Method for producing edible filamentous fungus body

Abstract

Due to the constituent components of the cell walls of filamentous fungi, the texture when eating a filamentous fungus body resembles a paper-like texture, and applications other than meat analogs are limited. Additionally, filamentous fungus bodies have characteristics of absorbing water but also readily releasing water, and having no binding property between mycelia, and thus even when a crushing treatment is performed, it has been difficult to process the result into foodstuffs that have a feeling of unity due only to a filamentous fungus culture product, or into foodstuffs that have uniform and smooth paste-like physical properties. This edible filamentous fungus body having an adjusted texture can be provided by treating a filamentous fungus body with a cell wall degrading enzyme.

Description

Method for producing edible filamentous fungal cells

[0001] The present invention relates to a method for producing edible filamentous fungal cells, edible filamentous fungal cells produced using the method, and foods containing the edible filamentous fungal cells.

[0002] As one solution to the global food problem, technology for processing microorganisms into meat substitutes has been attracting attention. In particular, filamentous fungi, due to their thread-like structure, form aggregates (mycelium) as fibrous masses with a texture similar to meat, making them promising raw materials for meat substitutes. Among filamentous fungi, filamentous fungi of the genus Fusarium have attracted attention as raw materials for meat substitutes due to their meat-like texture, and are already sold in several countries under the label of mycoprotein. Research and development of filamentous fungi other than Fusarium is also underway. Aspergillus oryzae, Japan's national fungus, has long been used in the production of fermented foods such as miso, soy sauce, and sake. Because of its guaranteed food safety, it has also attracted attention as an alternative protein, and technology for mass-producing koji as a protein source is being investigated (Patent Document 1). However, when filamentous fungi are consumed as is, the texture derived from the fibrous mycelium is too strong, making it difficult to apply to foods other than meat substitutes, which have a strong texture.

[0003] Enzymatic treatment of food ingredients has been carried out for various purposes. β-Glucanase is known to degrade and solubilize yeast cell walls, thereby increasing the yield of yeast extract. Various β-glucanase preparations are commercially available (Denazyme (registered trademark) GEL-L1 / R, Nagase & Co.; Tunicase SD-FN, Amano Enzyme; Filtrase NL, DSM). Breaking down the cell walls of microorganisms facilitates extraction of the cell body contents. For this purpose, treatment of basidiomycete mycelia with β1,3-glucanase, chitinase, and / or cellulase has been disclosed (Patent Document 2: Japanese Patent No. 2908457). Furthermore, a method for measuring rice koji (rice koji) cell mass has been disclosed, in which koji mold is degraded using a cell wall-lytic enzyme and the N-acetylglucosamine released from the fungal bodies is measured (Non-Patent Document 1: Journal of the Brewing Society of Japan Vol. 82, No. 2, pp. 130-133 (1987)).

[0004] Patent No. 7433800 Patent No. 2908457

[0005] Journal of the Brewing Society of Japan Vol.82, No.2, p.130-133 (1987)

[0006] It has been discovered that the texture derived from the fiber of mycelia is derived from insoluble dietary fibers such as glucan and chitin, which are components of the cell wall of filamentous fungi. Such insoluble dietary fibers have a paper-like texture when placed in the mouth. Furthermore, insoluble dietary fibers absorb water but are prone to release water, and have poor adhesion between mycelia. Therefore, even after crushing, it has been difficult to process filamentous fungal cultures alone into foods with a cohesive texture or a uniform, smooth paste-like texture.

[0007] The present inventors have conducted extensive research to improve the applicability of filamentous fungal cells as food ingredients and have come up with the idea of ​​enzymatically decomposing cell wall components. More specifically, they have discovered that treating filamentous fungal cells with a cell wall-degrading enzyme can provide edible filamentous fungal cells with a modified texture, leading to the present invention. The present invention therefore relates to the following: [1] A method for producing edible filamentous fungal cells with a modified texture, comprising treating a filamentous fungal culture with a cell wall-degrading enzyme. [2] The method according to Item 1, wherein the filamentous fungus is a microorganism selected from the group consisting of the genera Aspergillus, Fusarium, Geotrichum, Penicillium, Neurospora, Monascus, Paecilomyces, and Rhizopus. [3] The method according to item 1, wherein the filamentous fungus is one or more filamentous fungi selected from the group consisting of Aspergillus oryzae, Aspergillus kawachii, Aspergillus awamori, Aspergillus sojae, Aspergillus glaucus, Aspergillus tamari, Aspergillus luchuensis, and Aspergillus niger. [4] The method according to any one of items 1 to 3, wherein the cell wall-degrading enzyme is glucanase and / or chitinase. [5] The production method according to any one of items 1 to 4, wherein the treatment with the cell wall-degrading enzyme comprises treating with 16 to 8,000 units of the cell wall-degrading enzyme per 100 g of dry weight of filamentous fungal cells. [6] The production method according to any one of items 1 to 5, wherein the treatment with the cell wall-degrading enzyme comprises treating with 16 to 8,000 units of glucanase and 32 to 320 units of chitinase per 100 g of dry weight of filamentous fungal cells. [7] The production method according to any one of items 1 to 6, wherein filamentous fungal cells separated and recovered from a solid culture or a liquid culture are subjected to the cell wall-degrading enzyme treatment as a filamentous fungal culture.[8] The production method according to any one of items 1 to 7, wherein edible filamentous fungal cells are produced by treating at 0°C to 70°C for 0.5 to 48 hours, wherein the shear force value measured using a shear force measuring device manufactured by Instron (registered trademark) is less than 30 N. [9] Edible filamentous fungal cells produced by the production method according to any one of items 1 to 8.

[10] The edible filamentous fungal cells according to item 9, wherein the shear force value measured using a shear force measuring device manufactured by Instron (registered trademark) is 1 to 30 N.

[11] A food product comprising the edible filamentous fungal cells according to item 9.

[0008] Treatment with cell wall-degrading enzymes can improve the paper-like texture of fungal cells, and also improve the physical properties of the fungal cells, such as shear resistance, viscoelasticity, and water release, thereby increasing the applicability of the fungal cells as edible materials.

[0009] FIG. 1 shows the results of sensory evaluation of filamentous fungal cells when eaten after each treatment (C: untreated, T1: 0.01% β-glucanase treatment, T2: 0.1% β-glucanase treatment, T3: 1.0% β-glucanase treatment, T4: 0.1% β-glucanase short-term treatment, T5: 0.1% β-glucanase low-temperature treatment, T6: chitinase treatment, T7: crushing treatment) (A: release of water after one chew, B: remaining in the mouth after one chew, C: remaining in the mouth after 50 chews). FIG. 2 shows the results of shear force titers measured by instrumental analysis of filamentous fungal cells after each treatment (C: untreated, T1: 0.01% β-glucanase treatment, T2: 0.1% β-glucanase treatment, T3: 1.0% β-glucanase treatment, T4: 0.1% β-glucanase short-term treatment, T5: 0.1% β-glucanase low-temperature treatment, T6: chitinase treatment, T7: disruption treatment).

[0010] The present invention relates to a method for producing edible filamentous fungal cells with a controlled texture, which comprises treating a filamentous fungal culture with a cell wall-degrading enzyme. Treatment with the cell wall-degrading enzyme improves the paper-like texture. Furthermore, by modifying the treatment conditions, it is possible to provide edible filamentous fungal cells with desired physical properties, such as shear resistance, viscoelasticity, and low water release.

[0011] The filamentous fungi are not limited as long as they are edible as foods, and examples include filamentous fungi belonging to the genera Aspergillus, Fusarium, Geotrichum, Penicillium, Neurospora, Monascus, Paecilomyces, and Rhizopus. Among these filamentous fungi, microorganisms of the genera Aspergillus and Fusarium are preferred, particularly from the viewpoint of edibility. Among these, filamentous fungi of the genus Aspergillus are particularly preferred because they have been used as foods for a long time. Examples of such filamentous fungi of the genus Aspergillus include Aspergillus oryzae, Aspergillus kawachii, Aspergillus awamori, Aspergillus sojae, Aspergillus glaucus, Aspergillus tamari, Aspergillus luchuensis, and Aspergillus niger. A single type of filamentous fungus may be used, or two, three, or more types of other filamentous fungi may be used in combination, or the filamentous fungus may be used in combination with a microorganism other than a filamentous fungus.

[0012] The filamentous fungal culture may contain filamentous fungal cells and the culture medium after culture, or may contain only the recovered filamentous fungal cells. The medium may be a liquid medium and / or a solid medium. The solid medium and the filamentous fungal culture cultured in the liquid medium may be subjected to enzyme treatment as is, or the cells obtained by separating the cells may be subjected to enzyme treatment as is. When subjected to enzyme treatment as is, an enzyme may be added to the culture at a certain point during the culture. To separate the cells from the solid medium, the grown cells can be peeled off from the solid medium. However, since the cells are connected in a sheet-like form, they must be crushed using a mixer or the like to be homogenized. Furthermore, if the cells and the solid medium are in close contact and separation is difficult, they can be crushed together with the medium for use. In this case, the edible filamentous fungal cells contain medium components. From the perspective of using the filamentous fungal cells as a food material, the filamentous fungal culture is preferably a filamentous fungal culture cultured in a liquid medium. A filamentous fungal culture cultured in a liquid medium can be easily separated and recovered from the liquid medium by separation treatments such as centrifugation, filtration, and squeezing. The filamentous fungal cells recovered from the liquid medium may be subjected to pretreatments such as washing, disruption, and solution replacement before treatment with a cell wall-degrading enzyme. Pretreatment can enhance reactivity to cell wall-degrading enzyme treatment. Therefore, the production method of the present invention may include one or more pretreatment steps selected from the group consisting of a step of recovering filamentous fungal cells, a washing step, a disruption step, and a solution replacement step before treatment with the cell wall-degrading enzyme. The edible filamentous fungal cells of the present invention may refer to the filamentous fungal cells themselves, or may include medium components and the like contained during the production process. When the edible filamentous fungal cells contain medium components and the like, they can be referred to as an edible composition containing edible filamentous fungal cells. In addition to edible filamentous fungal cells, the edible composition may contain any component, such as a medium component, a sweetener, a colorant, a thickener, a stabilizer, a gelling agent, an antioxidant, a color former, a bleaching agent, an acidulant, a seasoning, a coagulant, an emulsifier, a leavening agent, a nutrient fortifier, a pH stabilizer, a flavoring, and the like.

[0013] Treating with a cell wall-degrading enzyme can produce edible filamentous fungal cells with an adjusted texture. When subjected to sensory evaluation, it is preferable that the paper-like texture observed before the cell wall-degrading enzyme treatment is reduced. The paper-like texture refers to a texture resembling chewing paper due to the fibers. While not intending to be limited by theory, approximately half of the solid content of filamentous fungi is composed of fibrous cell walls, and the cell walls contained in the hyphae of filamentous fungal mycelia are thought to be the cause of the paper-like texture. Cell walls are composed of various polymers, including glucan, chitin, galactosaminogalactan, and galactomannan, among others. Among these, glucan and chitin, which account for a high proportion of the components, are thought to contribute to the paper-like texture. Glucans contained in filamentous fungal cells are broadly classified into α-1,3-glucan and β-1,3-glucan, each of which may further have branches, such as α-1,6 and / or β-1,6 branches. The enzyme treatment conditions can be adjusted to a level where the paper-like texture is no longer present. The paper-like texture can also be evaluated by shear force titer. For example, a shear force titer of 30 N or more measured using a shear force measuring device manufactured by Instron (registered trademark) can be said to be paper-like. Treatment with a cell wall-degrading enzyme can reduce the shear force titer to less than 30 N, more preferably to 5 N or more, and even more preferably to 10 N or more. Decomposition of the cell wall increases affinity with water, making it possible to prepare smooth, paste-like filamentous fungal cells.

[0014] The cell wall-degrading enzyme may be any enzyme capable of degrading cell wall components, more preferably an enzyme capable of degrading glucan and / or chitin. Examples of such enzymes include glucanase and chitinase. Examples of glucanase include α-glucanase and β-glucanase, with β-glucanase being preferred from the perspective of industrial use. Unless otherwise specified, α-glucanase and β-glucanase refer to α-1,3-glucanase and β-1,3-glucanase, respectively. These enzymes may be derived from any organism, and commercially available enzymes can be used. These enzymes may be used alone or in combination with other enzymes. Examples of other enzymes include proteases, peptidases, cellulases, amylases, xylases, pectinases, mannanases, lipases, etc. From the perspective of adjusting texture, glucanases, particularly a combination of β-glucanase and chitinase, can be used. The combination of glucanase and chitinase can decompose glucan and chitin, which are the main cell wall materials of filamentous fungi, and therefore can generally be used on any type of filamentous fungal cells.

[0015] The enzymatic treatment step using a cell wall-degrading enzyme is carried out by applying the cell wall-degrading enzyme to a filamentous fungal culture containing filamentous fungal cells. The concentration of the enzyme can be adjusted appropriately depending on the enzyme activity and the desired texture. For example, 10 to 10,000 units of enzyme can be applied per 100 g of dried fungal cells. The lower limit can be appropriately selected from 16, 50, and 100 units, and the upper limit can be appropriately selected from 8,000, 5,000, 2,000, 1,000, and 500 units, allowing the concentration range to be set as desired. For example, when β-glucanase is used, the treatment can be carried out with any consecutive combination of units of the cell wall-degrading enzyme selected from 16 to 50, 50 to 100, 100 to 320, 320 to 500, 500 to 1000, 1000 to 2000, 2000 to 5000, 5000 to 8000, and 8000 to 10,000 units per 100 g of dry weight of filamentous fungal cells. When chitinase is used, the treatment can be carried out with 32 to 320 units of the cell wall-degrading enzyme selected from 32 to 50, 50 to 100, 100 to 150, 150 to 200, 200 to 250, and 250 to 320 units per 100 g of dry weight of filamentous fungal cells. Two or more enzymes can be used simultaneously or sequentially for the enzymatic treatment. When performing consecutive enzyme treatments, multiple enzymes are added at different times, and a washing step may be included after each enzyme treatment. The amount of enzyme and the conditions for the enzyme treatment can be appropriately changed depending on the enzyme used and the physical properties, including the desired texture, and must be set appropriately within a range that does not cause denaturation, such as spoilage, of the filamentous fungal cells. The treatment temperature may be selected within a range in which the enzyme is active and can be selected from a range of 0°C to 70°C. The treatment time can be selected appropriately within a range in which the desired texture is achieved, and can be, for example, from 10 minutes to several days. While the reaction time can be shortened by performing the treatment near the optimal temperature of the enzyme, a temperature lower than the optimum can also be used to suppress denaturation, such as spoilage, of the filamentous fungal culture.Relatively low temperatures such as 0°C to 10°C are preferred because they can suppress denaturation of the filamentous fungal culture, but they require a correspondingly long reaction time, for example, 12 hours to 2 days. The reaction can also be carried out at around room temperature, 10 to 30°C, and can be carried out in a shorter reaction time, for example, 10 minutes to several hours. At 30°C to 70°C, a high reaction rate can be achieved as long as the enzyme is not denatured. When culturing at such high temperatures, the reaction can be carried out, for example, for 10 minutes to several hours. During the enzyme treatment step, a step of aliquoting a portion of the filamentous fungal culture and evaluating its texture may be carried out. This allows the enzyme treatment step to be terminated when the desired texture is obtained. The enzyme-treated filamentous fungal cells can be provided as edible filamentous fungal cells with an adjusted texture.

[0016] After the cell wall-degrading enzyme treatment step, a post-treatment step such as inactivation or washing may be included to stop the enzymatic reaction. The enzyme can be inactivated by heating the enzyme treatment solution. The enzymatic reaction can also be stopped by changing the pH of the enzyme reaction solution or by adding an enzyme inhibitor.

[0017] By appropriately selecting the conditions for the enzyme treatment, edible filamentous fungal cells can have the desired texture and / or physical properties. The edible filamentous fungal cells that have been subjected to the enzyme treatment according to the present invention have a reduced or eliminated paper-like texture. Edible filamentous fungal cells with a reduced or eliminated paper-like texture have a shear force titer of 30 N or less, preferably 25 N or less, and more preferably 20 N or less, as measured using a shear force measuring device manufactured by Instron (registered trademark). The lower limit of the shear force titer is not particularly limited, and examples include 1 N or more, 2 N or more, and 5 N or more. More specifically, any continuous combination of ranges selected from 1 N to 2 N, 2 N to 5 N, 5 N to 10 N, 10 N to 20 N, 20 N to 25 N, and 25 N to 30 N can be selected. The desired physical properties include a state without a papery texture, a physical property that melts easily in the mouth while retaining a fibrous texture, and a smooth, paste-like physical property. These properties can be expressed, for example, in terms of shear resistance, water-releasing properties, viscoelasticity, spinnability, breakability, cohesiveness, and adhesiveness, and can be evaluated using appropriate evaluation equipment and methods. As mentioned above, although it is possible to identify edible filamentous fungal cells that have undergone enzyme treatment using objective indicators of physical properties such as shear strength or subjective indicators such as sensory tests, it is usually impractical to directly identify a substance by its structure or properties. Therefore, it can be said that it is appropriate to identify the edible filamentous fungal cells of the present invention using the production method of the present invention.

[0018] Edible filamentous fungal cells or edible compositions can be used as food ingredients or added to foods. Since filamentous fungal cells have a solid content of approximately 30% protein, they can be used as a nutritional food ingredient or added to various foods for nutritional enrichment. By adjusting the conditions of the enzyme treatment, the texture can be adjusted as desired, and the cells can be made to melt in the mouth while retaining a fibrous texture, or processed into a paste. Conventional edible filamentous fungal cells have a problem in that their blending amount cannot be increased to reduce the paper-like texture. However, by adjusting the texture as desired, there is no longer a limit on the blending amount, and higher blending amounts can be incorporated. Edible filamentous fungal cells have the high nutritional value of filamentous fungi, particularly their high protein content, and can be used in a variety of foods due to their adjustable texture. The edible filamentous fungal cells or edible composition of the present invention can be used as, but is not limited to, soybean substitute foods, meat product substitute foods, dairy product substitute foods, wheat substitute foods, rice substitute foods, fish substitute foods, egg product substitute foods, etc., and can also be used as a new edible filamentous fungal cell food that is not a substitute food. Edible filamentous fungal cell foods can be used as seasonings, beverages, food, etc. They can also be used as animal feed for livestock, pets, farmed fish, etc.

[0019] All documents mentioned herein are incorporated by reference in their entirety.

[0020] The following examples of the present invention are for illustrative purposes only and do not limit the technical scope of the present invention. The technical scope of the present invention is limited only by the claims. The present invention may be modified, for example, by adding, deleting, or substituting components of the present invention, provided that the modifications do not depart from the spirit of the present invention.

[0021] Example 1: Treatment of filamentous fungal cells 1. Preparation of filamentous fungal cells Aspergillus oryzae was cultured in a jar fermenter in a total of 3 L of synthetic liquid medium containing glucose (Fujifilm Wako Pure Chemical Industries, Ltd.) as a carbon source and ammonium sulfate (Fujifilm Wako Pure Chemical Industries, Ltd.) as a nitrogen source at 30°C for 48 hours under an aerated environment. After cultivation, the filamentous fungal cells were heat-sterilized, collected by vacuum filtration, and washed with water to obtain filamentous fungal cells.

[0022] 2. The obtained filamentous fungal cells were used as a control (C: untreated) and were subjected to the following treatments to obtain a disruption treatment group and an enzyme treatment group. For the disruption treatment group, the filamentous fungal cells were physically disrupted for 1 minute using a food processor (Panasonic) in high-speed mode (T7). For the glucanase treatment group, Denatzyme GEL L1 / R (Nagase Sangyo Kaisha, Grade 400 units / mL) was added at concentrations of 0.01% (T1), 0.1% (T2), and 1.00% (T3) and incubated at 55°C for 6 hours. Furthermore, a short-term treatment group (T4) was prepared by incubating the cells at 55°C for 3 hours with a 0.1% enzyme concentration, and a low-temperature treatment group (T5) was prepared by incubating the cells at 4°C for 24 hours. For the chitinase-treated group, Denatzyme CBB-P1 / R (Nagase & Co., Ltd.: Grade 16 units / g) was added at a concentration of 0.1%, and the reaction was carried out at 55°C for 6 hours (T6). After the reaction, the sample was taken and heated in boiling water for 10 minutes to inactivate the enzyme, and the sample was stored at 4°C until analysis.

[0023] 3. Sensory Evaluation Eight trained sensory evaluators conducted a sensory evaluation at the time of eating. The treated samples were placed in the mouth, and the water release property and the remaining property in the mouth after one chewing and 50 chewings were scored based on the following criteria. The results are shown in Figure 1.

[0024] 1A-C, comparing the residual texture after the first and 50th chewing, although there is a slight decrease in residual texture, there is little difference between the first and 50th chews, indicating that the papery texture is unlikely to change with chewing. Regarding water syneresis, the T7 group showed a tendency to be low, while the other groups showed a similar tendency to residual texture. In the T7 group, the mycelium was physically cut, but the cell wall remained, which is thought to decrease water syneresis, but have little effect on residual texture after chewing.

[0025] 4. Instrumental Analysis The shear strength of the samples after the treatment was measured using an Instron shear force measuring device (model number: 5942). The results are shown in Figure 2.

[0026] Comparing Figure 1 and Figure 2, both have in common that C (untreated) had the highest value, and the value decreased depending on the enzyme concentration from T1 to T3. In the chitinase-treated group (T6), the shear force value did not decrease as easily (Figure 2), but the evaluation generally showed the same trend as the sensory evaluation in Figure 1, and the paper-like texture can be evaluated based on the shear force value measured using a shear force measuring device manufactured by Instron (registered trademark).

Claims

1. A method for producing edible filamentous fungal cells with a controlled texture, comprising a step of treating a culture of filamentous fungi with a cell wall-degrading enzyme.

2. The method according to claim 1, wherein the filamentous fungus is a microorganism selected from the group consisting of Aspergillus, Fusarium, Geotrichum, Penicillium, Neurospora, Monascus, Paecilomyces and Rhizopus.

3. The production method according to claim 1, wherein the filamentous fungus is one or more filamentous fungi selected from the group consisting of Aspergillus oryzae, Aspergillus kawachii, Aspergillus awamori, Aspergillus sojae, Aspergillus glaucus, Aspergillus tamari, Aspergillus luchuensis, and Aspergillus niger.

4. The method according to claim 1, wherein the cell wall-decomposing enzyme is glucanase and / or chitinase.

5. The method according to claim 1, wherein the treatment with the cell wall-degrading enzyme comprises treating with 16 to 8,000 units of the cell wall-degrading enzyme per 100 g of dry weight of the filamentous fungal cells.

6. The method of claim 1, wherein the treatment with the cell wall-degrading enzymes comprises treating with 16 to 8,000 units of glucanase and 32 to 320 units of chitinase per 100 g of dry weight of filamentous fungal cells.

7. The method according to claim 1, wherein the filamentous fungal cells separated and recovered from the solid or liquid culture are subjected to the cell wall-decomposing enzyme treatment as a filamentous fungal culture.

8. The method according to claim 1, wherein edible filamentous fungal cells are produced by treating at 0°C to 70°C for 0.5 to 48 hours, the shear force titer of which, as measured with an Instron (registered trademark) shear force measuring device, is less than 30 N.

9. Edible filamentous fungal cells produced by the method according to any one of claims 1 to 8.

10. The edible filamentous fungus cells according to claim 9, which have a shear force value of 1 to 30 N as measured by an Instron (registered trademark) shear force measuring device.

11. A food composition or food comprising the edible filamentous fungus cells of claim 9.

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