcohols include the following compounds: [Table 1]

[0013] Examples of aliphatic higher aldehydes include the following compounds:

Table 2

[0014] Examples of aromatic aldehydes include the following compounds:

Table 3

[0015] Examples of indole or its derivatives include the following compounds:

Table 4

[0016] Examples of ketones include the following compounds:

Table 5

[0017] Examples of fatty acids include the following compounds:

Table 6

[0018] Examples of lactones include the following compounds:

Table 7

[0019] Examples of terpene hydrocarbons include the following compounds:

Table 8

[0020] Examples of phenol ethers include the following compounds: [Table 9]

[0021] Examples of esters include the following compounds: [Table 10]

[0022] The oils and fats may be triglycerides, diglycerides, or monoglycerides, but edible oils and fats are preferred. Edible oils and fats may include vegetable oils, fish oils, or animal fats. Vegetable oils that can be used include olive oil, rapeseed oil (canola oil), almond oil, sesame oil, camellia oil, perilla oil, linseed oil, grapeseed oil, coconut oil, rice oil, palm oil, safflower oil, corn oil, shiso oil, and sunflower oil, with olive oil and rapeseed oil being preferred. Animal fats that can be used include oils and fats derived from animals such as cattle, pigs, sheep, chickens, goats, and whales. Fish oils that can be used include shark oil and liver oil. The oils and fats added to the culture medium are added at a concentration of 0.1 to 20% by mass. From the viewpoint of promoting the loading of fragrances, a concentration of 0.5% by mass or more is preferred, and 1% by mass or more is more preferred. From the viewpoint of productivity, a concentration of 10% by mass or less is preferred, and 5% by mass or less is more preferred.

[0023] The mass ratio of fragrance to fat (fragrance / fat) is typically 0.01 to 0.1, from the perspective of promoting fragrance adhesion.

[0024] The step of culturing filamentous fungi in a culture medium containing fragrance and oil may be performed at any point in the filamentous fungal culture process. That is, filamentous fungi may be cultured in a culture medium containing fragrance and oil throughout all stages of the filamentous fungal culture process, or it may be performed at any stage selected from some stages, such as the initial, middle, and final stages of culture. As an example, after culturing filamentous fungi, the step of culturing filamentous fungi in a culture medium containing fragrance and oil may be performed by adding fragrance and oil to the culture medium for 1 hour to 7 days and continuing the culture before harvesting the filamentous fungi. More preferably, before harvesting the filamentous fungi, the step of culturing filamentous fungi in a culture medium containing fragrance and oil may be performed for 12 hours or more, and even more preferably for 24 hours or more. From the viewpoint of manufacturing efficiency, the step of culturing filamentous fungi in a culture medium containing fragrance and oil is preferably within 7 days, and more preferably within 3 days.

[0025] The culture of filamentous fungi may be carried out by methods known in the art, and may be performed by either a solid culture method or a liquid culture method, but the liquid culture method is preferred. In either the solid culture method or the liquid culture method, any medium in which filamentous fungi can grow can be used. As an example of solid culture, grains such as rice, wheat, potatoes, or legumes can be steamed, the temperature is controlled, conidia of the filamentous fungi are sprinkled on them, and the mixture is stirred to ensure uniformity and allowed to stand for cultivation. Alternatively, a liquid medium gelled with agar or gelatin can be used as a solid culture medium. As for liquid culture, synthetic media with a carbon source, nitrogen source, inorganic salts, vitamins, minerals, and amino acids added, or commercially available media such as YDP medium, PDA medium, and SCD medium may be used. As carbon sources, monosaccharides, disaccharides, polysaccharides, starch, molasses, etc. can be used, and more specifically, glucose, maltose, sucrose, etc. can be used. As nitrogen sources, ammonia, ammonium sulfate, ammonium phosphate, ammonium carbonate, ammonium acetate, peptone, yeast extract, casein hydrolysate, bran, meat extract, etc., can be used. As inorganic salts, potassium salts, magnesium salts, sodium salts, phosphates, manganese salts, iron salts, zinc salts, copper salts, etc., can be used. In addition, a liquid culture medium can be prepared by grinding grains such as rice, wheat, potatoes, or legumes, mixing them with water, and boiling them. The liquid culture medium may contain solid components. 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 preferably 1 to 10 days, preferably 3 to 7 days, more preferably 3 to 4 days. In the case of liquid culture, shaking culture may be performed at 0 to 150 rpm. Known culture devices such as jar fermenters and plant fermenters may be used.

[0026] Culturing can be started by inoculating the culture medium with conidia of filamentous fungi, or by inoculating a pre-cultured culture of filamentous fungi. The culture medium may or may not be sterilized before inoculation. Sterilization methods such as heat sterilization, autoclave sterilization, and filter sterilization can be selected as appropriate. Autoclave sterilization is preferred from the viewpoint of preventing contamination. On the other hand, if a culture medium to which volatile fragrances have been added is subjected to autoclave sterilization, the volatile components may be lost, so autoclave sterilization may not be performed in some cases.

[0027] The cultured bacterial cells are recovered by any recovery method. Recovery methods include centrifugation, filtration, and compression. For example, the culture solution can be recovered by suction filtration using filter paper or gauze. The recovered bacterial cells are further washed with water or hot water at 40-50°C. The recovered bacterial cells may be sterilized to become dead cells or used as live cells. The recovered flavor-carrying bacterial cells can be identified by gas chromatography. The recovered bacterial cells can be molded, flavored, and processed into food products. Further flavoring may be added for enhanced taste.

[0028] The process involves culturing filamentous fungi in a culture medium containing fragrances and oils, thereby immobilizing the fragrances onto the fungal cells. Attempting to immobilize only the fragrances may hinder the growth of the fungi, depending on the type of fragrance. Furthermore, filamentous fungi cultured in a medium containing only fragrances may not achieve the desired amount of fragrance immobilization. Fragrance immobilization refers to the incorporation of the fragrance into any part of the fungal cell, such as the cell membrane, cell wall, and cytoplasm.

[0029] All references made herein are incorporated herein by citation in their entirety.

[0030] The embodiments of the present invention described below are for illustrative purposes only and do not limit the technical scope of the invention. The technical scope of the invention is limited solely by the claims. Modifications to the invention, such as additions, deletions, and substitutions of constituent elements of the invention, can be made without departing from the spirit of the invention. [Examples]

[0031] 1. Preparation of liquid culture medium Trypto-sawyer broth (manufactured by Nissui Pharmaceutical Co., Ltd.) was dissolved in water to prepare a liquid culture medium.

[0032] 2. Fragrance transfer to filamentous fungi Test Example 1: Fragrance Loading onto Aspergillus oryzae (1) Preparation of inoculum for inoculation Cellular stocks of Aspergillus oryzae were spread onto slant plates of potato dextrose agar (Becton Dickinson), grown at 28°C for more than one week, and then suspended in physiological saline containing 0.5% tween20. Conidia were obtained by filtration using a cell strainer.

[0033] (2) Preparation of liquid culture medium for fragrance support Oils and / or fragrances were added to the liquid culture medium according to the table below to prepare the fragrance-supporting liquid culture media for Examples 1-11 and Comparative Examples 1-4 (units in the table: mass%). [Table 11]

[0034] (3) Main culture 100 mL of the aforementioned fragrance-supporting liquid culture medium was placed in a 300 mL baffled Erlenmeyer flask and autoclaved at 121 °C for 15 minutes. After inoculating with bacterial conidia, the flask was cultured in a 28 °C incubator at a rotation speed of 135 rpm for 3 days with swirling and shaking.

[0035] (4) Recovery After cultivation was complete, the culture solution was filtered by suction using filter paper, and the culture was washed twice each with water and 40-50°C hot water to remove surface culture medium components and oil. Then, water was removed by suction and pressing to obtain Aspergillus oryzae cells.

[0036] (5) Extraction and measurement of volatile components Volatile components were extracted using a multi-functional autosampler (Gerstel MPS2). 0.5 g of dried bacterial cells were placed in a 20 mL vial and equilibrated at 60°C for 10 minutes at 500 rpm. Next, the vial was heated at 60°C for 10 minutes to extract the volatile components, which were then adsorbed onto SPME fiber (Sigma-Aldrich DVB / Carboxen / PDMS). The adsorbed volatile components were injected into a gas chromatograph-mass spectrometer (Agilent Technologies 7890B GC / 5977A MSD) and measured using the following settings: • Gas chromatograph (GC) Injection method: Splitless, 2 minutes Inlet temperature: 270℃ Column: DB-WAX UI 30m, 0.25mm, 0.25μm Oven temperature: 40°C (hold for 5 minutes), increase temperature by 10°C / minute, then 250°C (hold for 10 minutes) Column flow rate: 2.0 mL / min (constant flow mode) ·Mass spectrometry (MS) Transfer line temperature: 280℃ Ionization mode: EI Electron energy: 70 eV Ion source temperature: 230℃ Measurement mode: Scan (m / z 43-300)

[0037] (6) Results (i) Examples 1 and 2: Ethyl octanoate and canola oil Ethyl octanoate (12.6 min) was detected from the fragrance-supported Aspergillus oryzae cells. The amount of ethyl octanoate detected from the fragrance-supported Aspergillus oryzae cells was approximately 160 times greater when 0.1% ethyl octanoate was included (Example 2) compared to when 0.01% ethyl octanoate was included (Example 1).

[0038] (ii) Examples 3 and 4: Ethyl octanoate and olive oil Ethyl octanoate (12.6 min) was detected from the fragrance-supported Aspergillus oryzae cells. The amount of ethyl octanoate detected from the fragrance-supported Aspergillus oryzae cells was approximately 160 times greater when 0.1% ethyl octanoate was included (Example 4) compared to when 0.01% ethyl octanoate was included (Example 3).

[0039] (iii) Examples 5 and 6: 2-nonanone and canola oil 2-nonanone (11.9 min) was detected from the fragrance-supporting Aspergillus oryzae cells. The amount of 2-nonanone detected from the fragrance-supporting Aspergillus oryzae cells was approximately 35 times greater when 2-nonanone was included at a concentration of 0.1% (Example 6) compared to when 2-nonanone was included at a concentration of 0.01% (Example 5).

[0040] (iv) Examples 7 and 8: 2-nonanone and olive oil 2-nonanone (11.9 min) was detected from the fragrance-supporting Aspergillus oryzae cells. The amount of 2-nonanone detected from the fragrance-supporting Aspergillus oryzae cells was approximately 40 times greater when 2-nonanone was included at a concentration of 0.1% (Example 8) compared to when 2-nonanone was included at a concentration of 0.01% (Example 7).

[0041] (v) Examples 9 and 10: Vanillin and Canola Oil A trace amount of vanillin (24.2 min) was detected from the fragrance-supporting koji mold cells. The amount of vanillin detected from the fragrance-supporting koji mold cells was similar to the amount detected when 0.01% vanillin was included (Example 9) and when 0.1% vanillin was included (Example 10).

[0042] (vi) Example 11: 0.01% maltol and canola oil A trace amount of maltol (18.9 min) was detected in the flavoring-carrying koji mold cells.

[0043] (vii) Comparative Examples 1-4: No oils or fats were added, and only 0.1% fragrance (ethyl octanoate, 2-nonanone, vanillin, maltol) was added. The koji could not be confirmed to be multiplying and died.

[0044] Test Example 2: Fragrance Transfer to Fusarium Filamentous Fungi (1) Preculture 100 mL of the aforementioned liquid medium was placed in a 300 mL baffled Erlenmeyer flask and autoclaved at 121°C for 15 minutes. Under sterile conditions, a suspension of Fusarium venenatum was inoculated, and the culture was incubated at 28°C incubator at 135 rpm with swirling and shaking for 2 days.

[0045] (2) Preparation of liquid culture medium for fragrance support Oils and / or fragrances were added to the liquid culture medium according to the table below to prepare the fragrance-supporting liquid culture media for Examples 12-13 and Comparative Example 5 (units in the table: mass%). [Table 12]

[0046] (3) Main culture 100 mL of the aforementioned fragrance-supporting liquid culture medium was placed in a 300 mL baffled Erlenmeyer flask and autoclaved at 121 °C for 15 minutes. 1 mL of the pre-culture was inoculated, and the culture was incubated at 28 °C with a rotation speed of 135 rpm for 3 days. The Aspergillus oryzae was recovered, its volatile components extracted, and measured using the same method as for Aspergillus oryzae.

[0047] (4) Results (i) Examples 12 and 13: Ethyl octanoate and olive oil Ethyl octanoate (12.6 min) was detected from fragrance-supported Fusarium filamentous fungal cells. The amount of ethyl octanoate detected from fragrance-supported Fusarium filamentous fungal cells was approximately four times higher when 0.1% ethyl octanoate was included (Example 13) compared to when 0.01% ethyl octanoate was included (Example 12).

[0048] (ii) Comparative Example 5: No oil or fat was added, only 0.1% ethyl octanoate was added. No proliferation of Fusarium filamentous fungi was observed, and they died.

[0049] Test Example 3: Fragrance Loading onto Mucor Filamentous Fungi (1) Preculture 100 mL of the liquid culture medium was placed in a 300 mL baffled Erlenmeyer flask and autoclaved at 121 °C for 15 minutes. Under sterile conditions, a suspension of Mucor circinelloides bacteria was inoculated, and the culture was incubated at 28 °C with shaking at 135 rpm for 2 days.

[0050] (2) Preparation of liquid culture medium for fragrance support Oils and / or fragrances were added to the liquid culture medium according to the table below to prepare the fragrance-supporting liquid culture media for Comparative Examples 6 to 8 below (units in the table: mass%). [Table 13] The liquid culture media for fragrance support in Comparative Examples 6-8 are identical to the liquid culture media for fragrance support in Examples 12 and 13 of Test Example 2, and Comparative Example 5.

[0051] (3) Main culture 100 mL of the aforementioned fragrance-supporting liquid culture medium was placed in a 300 mL baffled Erlenmeyer flask and autoclaved at 121 °C for 15 minutes. 1 mL of the pre-culture was inoculated, and the culture was incubated at 28 °C with a rotation speed of 135 rpm for 3 days. The Aspergillus oryzae was recovered, its volatile components extracted, and measured using the same method as for Aspergillus oryzae. (4) Results (i) Comparative Examples 6 and 7: Ethyl octanoate and olive oil The growth of Mucor filamentous fungi was confirmed, but the presence of fragrance was not detected.

[0052] (ii) Comparative Example 8: No oil or fat was added, only 0.1% ethyl octanoate was added. The growth of Mucor filamentous fungi was confirmed, but the presence of fragrance was not detected.

Claims

1. A method for producing fragrance-carrying filamentous fungi, The process includes cultivating filamentous fungi in a culture medium containing fragrances and oils, The filamentous fungus is a filamentous fungus belonging to the genus Aspergillus or Fusarium. The manufacturing method wherein the fragrance has an n-octanol / water partition coefficient of 2 to 6.

2. The manufacturing method according to claim 1, wherein the concentration of the fragrance in the culture medium is 0.01% by mass to 0.1% by mass.

3. The manufacturing method according to claim 1 or 2, wherein the concentration of oil and fat in the culture medium is 0.1% by mass to 20% by mass.

4. The manufacturing method according to any one of claims 1 to 3, wherein the fragrance is selected from the group consisting of aliphatic higher alcohols, aliphatic higher aldehydes, aromatic aldehydes, indoles or derivatives thereof, ketones, fatty acids, lactones, terpene hydrocarbons, phenol ethers, and esters.

5. The manufacturing method according to any one of claims 1 to 4, wherein the oil and fat is selected from the group consisting of olive oil, rapeseed oil, almond oil, sesame oil, camellia oil, perilla oil, linseed oil, grapeseed oil, coconut oil, rice oil, palm oil, safflower oil, corn oil, shiso oil, and sunflower oil.

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