Method for culturing Aspergillus oryzae, culture kit, method for producing mycoprotein, and mycoprotein

Abstract

This invention provides a method for culturing Aspergillus oryzae, a culture kit, a method for producing mycoprotein, and mycoprotein, all of which result in a culture of Aspergillus oryzae that is rich in γ-aminobutyric acid compared to ordinary Aspergillus oryzae, yields a large amount of microbial cells, and improves the mushroom-like flavor of the purified mycoprotein. [Solution] A method for culturing γ-aminobutyric acid-enriched Aspergillus oryzae is provided, characterized by adding glutamic acid and / or its salt and a vitamin B6-containing component to a culture medium for culturing the Aspergillus oryzae. A culture kit for γ-aminobutyric acid-enriched Aspergillus oryzae is also provided, comprising Aspergillus oryzae and a culture medium for culturing the Aspergillus oryzae, wherein the culture medium contains glutamic acid and / or its salt and a vitamin B6-containing component. Furthermore, a γ-aminobutyric acid-enriched mycoprotein and a method for producing the same are also provided, characterized by having a γ-aminobutyric acid content of 400 mg or more per 100 g dry weight of mycoprotein.

Description

Technical Field

[0001] The present invention relates to a method for culturing γ-aminobutyric acid-enriched koji mold, a culture kit, a method for producing γ-aminobutyric acid-enriched mycoprotein, and γ-aminobutyric acid-enriched mycoprotein.

Background Art

[0002] Currently, due to the increasing health awareness of people and the changing food preferences, the demand for proteins and amino acids among the nutrients contained in foods is increasing year by year.

[0003] Regarding proteins, so-called next-generation proteins that take into account environmental load and animal welfare have attracted attention compared to conventional animal proteins contained in meat and fish.

[0004] Examples of foods containing next-generation proteins include meat-like foods containing non-animal proteins. As sources of such proteins, various production processes have been developed, such as plants like soybeans, chemical cultures, insects, algae, etc.

[0005] In particular, research and development of proteins using microorganisms has been promoted as one of such next-generation proteins. Among them, alternative proteins (mycoproteins) produced by fungi are currently attracting attention.

[0006] Mycoprotein is a product obtained by processing the mycelium itself formed by fungi during culture as a nutrient (protein) for food. Foods containing mycoprotein have already been manufactured and sold overseas.

[0007] In Japan, it is expected that many consumers will show resistance to eating fungi themselves. Among them, koji molds such as Aspergillus are well known to be used in the production of soy sauce and miso, and the fermentation of sake and salt koji, and it is considered that the resistance of consumers is low.

[0008] In addition, Aspergillus oryzae has undergone whole-genome sequencing, its detoxification through selective breeding has been proven, and it also has advantages such as a short production period and low environmental impact. Therefore, mycoproteins derived from Aspergillus oryzae are considered to be very useful as next-generation proteins in Japan.

[0009] On the other hand, gamma-aminobutyric acid (GABA) is attracting attention as a nutrient contained in these koji molds and fermented foods made from koji mold. Gamma-aminobutyric acid has been reported to have functions such as improving blood pressure, reducing stress, improving sleep, and improving cognitive function, and various foods and beverages containing gamma-aminobutyric acid as a functional ingredient have been developed and are being sold.

[0010] As a prior art document regarding the cultivation method of such γ-aminobutyric acid-enriched koji mold, for example, Patent Document 1 discloses a method for producing γ-aminobutyric acid-enriched koji, which includes the steps of (A) immersing a koji raw material in a solution containing glutamic acid and heating it, and (B) adding koji mold after step (A) and producing koji. Furthermore, in the above production method, it is preferable to add pyridoxal phosphate to the solution containing glutamic acid, and a method for producing a γ-aminobutyric acid-enriched high-salt food is also described, which includes the steps of (A) immersing a koji raw material in a solution containing glutamic acid and heating it, (B) adding koji mold after step (A) and producing koji, and (C) brewing after step (B) with a salt concentration of 5% by mass or more. [Prior art documents] [Patent Documents]

[0011] [Patent Document 1] Japanese Patent Publication No. 2004-147560 [Overview of the project] [Problems that the invention aims to solve]

[0012] The pyridoxal phosphate (vitamin B6) described in Patent Document 1 acts as a coenzyme for glutamate decarboxylase (GAD), a decarboxylase, in the mechanism of synthesizing γ-aminobutyric acid from glutamic acid in Aspergillus oryzae cells. Therefore, it promotes the activity of GAD present in Aspergillus oryzae added in step (B), and can produce more γ-aminobutyric acid.

[0013] However, when cultivating Aspergillus oryzae rich in γ-aminobutyric acid, vitamin B6 (hereinafter also referred to as VB6), which is an additive to the culture medium described in Patent Document 1, is very expensive per unit volume. Considering the amount of Aspergillus oryzae cells recovered after cultivation and the amount of mycoprotein purified therefrom, cost issues remained.

[0014] Furthermore, a distinctive mushroom-like smell and taste were observed, particularly in food compositions prepared using ingredients containing mycoproteins derived from Aspergillus oryzae.

[0015] It was anticipated that many consumers would dislike the mushroom-like smell and taste, just as they would dislike real mushrooms. Therefore, improving the mushroom-like smell and taste was a challenge when manufacturing and selling food compositions containing mycoprotein.

[0016] This invention has been made in view of these circumstances, and aims to provide a method for culturing Aspergillus oryzae, a culture kit, a method for producing mycoprotein, and mycoprotein, which are richer in γ-aminobutyric acid than ordinary Aspergillus oryzae, yield a larger amount of microbial cells, and have an improved mushroom-like flavor in the purified mycoprotein. [Means for solving the problem]

[0017] The inventors conducted thorough research and discovered that by adding glutamic acid and / or its salts and a vitamin B6-containing component to the culture medium for culturing Aspergillus oryzae, the culture medium becomes rich in γ-aminobutyric acid, yields a large amount of microbial cells, and improves the mushroom-like flavor of the purified mycoprotein, thus completing the present invention. In other words, the present invention provides the following:

[0018] (1) The first aspect of the present invention is a method for culturing γ-aminobutyric acid-enriched Aspergillus koji, which is characterized by adding glutamic acid and / or its salt and a vitamin B6-containing component to a medium for culturing Aspergillus koji.

[0019] (2) The second aspect of the present invention is the method for culturing γ-aminobutyric acid-enriched Aspergillus koji according to (1), wherein the vitamin B6-containing component is sake lees, soy protein or sesame.

[0020] (3) The third aspect of the present invention is the method for culturing γ-aminobutyric acid-enriched Aspergillus koji according to (1), wherein the Aspergillus koji belongs to the genus Aspergillus.

[0021] (4) The fourth aspect of the present invention is the method for culturing γ-aminobutyric acid-enriched Aspergillus koji according to (1), wherein the addition amount of the glutamic acid and / or its salt to the medium is 0.085 to 10% as glutamic acid, and the addition amount of the vitamin B6-containing component is 0.1 to 10%.

[0022] (5) The fifth aspect of the present invention is a culture kit for γ-aminobutyric acid-enriched Aspergillus koji, which includes Aspergillus koji and a medium for culturing the Aspergillus koji, and the medium contains glutamic acid and / or its salt and a vitamin B6-containing component.

[0023] (6) The sixth aspect of the present invention is a method for producing γ-aminobutyric acid-enriched mycoprotein, which includes a step of adding glutamic acid and / or its salt and a vitamin B6-containing component to a medium and then adding and culturing Aspergillus koji, a step of recovering the cells from the medium, and a step of purifying mycoprotein, which is a mycelium, from the cells.

[0024] (7) The seventh aspect of the present invention is γ-aminobutyric acid-enriched mycoprotein, characterized in that the content of γ-aminobutyric acid in 100 g of the dry weight of mycoprotein is 400 mg or more.

Advantages of the Invention

[0025] According to the present invention, there can be provided a method for culturing Aspergillus oryzae, a culture kit, a method for producing mycoprotein, and mycoprotein, which are rich in γ-aminobutyric acid as compared with ordinary Aspergillus oryzae, have a large amount of recovered cells, and have an improved mushroom-like flavor of the purified mycoprotein.

Embodiments for Carrying Out the Invention

[0026] Hereinafter, embodiments for carrying out the present invention (hereinafter simply referred to as "the present embodiment") will be described in detail. The following present embodiment is an exemplification for explaining the present invention, and is not intended to limit the present invention to the following content. The present invention can be appropriately modified and implemented within the scope of its gist.

[0027] <Method for Culturing γ-Aminobutyric Acid-Enriched Aspergillus oryzae and Method for Producing γ-Aminobutyric Acid-Enriched Mycoprotein> The method for culturing γ-aminobutyric acid-enriched Aspergillus oryzae according to an embodiment of the present application is characterized in that a glutamic acid and / or its salt and a vitamin B6-containing component are added to a medium for culturing Aspergillus oryzae. Further, the method for producing γ-aminobutyric acid-enriched mycoprotein according to another embodiment of the present application includes a step of adding Aspergillus oryzae and culturing after adding a glutamic acid and / or its salt and a vitamin B6-containing component to a medium, a step of recovering cells from the medium, and a step of purifying mycoprotein, which is a mycelium, from the cells.

[0028] The koji mold targeted by the culture method according to this embodiment and the production method according to the other embodiment is not particularly limited, but it is preferably of the genus Aspergillus. More specifically, it is preferably one of Aspergillus oryzae, Aspergillus sojae, Aspergillus awamori, Aspergillus luchuensis, or Aspergillus kawachii, or a mixture thereof. The following describes in detail each step of the culture method and manufacturing method.

[0029] (Preparation of culture medium) In the culture method according to this embodiment and the manufacturing method according to another embodiment, glutamic acid and / or a salt thereof are added to the culture medium for culturing Aspergillus oryzae.

[0030] The glutamic acid and / or salt thereof to be added is not particularly limited as long as it is a known glutamic acid and / or salt thereof used in culture, but examples include glutamic acid, sodium glutamate, magnesium glutamate, calcium glutamate, or mixtures thereof, with sodium glutamate being preferred.

[0031] The amount of glutamic acid and / or its salt added to the culture medium is preferably 0.085 to 10% in terms of glutamic acid, and more preferably 0.425 to 5%. When the amount added is within the above range, both the γ-aminobutyric acid content contained in Aspergillus oryzae and the amount of Aspergillus oryzae cells recovered are significantly increased, and the mushroom-like flavor of the purified mycoprotein is also more clearly improved.

[0032] Furthermore, the form of glutamic acid and / or its salt added is not particularly limited, as long as it is the form that additives normally take, such as a solid (powder), extract, or protein hydrolysate solution.

[0033] In addition, a vitamin B6-containing component is added to the culture medium in addition to glutamic acid and / or its salts.

[0034] Here, "vitamin B6-containing ingredients" refers to foods or additives that are rich in vitamin B6 and have a low unit price. Among such vitamin B6-containing ingredients, sake lees, soy protein, or sesame seeds are preferred. According to the Standard Tables of Food Composition in Japan (8th Revised and Enlarged 2023), the amino acid content per 100g is 16,000mg for sake lees, 38,000mg for soy protein, and 22,000mg for sesame seeds. Furthermore, the vitamin B6 content per 100g is 0.94mg for sake lees, 0.51mg for dried soybeans, and 0.60mg for sesame seeds. In addition, all of these are very inexpensive per unit volume compared to vitamin B6.

[0035] Furthermore, in addition to the reasons mentioned above, it has been confirmed that adding vitamin B6-containing components, particularly sake lees, soy protein, or sesame, together with glutamic acid and / or its salts to the culture medium synergistically increases both the γ-aminobutyric acid content of Aspergillus oryzae and the amount of Aspergillus oryzae cells recovered, and also clearly improves the mushroom-like flavor of the purified mycoprotein. For the reasons stated above, vitamin B6-containing components, particularly sake lees, soy protein, or sesame, are suitable additives to be added to the culture medium when culturing gamma-aminobutyric acid-enriched Aspergillus oryzae.

[0036] Furthermore, the amount of the above-mentioned vitamin B6-containing component added to the culture medium is preferably 0.01 to 10%, and more preferably 0.1 to 5%. When the amount added is within the above numerical range, both the γ-aminobutyric acid content contained in the Aspergillus oryzae and the amount of Aspergillus oryzae cells recovered are significantly increased, and the mushroom-like flavor of the purified mycoprotein is also more clearly improved.

[0037] Furthermore, the form of the added vitamin B6-containing component is not particularly limited, as long as it is in the form that additives normally take, such as a solid (powder), extract, or protein hydrolysate solution.

[0038] Furthermore, the culture medium to which the aforementioned glutamic acid and / or its salt and the vitamin B6-containing component are added preferably contains a carbohydrate source and a nitrogen source as a base medium. The carbohydrate source and nitrogen source are not particularly limited as long as they are substances that can supply carbohydrates and nitrogen, but the carbohydrate source is preferably glucose and the nitrogen source is preferably malt. Furthermore, while the form of the culture medium is not particularly limited, it is preferable to use a liquid culture medium in order to facilitate the subsequent recovery and purification processes.

[0039] Furthermore, as an invention according to another embodiment, this application may also include a culture kit for γ-aminobutyric acid-enriched Aspergillus oryzae, comprising the above-mentioned Aspergillus oryzae and a culture medium for culturing Aspergillus oryzae, wherein the culture medium is characterized by containing glutamic acid and / or its salts and a vitamin B6-containing component.

[0040] (Culture process) Next, add Aspergillus oryzae to the culture medium described above, and then perform the culturing. Regarding the genus and species of Aspergillus oryzae, those described above are preferable. The amount of bacteria to add is 1.0 × 10⁶ of the culture medium. 2 It is preferable that the concentration be CFU / mL or higher, and 1.0 × 10 3 ~1.0×10 10 A CFU / mL concentration is more preferable.

[0041] The culture conditions are not particularly limited as long as they are similar to those for the normal culture of Aspergillus oryzae, but 15-40°C and 18-240 hours are preferred. Furthermore, shaking culture at 100-800 rpm is preferred.

[0042] (Recovery process) Next, the bacterial cells are collected from the culture medium. This can be done in the same way as with normal bacterial cell collection; the cells are collected by filtering the culture medium.

[0043] (purification process) Finally, γ-aminobutyric acid-enriched mycoprotein is obtained through a process of purifying mycoprotein, which is the mycelium, from the Aspergillus oryzae cells. In this case, since the mycelium formed by the Aspergillus oryzae is itself a mycoprotein, no chemical extraction procedures are particularly necessary. Furthermore, additional purification may be performed depending on the culture conditions.

[0044] <γ-aminobutyric acid-enriched mycoprotein> The gamma-aminobutyric acid-enriched mycoprotein produced by the manufacturing method described above preferably contains 400 mg or more of gamma-aminobutyric acid per 100 g of dry weight of mycoprotein.

[0045] Furthermore, the manufactured gamma-aminobutyric acid-enriched mycoprotein may be added as an additive to existing foods, beverages, or supplements, or it may be used as the main ingredient in food compositions. For example, when manufacturing a food composition, the gamma-aminobutyric acid-enriched mycoprotein according to this embodiment is combined with seasonings and binding agents, and then subjected to heat treatment processes such as baking, steaming, or boiling to obtain the food composition. Depending on the sales format, subsequent processes such as freezing, bagging, or shrink wrapping may also be used.

[0046] Although the present invention has been described above using embodiments, it goes without saying that the technical scope of the present invention is not limited to the scope described in the above embodiments. It will be obvious to those skilled in the art that various modifications or improvements can be made to the above embodiments. Furthermore, it is clear from the claims that such modified or improved forms may also be included in the technical scope of the present invention. [Examples]

[0047] The present invention will be described in detail below with reference to examples. However, the present invention is not limited in any way to the examples shown below.

[0048] <Experiment 1: Examination of the amount of MSG to add to the culture medium> First, before conducting a test to culture koji mold with added vitamin B6-containing components, we performed a test to determine the appropriate amount of MSG to add. As shown in Table 3, MSG (monosodium glutamate) was added to the basal culture media of samples (test plots) No. 1 to 8, each containing 2.0% glucose as a carbohydrate source and 0.8% malt extract as a nitrogen source (No. 1 did not contain MSG).

[0049] Next, each sample was inoculated with Aspergillus oryzae (Japanese koji mold), cultured with shaking under the conditions shown in Table 1, and the culture medium was filtered to collect the fungal cells. [Table 1]

[0050] Finally, the mass of the recovered bacterial cells (bacterial cell volume) was measured, followed by the γ-aminobutyric acid content per 100g of bacterial cells in each sample, which was then measured using LC / MS / MS.

[0051] (Measurement by LC / MS / MS) First, 0.1 g of freeze-dried Aspergillus oryzae was added to 9.9 mL of pure water and homogenized. Next, 1 mL of 4N perchloric acid was added and the mixture was centrifuged. An equal volume of saturated hexane / acetonitrile was added to the recovered supernatant and centrifuged again. The recovered supernatant was filtered through a 0.2 μm filter, and the supernatant after filtering was diluted 10-fold with 0.1 N HCl and used as the measurement solution.

[0052] Next, liquid chromatography (LC) was performed. The LC conditions are shown below. Column: Intrada Amino Acid (150 x 3 mm, Imtakt) Mobile phase A: 100 mM ammonium formate / water Mobile phase B: 0.3% formic acid / acetonitrile Column temperature: 60℃ Injection volume: 5μL Flow rate: 0.5mL / min Gradient: Conditions shown in Table 2 below [Table 2]

[0053] Finally, mass spectrometry (MS) was performed. The MS conditions are as follows. Ionization method: Electrospray ionization (ESI), positive mode Monitoring ions: Precursor ion (m / z): 103.97, Product ion (m / z): 86.95

[0054] The measurement results and the culture medium components of each sample are shown in Table 3. [Table 3]

[0055] As shown in Table 3, the amount of bacterial cells recovered and the γ-aminobutyric acid content of each sample confirmed that an MSG concentration of 0.5-2.0% is suitable for addition to the culture medium.

[0056] <Experiment 2: Examination of the amount of MSG to add to the culture medium> Next, koji mold was recovered from culture media in which both MSG and VB6 were added, and a test was conducted to compare the amount of recovered cells and the γ-aminobutyric acid content with media in which only MSG was added, and media in which neither MSG nor VB6 was added. Samples (test plots) No. 9-13, each containing 2.0% glucose as a carbohydrate source and 0.8% malt extract as a nitrogen source, were given basal culture media with MSG and VB6 (vitamin B6 compound) added, as shown in Table 5 (no additives were added to No. 9, and only MSG was added to Nos. 10-12). The glucose, malt extract, and MSG used were the same as in Experiment 1.

[0057] Next, each sample was inoculated with the same Aspergillus oryzae as in Experiment 1, cultured with shaking under the culture conditions shown in Table 4, and the culture medium was filtered to collect the microbial cells. [Table 4]

[0058] Finally, the mass of the recovered bacterial cells (bacterial cell volume) was measured, followed by the measurement of the γ-aminobutyric acid content per 100g of bacterial cells in each sample using LC / MS / MS. The LC / MS measurement method and conditions were the same as those used in Experiment 1.

[0059] The measurement results and the culture medium components of each sample are shown in Table 5. [Table 5]

[0060] As shown in Table 5, it was confirmed that the addition of MSG and VB6 increased the amount of microbial cells recovered and the γ-aminobutyric acid content in cultured Aspergillus oryzae.

[0061] <Test 3: Examination of sake lees extract as a vitamin B6-containing ingredient> Next, koji mold was recovered from culture media supplemented with MSG and sake lees extract as a vitamin B6-containing component. A test was then conducted to compare the amount of microbial cells recovered and the γ-aminobutyric acid content with media supplemented with only sake lees extract, media supplemented with only MSG, and media without either MSG or sake lees extract. Samples (test plots) No. 14-17, each containing 2.0% glucose as a carbohydrate source and 0.8% malt extract as a nitrogen source, were given basal culture media. As shown in Table 7, MSG and sake lees extract (concentrated sake lees extract) were added to these media (no additives were added to No. 14, only MSG was added to No. 15, and only sake lees extract was added to No. 16). The glucose, malt extract, and MSG used were the same as in Experiment 1.

[0062] Next, each sample was inoculated with the same Aspergillus oryzae as in Experiment 1, cultured with shaking under the culture conditions shown in Table 6, and the culture medium was filtered to collect the microbial cells. [Table 6]

[0063] Finally, the mass of the recovered bacterial cells (bacterial cell volume) was measured, followed by the measurement of the γ-aminobutyric acid content per 100g of bacterial cells in each sample using LC / MS / MS. The LC / MS measurement method and conditions were the same as those used in Experiment 1.

[0064] The measurement results and the culture medium components of each sample are shown in Table 7. [Table 7]

[0065] As shown in Table 7, it was confirmed that adding both MSG and sake lees extract increased the amount of microbial cells recovered and the γ-aminobutyric acid content in cultured Aspergillus oryzae.

[0066] <Test 4: Comparison of the concentration of sake lees extract added> Next, different concentrations of sake lees extract were added to each culture medium and incubated. Aspergillus oryzae was then recovered from each medium, and the amount of microbial cells recovered and the γ-aminobutyric acid content were compared. Samples (test plots) No. 18-23, each containing 2.0% glucose as a carbohydrate source and 0.8% malt extract as a nitrogen source, were supplemented with MSG and sake lees extract as shown in Table 9 (only MSG was added to No. 18, and only sake lees extract was added to No. 23). The glucose, malt extract, and MSG used were the same as in Experiment 1, and the sake lees extract used was the same as in Experiment 3.

[0067] Next, each sample was inoculated with the same Aspergillus oryzae as in Experiment 1, cultured with shaking under the culture conditions shown in Table 8, and the culture medium was filtered to collect the microbial cells. [Table 8]

[0068] Finally, the mass of the recovered bacterial cells (bacterial cell volume) was measured, followed by the measurement of the γ-aminobutyric acid content per 100g of bacterial cells in each sample using LC / MS / MS. The LC / MS measurement method and conditions were the same as those used in Experiment 1.

[0069] The measurement results and the culture medium components of each sample are shown in Table 9. [Table 9]

[0070] As shown in Table 9, it was confirmed that adding 0.1% or more of sake lees extract increased the amount of microbial cells recovered and the γ-aminobutyric acid content in cultured koji mold, although there was some variation in the γ-aminobutyric acid content.

[0071] <Test 5: Comparison of MSG concentration when sake lees extract is added> Next, a test was conducted in which different concentrations of MSG were added to each culture medium along with a fixed concentration of sake lees extract, and koji mold was recovered from each medium. The amount of microbial cells recovered and the γ-aminobutyric acid content of each medium were then compared. Samples (test plots) No. 24-33, each containing 2.0% glucose as a carbohydrate source and 0.8% malt extract as a nitrogen source, were supplemented with MSG and sake lees extract as shown in Table 11 (only sake lees extract was added to No. 25, and neither was added to No. 24). The glucose, malt extract, and MSG used were the same as in Experiment 1, and the sake lees extract used was the same as in Experiment 3.

[0072] Next, each sample was inoculated with the same Aspergillus oryzae as in Experiment 1, cultured with shaking under the culture conditions shown in Table 10, and the culture medium was filtered to collect the microbial cells. [Table 10]

[0073] Finally, the mass of the recovered bacterial cells (bacterial cell volume) was measured, followed by the measurement of the γ-aminobutyric acid content per 100g of bacterial cells in each sample using LC / MS / MS. The LC / MS measurement method and conditions were the same as those used in Experiment 1.

[0074] The measurement results and the culture medium components of each sample are shown in Table 11. [Table 11]

[0075] As shown in Table 11, adding 0.1% or more MSG along with sake lees extract resulted in a tendency for the amount of microbial cells recovered to decrease above a certain amount, but it was confirmed that the amount of microbial cells recovered and the γ-aminobutyric acid content in the cultured koji mold increased.

[0076] <Test 6: Comparison of sake lees extract and vitamin B6 compound> Next, each culture medium was cultured with MSG, along with either a vitamin B compound (VB6) or sake lees extract. Aspergillus oryzae was then recovered from each medium, and the amount of microbial cells recovered and the γ-aminobutyric acid content were compared. Samples (test plots) No. 34-38, each containing 2.0% glucose as a carbohydrate source and 0.8% malt extract as a nitrogen source, were given MSG, VB6, and / or sake lees as shown in Table 13 (no additives were given to No. 34). The same glucose, malt extract, and MSG were used as in Test 1, the same VB6 as in Test 2, and the same sake lees extract as in Test 3.

[0077] Next, each sample was inoculated with the same Aspergillus oryzae as in Experiment 1, cultured with shaking under the culture conditions shown in Table 12, and the culture medium was filtered to collect the microbial cells. [Table 12]

[0078] Finally, the mass of the recovered bacterial cells (bacterial cell volume) was measured, followed by the measurement of the γ-aminobutyric acid content per 100g of bacterial cells in each sample using LC / MS / MS. The LC / MS measurement method and conditions were the same as those used in Experiment 1.

[0079] The measurement results and the culture medium components of each sample are shown in Table 13. [Table 13]

[0080] As shown in Table 13, compared to samples No. 35-36, which only had MSG added, and sample No. 37, which had MSG and sake lees extract (a vitamin B6-containing component) added separately, sample No. 38, which had MSG and sake lees extract added, showed a significant increase in both the amount of bacterial cells recovered and the γ-aminobutyric acid content. In particular, compared to No. 34, which had nothing added to the basal medium, No. 38 showed more than three times the amount of microbial cells recovered and more than four times the amount of γ-aminobutyric acid. These results confirm that adding glutamic acid and / or its salts and vitamin B6-containing components to the culture medium results in a richer γ-aminobutyric acid content in the cultured Aspergillus oryzae and a larger amount of microbial cells recovered.

[0081] <Test 7: Comparison based on the type of sake lees added> Next, sake lees, which is a different form from the extract, was added to each culture medium and cultured. Aspergillus oryzae was then recovered from each culture medium, and the amount of microbial cells recovered and the γ-aminobutyric acid content were compared. Samples (test plots) No. 39-44, each containing 2.0% glucose as a carbohydrate source and 0.8% malt extract as a nitrogen source, were given basal culture media to which MSG and either sake lees powder or edible sake lees were added, as shown in Table 15 (No. 40 had only MSG added, No. 41 had only sake lees powder added, No. 43 had only edible sake lees added, and No. 39 had neither added). The glucose, malt extract, and MSG used were the same as in Experiment 1.

[0082] Next, each sample was inoculated with the same Aspergillus oryzae as in Experiment 1, cultured with shaking under the culture conditions shown in Table 14, and the culture medium was filtered to collect the microbial cells. [Table 14]

[0083] Finally, the mass of the recovered bacterial cells (bacterial cell volume) was measured, followed by the measurement of the γ-aminobutyric acid content per 100g of bacterial cells in each sample using LC / MS / MS. The LC / MS measurement method and conditions were the same as those used in Experiment 1.

[0084] The measurement results and the culture medium components of each sample are shown in Table 15. [Table 15]

[0085] As shown in Table 15, it was confirmed that adding sake lees powder or edible sake lees along with MSG increased the amount of microbial cells recovered and the γ-aminobutyric acid content in cultured koji mold to a similar extent as sake lees extract.

[0086] <Test 8: Comparison of the concentration of sake lees powder added> Next, different concentrations of sake lees powder were added to each culture medium and incubated. Aspergillus oryzae was then recovered from each medium, and the amount of microbial cells recovered and the γ-aminobutyric acid content were compared. Samples (test plots) No. 45-52, each containing 2.0% glucose as a carbohydrate source and 0.8% malt extract as a nitrogen source, were given basal culture media with MSG and sake lees powder added, as shown in Table 17 (No. 46 received only MSG, Nos. 47, 49, and 51 received only sake lees powder, and No. 45 received none of the additives). The glucose, malt extract, and MSG used were the same as in Experiment 1.

[0087] Next, each sample was inoculated with the same Aspergillus oryzae as in Experiment 1, cultured with shaking under the culture conditions shown in Table 16, and the culture medium was filtered to collect the microbial cells. [Table 16]

[0088] Finally, the mass of the recovered bacterial cells (bacterial cell volume) was measured, followed by the measurement of the γ-aminobutyric acid content per 100g of bacterial cells in each sample using LC / MS / MS. The LC / MS measurement method and conditions were the same as those used in Experiment 1.

[0089] The measurement results and the culture medium components of each sample are shown in Table 17. [Table 17]

[0090] As shown in Table 17, it was confirmed that the amount of microbial cells recovered and the γ-aminobutyric acid content in the cultured koji mold increased in proportion to the amount of sake lees powder added to the culture medium.

[0091] <Experiment 9: Investigation of culture temperature during Aspergillus oryzae cultivation 1> Next, the culture temperature was set to 25°C, different from that used in experiments 1-8, and the amount of bacterial cells recovered and the γ-aminobutyric acid content were compared with the results from previous experiments. Samples (test plots) No. 53-55, each containing 2.0% glucose as a carbohydrate source and 0.8% malt extract as a nitrogen source, were supplemented with MSG and sake lees extract as shown in Table 19 (only MSG was added to No. 54, and neither was added to No. 53). The glucose, malt extract, and MSG used were the same as in Experiment 1, and the sake lees extract used was the same as in Experiment 3.

[0092] Next, each sample was inoculated with the same Aspergillus oryzae as in Experiment 1, cultured with shaking under the culture conditions shown in Table 18, and the culture medium was filtered to collect the cells. The culture temperature was set to 25°C, different from that of Experiments 1-8, and the culture period was 3 days. [Table 18]

[0093] Finally, the mass of the recovered bacterial cells (bacterial cell volume) was measured, followed by the measurement of the γ-aminobutyric acid content per 100g of bacterial cells in each sample using LC / MS / MS. The LC / MS measurement method and conditions were the same as those used in Experiment 1.

[0094] The measurement results and the culture medium components of each sample are shown in Table 19. [Table 19]

[0095] As shown in Table 19, even when the culture temperature was set to 25°C, it was confirmed that the amount of microbial cells recovered and the γ-aminobutyric acid content in cultured Aspergillus oryzae increased in the culture medium to which MSG and sake lees extract were added.

[0096] <Test 10: Suppression of mushroom-like flavor in Aspergillus oryzae mycoprotein cultured in sake lees-added medium> Next, mycoproteins were produced from koji mold cultured in a sake lees-added medium, and a test was conducted to confirm whether the mushroom-like flavor was suppressed compared to mycoproteins derived from koji mold cultured in a normal medium. In basal culture media and sake lees-added culture media (300 mL each) containing the additives shown in Table 20, the same Aspergillus oryzae as in Experiment 1 was introduced, with an initial spore count of 10. 3 The culture medium was inoculated to a CFU / mL concentration, cultured at 30°C, 200 rpm, and 0.2 SLM with shaking, and the cells were collected by filtering the medium. The same glucose, malt extract, and MSG were used as in Experiment 1, and the same sake lees extract was used as in Experiment 3. [Table 20]

[0097] Next, using the microbial cells recovered from the sake lees-added culture medium as 100%, a mixture of 5% dried egg white and 0.4% refined salt was added and molded into a roughly cylindrical shape. Two such molded samples were prepared; one was baked on a hot plate for 5 minutes, and the other was boiled at 80°C for 30 minutes, and both were prepared as food. For comparison, two similar samples were also prepared from koji mold cultured in the basic culture medium alone, and each was subjected to either baking or boiling treatment.

[0098] Finally, 7-8 evaluators rated the mushroom-like flavor of each food item on a 10-point scale. The mushroom-like smell and taste of mycoprotein derived from Aspergillus oryzae cultured in a basic culture medium (and food items containing it) were assigned a score of 5, while the flavor of mycoprotein derived from Aspergillus oryzae cultured in a culture medium supplemented with sake lees was quantified, including the standard deviation. The results are shown in Table 21. [Table 21]

[0099] As shown in Table 21, when koji mold was cultured in a sake lees-added medium containing sake lees and MSG, sensory evaluation confirmed an improvement in the mushroom-like flavor of the koji mold when consumed, based on the mycoproteins contained in the medium. As a result, the mushroom-like flavor improved with both baking and boiling treatments, but the boiled food showed a particularly significant improvement in mushroom-like flavor.

[0100] <Test 11: Examination of soy protein as a vitamin B6-containing component> Next, koji mold was recovered from a culture medium supplemented with MSG and soy protein as a vitamin B6-containing component, and the amount of recovered cells and γ-aminobutyric acid content were compared with that of koji mold recovered from the basal medium. As a reference, koji mold was also recovered from a culture medium supplemented with MSG and sake lees extract, and the amount of recovered cells and γ-aminobutyric acid content were similarly measured. In sake lees-added culture media and soybean protein-added culture media, which contained the additives shown in Table 22, the same Aspergillus oryzae as in Experiment 1 was introduced with an initial spore count of 1.0 × 10⁶. 3The culture medium was inoculated to a CFU / mL level, cultured with shaking at 30°C, 200 rpm, and 0.2 SLM, and the cells were collected by filtering the medium. The same glucose, malt extract, and MSG were used as in Experiment 1, and the same sake lees extract was used as in Experiment 3. In Table 22, "VB6 source" refers to the amount of sake lees or soy protein added to the culture medium. [Table 22]

[0101] Next, the mass of the recovered bacterial cells (bacterial cell volume) was measured, followed by the measurement of the γ-aminobutyric acid content per 100g of bacterial cells in each sample using LC / MS / MS. The LC / MS measurement method and conditions were the same as those used in Experiment 1.

[0102] The results of each of the above measurements are shown in Table 23. For reference, the results of culturing using only the basal medium are also shown in Table 23. [Table 23]

[0103] As shown in Table 23, a tendency for increased cell yield and γ-aminobutyric acid content was observed in the soy protein supplemented medium as well. When directly comparing the sake lees supplemented medium and the soy protein supplemented medium, the sake lees supplemented medium tended to have a higher γ-aminobutyric acid content, while the soy protein supplemented medium tended to have a higher cell yield.

[0104] <Test 12: Comparison of differences in the type and concentration of added soy protein> Next, different concentrations of soy protein were added to each culture medium, and the cultures were incubated. Aspergillus oryzae was then recovered from each medium, and the amount of microbial cells recovered and the γ-aminobutyric acid content were compared. Samples (test plots) No. 56-63, each containing 2.0% glucose as a carbohydrate source and 0.8% malt extract as a nitrogen source, were supplemented with MSG and soy protein as shown in Table 25 (No. 57 had only MSG added, Nos. 58, 60, and 62 had only soy protein added, and No. 56 had none added). The glucose, malt extract, and MSG were the same as in Test 1, while the soy protein used was purchased from a different manufacturer than in Test 11.

[0105] Next, each sample was inoculated with the same Aspergillus oryzae as in Experiment 1, cultured with shaking under the culture conditions shown in Table 24, and the culture medium was filtered to collect the microbial cells. [Table 24]

[0106] Finally, the mass of the recovered bacterial cells (bacterial cell volume) was measured, followed by the measurement of the γ-aminobutyric acid content per 100g of bacterial cells in each sample using LC / MS / MS. The LC / MS measurement method and conditions were the same as those used in Experiment 1.

[0107] The measurement results and the culture medium components of each sample are shown in Table 25. [Table 25]

[0108] As shown in Table 25, it was confirmed that adding soy protein along with MSG resulted in similar increases in both the amount of bacterial cells recovered and the γ-aminobutyric acid content, even with soy protein purchased from other manufacturers. In particular, compared to No. 56, which had neither MSG nor soy protein added, No. 59 and No. 61 showed increases in bacterial cell recovery by approximately 1.3 to 3.4 times, and in γ-aminobutyric acid content by approximately 3 times. From these results, it was confirmed that adding soy protein along with MSG is effective not only for specific types of soy protein, but also for other types of soy protein.

[0109] <Test 13: Suppression of mushroom-like flavor in Aspergillus oryzae mycoprotein cultured in soy protein supplemented medium> Next, mycoproteins were produced from Aspergillus oryzae cultured in a soy protein-supplemented medium, and a test was conducted to confirm whether the mushroom-like flavor was suppressed compared to mycoproteins derived from Aspergillus oryzae cultured in a normal medium. In basal culture media and soy protein supplemented culture media (300 mL each) containing the additives shown in Table 26, the same Aspergillus oryzae as in Experiment 1 was introduced to each medium with an initial spore count of 1.0 × 10⁶. 3 The culture medium was inoculated to a CFU / mL level, cultured with shaking at 30°C, 200 rpm, and 0.2 SLM, and the cells were collected by filtering the medium. The same glucose, malt extract, and MSG were used as in Experiment 1, and the same soy protein was used as in Experiment 11. [Table 26]

[0110] Next, using the bacterial cells recovered from the soy protein-supplemented medium as 100%, a mixture of 5% dried egg white and 0.4% refined salt was added and shaped into a roughly cylindrical form. These molded samples were then baked on a hot plate and prepared as food. For comparison, similar samples were prepared from Aspergillus oryzae cultured in the basic medium alone and subjected to the same baking process.

[0111] Finally, seven evaluators rated the mushroom-like flavor of each food item on a 10-point scale. The mushroom-like smell and taste of mycoprotein derived from Aspergillus oryzae cultured in a basic culture medium (and food items containing it) were assigned a score of 5, while the flavor of mycoprotein derived from Aspergillus oryzae cultured in a medium supplemented with soy protein was quantified, including the standard deviation. The results are shown in Table 27. [Table 27]

[0112] As shown in Table 27, when Aspergillus oryzae was cultured in a soy protein supplement medium containing both soy protein and MSG, sensory evaluation confirmed an improvement in the mushroom-like flavor of the cultured Aspergillus oryzae, as indicated by the mycoprotein contained in the cultured Aspergillus oryzae.

[0113] <Test 14: Examination of ground sesame seeds as a vitamin B6-containing ingredient> Next, koji mold was recovered from culture media that had been supplemented with MSG and ground sesame seeds as a vitamin B6-containing ingredient. A test was then conducted to compare the amount of microbial cells recovered and the γ-aminobutyric acid content with media that had ground sesame seeds only, media that had MSG only, and media that had neither MSG nor ground sesame seeds added. Samples (test plots) No. 64-67, each containing 2.0% glucose as a carbohydrate source and 0.8% malt extract as a nitrogen source, were given basal culture media with MSG and ground sesame seeds added, as shown in Table 29 (no additives were added to No. 64, only MSG was added to No. 65, and only ground sesame seeds were added to No. 66). The glucose, malt extract, and MSG used were the same as in Experiment 1.

[0114] Next, each sample was inoculated with the same Aspergillus oryzae as in Experiment 1, cultured with shaking under the culture conditions shown in Table 28, and the culture medium was filtered to collect the microbial cells. [Table 28]

[0115] Finally, the mass of the recovered bacterial cells (bacterial cell volume) was measured, followed by the measurement of the γ-aminobutyric acid content per 100g of bacterial cells in each sample using LC / MS / MS. The LC / MS measurement method and conditions were the same as those used in Experiment 1.

[0116] The measurement results and the culture medium components of each sample are shown in Table 29. [Table 29]

[0117] As shown in Table 29, it was confirmed that adding both MSG and ground sesame seeds increased the amount of microbial cells recovered and the γ-aminobutyric acid content in cultured Aspergillus oryzae.

[0118] <Experiment 15: Investigation of culture temperature during Aspergillus oryzae cultivation 2> Next, the culture temperature was set to 20°C, different from that used in tests 1-14, and the bacterial cell yield and γ-aminobutyric acid content were compared with the results from previous tests. Samples (test plots) No. 68-71, each containing 2.0% glucose as a carbohydrate source and 0.8% malt extract as a nitrogen source, were supplemented with MSG and either sake lees extract or ground sesame seeds, as shown in Table 31 (only MSG was added to No. 69, and neither was added to No. 68). The glucose, malt extract, and MSG used were the same as in Test 1, the sake lees extract used was the same as in Test 3, and the ground sesame seeds used was the same as in Test 14.

[0119] Next, each sample was inoculated with the same Aspergillus oryzae as in Experiment 1, cultured with shaking under the culture conditions shown in Table 30, and the culture medium was filtered to collect the cells. The culture temperature was set to 20°C, which was different from that of Experiments 1-14, and the culture period was 4 days. [Table 30]

[0120] Finally, the mass of the recovered bacterial cells (bacterial cell volume) was measured, followed by the measurement of the γ-aminobutyric acid content per 100g of bacterial cells in each sample using LC / MS / MS. The LC / MS measurement method and conditions were the same as those used in Experiment 1.

[0121] The measurement results and the culture medium components of each sample are shown in Table 31. [Table 31]

[0122] As shown in Table 31, even when the culture temperature was set to 20°C, it was confirmed that the amount of microbial cells recovered and the γ-aminobutyric acid content in cultured Aspergillus oryzae increased in all culture media that contained MSG and sake lees extract or ground sesame seeds.

[0123] <Test 16: Suppression of mushroom-like flavor in Aspergillus oryzae mycoprotein cultured in sesame-added medium> Finally, mycoproteins were produced from koji mold cultured in a sesame-added medium, and a test was conducted to confirm whether the mushroom-like flavor was suppressed compared to mycoproteins derived from koji mold cultured in a normal medium. In basal culture media and sesame-added culture media (300 mL each) containing the additives shown in Table 32, the same Aspergillus oryzae as in Experiment 1 was introduced to each medium with an initial spore count of 1.0 × 10⁶. 3 The culture medium was inoculated to a CFU / mL concentration, cultured with shaking at 30°C, 200 rpm, and 0.2 SLM, and the cells were collected by filtering the medium. The same glucose, malt extract, and MSG were used as in Experiment 1, and the same ground sesame seeds were used as in Experiment 14. [Table 32]

[0124] Next, using the microbial cells recovered from the sesame-added culture medium as 100%, a mixture of 5% dried egg white and 0.4% refined salt was added and shaped into a roughly cylindrical form. These molded samples were then baked on a hot plate and prepared as food. For comparison, similar samples were prepared from Aspergillus oryzae cultured in the basic culture medium alone and subjected to the same baking process.

[0125] Finally, seven evaluators rated the mushroom-like flavor of each food item on a 10-point scale. The mushroom-like smell and taste of mycoprotein derived from Aspergillus oryzae cultured in a basic culture medium (and the food item containing it) were assigned a score of 5, while the flavor of mycoprotein derived from Aspergillus oryzae cultured in a medium supplemented with ground sesame seeds was quantified, including the standard deviation. The results are shown in Table 33. [Table 33]

[0126] As shown in Table 33, when koji mold was cultured in a sesame-added medium containing ground sesame and MSG, sensory evaluation confirmed an improvement in the mushroom-like flavor of the koji mold when it was consumed, based on the mycoprotein contained in the cultured koji mold.

[0127] Based on the above examples, it has been confirmed that the present invention provides a method for culturing Aspergillus oryzae, a culture kit, a method for producing mycoprotein, and mycoprotein, which, compared to ordinary Aspergillus oryzae, are rich in γ-aminobutyric acid, yield a large amount of microbial cells, and have an improved mushroom-like flavor in the purified mycoprotein.

Claims

1. A method for culturing γ-aminobutyric acid-enriched Aspergillus oryzae, A method for culturing γ-aminobutyric acid-enriched Aspergillus oryzae, characterized by adding glutamic acid and / or its salt and a vitamin B6-containing component to a culture medium for culturing Aspergillus oryzae.

2. The method for culturing γ-aminobutyric acid-enriched koji mold according to claim 1, characterized in that the vitamin B6-containing component is sake lees, soy protein, or sesame.

3. A method for culturing γ-aminobutyric acid-enriched koji mold according to claim 1, characterized in that the koji mold is of the genus Aspergillus.

4. A method for culturing γ-aminobutyric acid-enriched Aspergillus oryzae according to claim 1, characterized in that the amount of glutamic acid and / or its salt added to the culture medium is 0.085 to 10% as glutamic acid, and the amount of the vitamin B6-containing component added is 0.1 to 10%.

5. Aspergillus oryzae and A culture medium for culturing the aforementioned koji mold, The culture medium is characterized by containing glutamic acid and / or a salt thereof, and a vitamin B6-containing component, and is a culture kit for gamma-aminobutyric acid-enriched Aspergillus oryzae.

6. A method for producing γ-aminobutyric acid-enriched mycoprotein, A step of adding glutamic acid and / or its salt and a vitamin B6-containing component to a culture medium, followed by adding Aspergillus oryzae and culturing, The process of recovering bacterial cells from the culture medium, The process of purifying mycoproteins, which are mycelial cells, from bacterial cells. A method for producing γ-aminobutyric acid-enriched mycoprotein, characterized by containing the following:

7. A gamma-aminobutyric acid-enriched mycoprotein characterized by having a gamma-aminobutyric acid content of 400 mg or more per 100 g of dry weight mycoprotein.

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