Method for producing filamentous fungal cells with reduced unpleasant taste and / or odor

Abstract

To provide a method for producing an edible filamentous fungus cell, by which the number of times of washing with water is reduced or an offensive taste and an offensive smell are suppressed to such an extent that a water-washing process is not required.SOLUTION: By allowing the protease to act during the liquid culture, the strange taste and / or strange odor can be reduced to such an extent that water washing is not required.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for producing filamentous fungal cells with reduced off-taste and / or off-odor, edible filamentous fungal cells produced using the production method, and foods containing the edible filamentous fungal cells. The present invention also relates to a method for reducing off-taste and off-odor in liquid-cultured filamentous fungal cells, and a method for defoaming liquid cultures. [Background technology]

[0002] As one solution to the global food problem, technology for processing microorganisms into meat substitutes has been attracting attention. Filamentous fungi, in particular, have a thread-like shape, and their aggregates (mycelium) form fibrous masses with a texture similar to meat, making them promising raw materials for meat substitutes. Among filamentous fungi, koji mold, 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. For efficiency reasons, culturing as many microorganisms as possible simultaneously is required, and the mainstream method is to cultivate filamentous fungi at high densities in liquid media. Research is currently underway into mass-production techniques for koji in liquid culture (Patent Document 1). In addition to koji mold, filamentous fungi of the genus Fusarium have also attracted attention as raw materials for meat substitutes due to their meat-like texture. They are already being sold in several countries under the label "mycoprotein." When koji mold is cultured in a liquid culture to produce koji fungus cells for food replacement, the collected cells have an unpleasant taste and / or odor, so they are washed with water after collection. In the process of washing the koji mold cells, they are washed multiple times using large amounts of water to suppress the unpleasant taste and odor so that they can be consumed as a food. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Patent No. 7433800 Summary of the Invention [Problem to be solved by the invention]

[0004] When edible filamentous fungal cells are produced on an industrial scale, the large amount of water used in the washing process places a heavy burden on the environment. Generally, the amount of wastewater generated during washing is several times the amount of culture unit, necessitating large-scale wastewater treatment facilities. The objective of this study was to reduce the number of water washings required for edible filamentous fungal cells produced by liquid culture, or to suppress the off-flavors and odors to the extent that the water washing process is not necessary. [Means for solving the problem]

[0005] The present inventors investigated the cause of the off-taste and / or off-odor exhibited by liquid-cultured filamentous fungal cells and found that proteins and peptides, which are fungal metabolites, are the cause of the off-taste and / or off-odor. They discovered that the action of protease during liquid culture can reduce the off-taste and / or off-odor to a level that does not require washing with water, and thus arrived at the present invention. Thus, the present invention relates to: [1-1] culturing a filamentous fungus in a liquid medium containing a protease; and a step of recovering the grown bacterial cells and removing the liquid medium to recover the bacterial cells; A method for producing edible filamentous fungal cells, comprising: [1-2] A method for reducing off-taste and / or off-odor in a filamentous fungal culture, comprising culturing the filamentous fungus in a liquid medium containing a protease. [1-3] A method for defoaming a filamentous fungal culture, comprising culturing the filamentous fungus in a liquid medium containing a protease. [2] The method according to any one of Items 1-1 to 1-3, 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 Items 1-1 to 1-3 or 1-2, 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 1-3, or Items 2 to 3, wherein the protease is a protease of EC.3.4.11 to EC.3.4.25. [5] The method according to any one of Items 1-1 to 1-3 or Items 2 to 4, wherein the protease has both endopeptidase and exopeptidase activity or contains both endopeptidase and exopeptidase activity. [6] The method according to any one of Items 1-1 to 1-3 or Items 2 to 5, wherein the protease is one or a combination of two or more selected from the group consisting of papain, pancreatin, trypsin, chymotrypsin, pepsin, cathepsin, elastase, subtilisin, streptoglycin, bromelain, zingibain, ficain, and actinidin. [7] The method according to any one of Items 1-1 to 1-3 or Items 2 to 6, which does not include a washing step with water after the culture. [8] The method according to any one of Items 1-1 to 1-3 or Items 2 to 7, wherein the protease is added after the liquid medium has been subjected to a sterilization treatment. [9] The method according to any one of Items 1-1 to 1-3 or Items 2 to 8, wherein the protease is contained in an amount of 0.001 to 10% by mass.

[10] The method according to any one of Items 1-1 to 1-3 or Items 2 to 9, wherein the protease has an activity temperature of 20°C to 50°C.

[11] The method according to any one of Items 1-1 to 1-3 or Items 2 to 10, wherein the protease has an activity of 10,000 U / g or more at its activity temperature.

[12] The method according to any one of Items 1-1 to 1-3 or Items 2 to 11, wherein the protease reduces the off-flavor of edible filamentous fungal cells.

[13] The method according to any one of Items 1-1 to 1-3 or Items 2 to 12, wherein the protease inhibits foam formation during culture.

[14] Edible filamentous fungal cells produced by the production method described in Items 1-1 to 1-3 or Items 2 to 13.

[15] A food composition or food comprising the edible filamentous fungus cells according to Item 14. [Effects of the Invention]

[0006] The edible filamentous fungal cells produced by the production method of the present invention have reduced off-taste and / or off-odor, and can be provided for food use without the need for washing or with reduced frequency of washing. [Brief explanation of the drawings]

[0007] [Figure 1] Figure 1 shows photographs comparing the foaming properties of the filtrate obtained after recovering the solid matter (mycelia) from the liquid culture of koji mold when protease was added (0.1% or 0.5% enzyme), and when no treatment or water was added, after aeration at 1 vvm for 2 minutes. [Figure 2] Figure 2 shows a graph comparing foaming properties depending on the timing of protease addition during liquid culture of koji mold. Protease was added before culture (0 hours), after 24 hours of culture (24 hours), and after 47 hours of culture (47 hours), for a total of three times (0 hours, 24 hours, and 47 hours). After 48 hours, the solid matter (mycelium) was recovered from the culture, and the recovered filtrate was aerated at 1 vvm for 2 minutes. The foaming properties of the recovered filtrate are shown as the height of the liquid surface. DETAILED DESCRIPTION OF THE INVENTION

[0008] The present invention provides the following: Cultivating a filamentous fungus in a liquid medium containing a protease; and a step of recovering the grown bacterial cells and removing the liquid medium to recover the bacterial cells; The present invention relates to a method for producing edible filamentous fungal cells, comprising: By culturing filamentous fungi in a medium containing a protease, it is possible to reduce the unpleasant taste and / or odor of the collected fungal cells. This allows for the provision of food-grade filamentous fungal cells without the need for water washing after the collected fungal cells have been washed, or without the water washing step. The production method of the present invention can also be characterized by not carrying out the water washing step.

[0009] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: Cultivating a filamentous fungus in a liquid medium containing a protease The present invention also relates to a method for reducing the off-taste and / or off-odor of filamentous fungal cells, comprising: Filamentous fungal cells cultured in liquid culture exhibit an off-taste and / or an off-odor depending on the type of filamentous fungus and / or the composition of the culture medium. This off-taste and / or off-odor can be reduced by culturing the filamentous fungus in a liquid medium containing a protease. The off-taste and / or off-odor can be evaluated by sensory evaluation, dividing the degree of the off-taste and / or off-odor into multiple stages and assigning a score. Alternatively, the off-taste and / or off-odor can be evaluated by identifying the substance causing the off-taste and / or off-odor and measuring its amount. The off-taste and / or off-odor refers to acridity, bitterness, odor, strong taste, etc. The present invention may also relate to a protease-containing agent for suppressing the off-taste and / or off-odor of filamentous fungal cells or cultures.

[0010] In yet another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: Cultivating a filamentous fungus in a liquid medium containing a protease The present invention also relates to a method for defoaming during culture, including the steps of: (1) defoaming the medium; (2) defoaming the medium; (3) suppressing foam formation; (4) treating the medium with a protease; (5) aerating the medium after filtering off the solids; and (6) evaluating foam formation. As an example, when 100 mL of filtrate is dispensed into a chromatographic tube with an inner diameter of 40 mm (chromatographic tube with a G4 filter and no stopcock) and aerated from the bottom at 1 vvm for 2 minutes, the foam height can be suppressed to 30% or less. In the case of such foam formation, foam formation can be suppressed even when culturing using an industrial-scale jar fermenter, and the addition of a separate defoaming agent is not required or the amount added can be reduced. The present invention may also relate to a defoaming agent containing a protease.

[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 species of filamentous fungus may be used, or two, three, or more species of filamentous fungi may be used in combination, or a microorganism other than a filamentous fungus may be used in combination.

[0012] The liquid medium may be a liquid medium commonly used for culturing filamentous fungi. It may be a laboratory medium for filamentous fungi or a liquid medium for industrial production. A liquid medium is typically a solution containing water as the primary solvent, a carbon source, a nitrogen source, minerals, and trace elements dissolved therein, and may also be a mixture containing solids in some cases. Examples of carbon sources include sugars such as glucose, lactose, dextrin, and blackstrap molasses. Examples of nitrogen sources include ammonium salts, amino acids, peptone, and yeast extract. Phosphates, sulfates, potassium, trace metals, and vitamins may be added as minerals and trace elements. More preferably, plant waste such as blackstrap molasses may be used. Furthermore, an antifoaming agent may be added to suppress foaming. Examples of antifoaming agents include silicones, fatty acid esters, phosphate esters, and glycerin fatty acid esters. The protease may be added to the liquid medium before inoculation, or it may be added to the liquid culture during or after the culture is completed. The protease may be added multiple times during the culture process. It is preferable to add the protease to the liquid medium after sterilization. The concentration of the protease can be appropriately selected depending on the level of off-flavor and off-odor to be achieved. Before adding the protease, a step of adjusting the pH to a pH at which the protease activity can be maintained may be included. The pH may vary depending on the type of protease, but can be adjusted to, for example, a pH range of 4 to 9, preferably 5 to 8, and more preferably 6 to 7. The pH is usually adjusted using a base such as sodium hydroxide or potassium hydroxide, or an acid such as hydrochloric acid or sulfuric acid, but the pH may also be adjusted by adding a buffer.

[0013] Filamentous fungi can be cultured in liquid media by any method, such as aeration culture, agitation culture, shaking culture, or static culture, depending on the type of filamentous fungus. The culture temperature can be set depending on the type of filamentous fungus to be cultured. The culture temperature for filamentous fungi is usually 20 to 50°C, preferably 25 to 40°C, and more preferably 25 to 35°C. From the perspective of industrial-scale cultivation, it is preferable to use a jar fermenter, in which case aeration and agitation culture is usually performed. During cultivation, the agitation speed and aeration rate may be determined so as to prevent foaming of the medium. Foaming in the medium can be caused by denatured proteins, so minimizing foaming can suppress off-flavors and / or unpleasant odors.

[0014] Since the protease is allowed to act during the cultivation of filamentous fungi, it is preferable to select a protease whose optimum temperature and pH are close to the culture conditions. From the viewpoint of closeness to the culture conditions, a protease active at 20°C to 50°C is used. Proteases can reduce off-flavors and / or odors by decomposing proteins or peptides contained in the medium. They can also suppress foaming. The concentration of the protease can be selected so that these effects can be achieved, and for example, it can be formulated at a concentration of 0.001 to 10% by mass. To fully exert its effects, the protease concentration is preferably 0.01% by mass or more, more preferably 0.05% by mass or more. To suppress the off-flavor and / or odor of the protease itself, the protease concentration is preferably 5% by mass or less, more preferably 1% by mass or less. The protease can be used within any combination of the upper and lower limits. The concentration of the protease can be adjusted so that it has an activity of 10,000 U / g or more at the active temperature. Furthermore, from the viewpoint of using filamentous fungal cells as food, it is preferable to use a food additive-grade protease. Reduction of off-tastes and / or off-odors by adding a protease can be evaluated by sensory evaluation. Filamentous fungal cells cultured without adding a protease and then recovered exhibit off-tastes and / or off-odors such as acridity, bitterness, or odor unless subjected to a water washing step. However, by culturing the cells with the addition of a protease, these off-tastes and / or off-odors can be reduced.

[0015] Any protease may be used, and either an exopeptidase or an endopeptidase may be used. A combination of an exopeptidase and an endopeptidase may be used, or an enzyme having both exopeptidase and endopeptidase activity may be used. Proteases can be classified into the following seven groups depending on the active center and the target amino acid residue: serine proteases, cysteine ​​proteases, threonine proteases, aspartic acid proteases, glutamic acid proteases, metalloproteases, and asparagine peptide lyases. Any one or a combination of these proteases may be used. Proteases belonging to enzyme classification groups EC.3.4.11 to EC.3.4.25 may be used.

[0016] The protease may be, for example, one selected from the group consisting of papain, pancreatin, trypsin, chymotrypsin, pepsin, cathepsin, elastase, subtilisin, streptoglycin, bromelain, zingibain, ficain, and actinidin, or a combination thereof. Food-additive grade enzymes are preferred, including papain, pancreatin, actinidin, and trypsin. For example, a combination of pancreatin and papain can be used. The protease P Amano 3SD used in the examples has both exopeptidase and endopeptidase activity.

[0017] Filamentous fungal cultures grown in liquid media can be easily separated and recovered from the liquid medium by separation processes such as centrifugation, filtration, and squeezing. The separated filamentous fungal cells may be subjected to additional processes such as washing, disruption, and enzyme treatment. Since culturing in a liquid medium containing protease reduces off-flavors and odors, washing is not necessarily required and may or may not be performed. Washing can be performed using ordinary water to obtain edible filamentous fungal cells. Additional enzyme treatments include treatment with cell wall-degrading enzymes, which can adjust texture, etc. 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 that are added 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.

[0018] Edible filamentous fungal cells can be used as a food ingredient or added to foods. Since approximately 30% of the solid content of filamentous fungal cells is protein, they can be used as a nutritional food ingredient or added to various foods for nutritional enrichment. By adjusting the conditions for additional enzymatic treatment with cell wall-degrading enzymes, the texture can be adjusted as desired, and they can be made to melt in the mouth while retaining a fibrous texture, or processed into a paste. 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. The edible filamentous fungal cells of the present invention can be used as a soybean substitute, meat substitute, dairy product substitute, wheat substitute, rice substitute, fish substitute, egg product substitute, etc., but are not intended to be limited thereto. The edible filamentous fungal cells can also be used in seasonings, beverages, food, etc. They can also be used as animal feed for livestock, pets, farmed fish, and other animals.

[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. [Example]

[0021] Example 1: Examination of the effectiveness of protease treatment 1. Preparation of Aspergillus oryzae Cultures Aspergillus oryzae was inoculated into a total of 3 L of synthetic liquid medium containing glucose (Fujifilm Wako Pure Chemical Industries, Ltd., special grade reagent) as the carbon source and ammonium sulfate (Fujifilm Wako Pure Chemical Industries, Ltd., food additive) as the nitrogen source, and cultured in a jar fermenter at 30°C for 48 hours under aeration.

[0022] 2. Processing After incubation, the culture medium was dispensed into 0.9 mL aliquots, and 0.1 mL of enzyme solution of the desired concentration was added. The enzyme solutions of the desired concentrations were 0.5%, 0.25%, 0.1%, and 0.05% by mass of papain (Nagase ChemteX), pancreatin (Fujifilm Wako Pure Chemical Industries), or protease P Amano 3SD (Amano Enzyme) based on the final volume of the liquid. A control solution was prepared by adding only water. After addition, the enzymes were reacted at 30°C for 1 hour, and then subjected to sensory evaluation.

[0023] 3. Sensory evaluation The culture solutions were subjected to sensory evaluation by four trained sensory evaluators. The control culture solution + water was held in the mouth and the taste was rated on a scale of 5. The other enzyme-treated solutions were evaluated based on the evaluation criteria in Table 1 below. The filamentous fungal cells washed with water are tasteless, and the substances that cause the off-taste exhibited by the filamentous fungal cells are components contained in the culture solution. Therefore, by performing a sensory evaluation of the culture solution, the effect of the enzyme in reducing the off-taste can be more sensitively evaluated. In the sensory evaluation, if the average score is 4 points or less, it is evaluated that the off-flavor has been reduced by the enzyme, and if it is 3 points or less, it is evaluated that the off-flavor has been reduced to the extent that the culture medium can be swallowed without any trouble.If the average score is 2 points or less, it is evaluated that the off-flavor has been significantly reduced to the extent that the culture medium can be swallowed easily. [Table 1] The results are shown in Table 2. [Table 2] The addition of enzymes, such as proteases, reduced the off-flavor of the medium. Papain reduced the off-flavor in a dose-dependent manner. On the other hand, pancreatin and Protease P Amano 3SD did not reduce the off-flavor in a dose-dependent manner. These enzymes themselves produce an off-flavor, and it is thought that this off-flavor appeared as the concentration increased. Regardless of which enzyme was used, the off-flavor derived from the medium could be reduced. Furthermore, although the effective concentration varied depending on the enzyme type, it was shown that an addition of 0.05% by mass or more was effective.

[0024] Example 2: Protease treatment of cultures after cultivation 1. Preparation of Aspergillus oryzae Cultures Aspergillus oryzae was inoculated into 500 mL of synthetic liquid medium containing glucose (Fujifilm Wako Pure Chemical Industries, Ltd., special grade reagent) as the carbon source and ammonium sulfate (Fujifilm Wako Pure Chemical Industries, Ltd., food additive) as the nitrogen source, and cultured at 30°C with a maximum shaking speed of 135 rpm for 48 hours.

[0025] 2. Processing After incubation, the pH of the culture was adjusted to 6.5 using NaOH, and 100 mL of enzyme solution at a predetermined concentration was added. The enzyme solutions were 0.1% by mass or 0.5% by mass of Protease P Amano 3SD (Amano Enzyme Inc.). Controls included a control group and a group containing 100 mL of water. The culture was shaken at 30°C and 135 rpm for 1 hour, after which the culture was collected and subjected to yield evaluation.

[0026] 3. Aspergillus yield evaluation The solid matter (bacterial cells) was collected from the culture by filtration, and the wet weight was measured. The solid matter (bacterial cells) was then dried, and the dry weight was measured, and the moisture content was measured. The results are shown in Table 3. In addition, sensory evaluation was performed by the experimenter on each sample, and it was confirmed that the off-taste and off-odor were reduced in both the 0.1% by mass and 0.5% by mass enzyme-treated groups. [Table 3] There was almost no difference in the koji mold yield due to the addition of enzymes.

[0027] 4. Evaluation of the foamability of the filtrate After collecting the solids (bacterial cells) from the culture, the filtrate was collected, and 100 mL of the filtrate was dispensed into a 40 mm inner diameter chromatography tube (with G4 filter and no stopcock). The medium was aerated from the bottom at 1 vvm for 2 minutes to evaluate the foaming properties of the medium. Figure 1 shows photographs of the culture before aeration, 1 minute after aeration, and 2 minutes after aeration. It was confirmed that the addition of enzymes significantly reduced foaming during aeration.

[0028] Example 3: Verification of the timing of adding protease 1. Preparation of Aspergillus oryzae Cultures To 500 mL of synthetic liquid medium with the same composition as used in Examples 1 and 2, 100 mL of enzyme solution containing 0.1% by mass of Protease P Amano 3SD (Amano Enzyme Inc.) was added before cultivation. This was used as the pre-culture addition group. In addition, 500 mL of synthetic liquid medium with the same composition was used, and the same enzyme solution was added after the completion of fungal cultivation. Furthermore, as a control group (no addition), 500 mL of synthetic liquid medium with the same composition was used for cultivation without enzyme addition. Aspergillus oryzae was inoculated into each liquid medium and cultured at 30°C with a maximum shaking speed of 135 rpm for 48 hours. The pH of the culture was adjusted to 6.5 using NaOH. Cultures from the pre-culture addition group and the control group were harvested 1 hour after pH adjustment. For the group containing the enzyme solution added after culture, after adjusting the pH, 100 mL of the enzyme solution was added, and the culture was shaken at 30°C and 135 rpm for 1 hour, and then the culture was harvested. Each harvested culture was subjected to yield evaluation.

[0029] 2. Evaluation of koji mold yield The solid matter (bacterial cells) was collected from the culture by filtration, and the wet weight was measured. The solid matter (bacterial cells) was then dried, and the dry weight was measured, and the moisture content was measured. The results are shown in Table 4. In addition, sensory evaluation was performed on each sample by the experimenter, and it was confirmed that off-tastes and off-odors were reduced in both the pre-culture addition group and the post-culture addition group. [Table 4] The addition of the enzyme did not decrease the yield of the cultured koji mold.

[0030] Example 4: Verification of the timing of adding protease 1. Preparation of Aspergillus oryzae Cultures Experiments were conducted using 450 mL of synthetic liquid medium with the same composition as used in Examples 1 to 3. For the pre-sterilization group, 50 mL of enzyme solution containing 0.1% by mass of Protease P Amano 3SD (Amano Enzyme Inc.) was added before sterilization and shaken for 1 hour. Afterwards, the medium was sterilized at 121°C for 15 minutes using an autoclave, and then inoculated. For the post-sterilization group (pre-culture group), the liquid medium was sterilized at 121°C for 15 minutes using an autoclave, and then 50 mL of enzyme solution containing 0.1% by mass of Protease P Amano 3SD (Amano Enzyme Inc.) was added, followed by inoculation. For the control group without addition, the liquid medium was sterilized and then inoculated. Shaking culture was conducted at 30°C and 135 rpm for 48 hours, and the cultures were harvested. The harvested cultures were subjected to yield evaluation.

[0031] 2. Evaluation of koji mold yield The solid matter (bacterial cells) was collected from the culture by filtration and the wet weight was measured. The solid matter (bacterial cells) was then dried and the dry weight was measured, and the moisture content was measured. The results are shown in Table 5. In addition, when the experimenter conducted a sensory evaluation of each sample, it was found that in the pre-sterilization addition group, the off-flavor was not suppressed due to the inactivation of the enzyme by sterilization, and instead an unusual sour taste and sour odor were exhibited. In the post-sterilization addition group, the off-flavor and off-odor were reduced. [Table 5] The post-sterilization group did not reduce the yield of the cultivated koji mold, whereas the pre-sterilization group reduced the yield.

[0032] Example 5: Verification of the timing of adding protease 1. Preparation of Aspergillus oryzae Cultures Experiments were conducted using 450 mL of synthetic liquid medium with the same composition as used in Examples 1 to 4. 50 mL of enzyme solution containing 0.1% by mass of Protease P Amano 3SD (Amano Enzyme Inc.) was added either once or all three times: before post-sterilization culture, after 24 hours of culture, and after 47 hours of culture. The pH was adjusted to 6 with NaOH before enzyme addition. For the three-time addition group, the enzyme concentrations at each addition were 0.04%, 0.04%, and 0.02% by mass. A control group was used without addition. The cultures harvested after 48 hours of culture were subjected to yield evaluation.

[0033] 2. Evaluation of koji mold yield The solid matter (bacterial cells) was collected from the culture by filtration, and the wet weight was measured. The solid matter (bacterial cells) was then dried, and the dry weight was measured, and the moisture content was measured. The results are shown in Table 6. [Table 6] The yield of the koji mold cultured was not reduced by the addition of the enzyme, and there was no difference in yield depending on the timing or number of times of addition.

[0034] 3. Evaluation of the foaming properties of the filtrate The filtrate after collecting the solid matter (bacterial cells) from the culture was collected and aerated at 1 vvm for 2 minutes to evaluate foamability using the same method as in Example 2. The foam height was measured every 10 seconds and plotted as a graph (Figure 2). The foam height was high in the control group (no addition) and the group added before culture (0 hours), but foaming was suppressed in the other groups.

[0035] Example 6: Verification of foaming properties in an industrial fermenter 1. Cultivation in a jar fermenter Experiments were conducted using 3 L of synthetic liquid medium with the same composition as used in Examples 1 to 5. At the start of cultivation, 30 mL of enzyme solution containing Protease P Amano 3SD (Amano Enzyme Inc.) was added. The enzyme solution was prepared to contain 0.05% and 0.15% by mass of enzyme relative to the medium. A control group was used without addition. The jar fermenter was equipped with an automatic antifoaming agent injection function, and when the foamed medium exceeded a predetermined height and touched the sensor, the antifoaming agent (Mitsubishi Chemical) was automatically injected. Cultivation was carried out for 48 hours at a maximum agitation speed of 600 rpm and 30°C. As a result, it was found that the addition of an antifoaming agent was unnecessary in the enzyme-added groups (both 0.05% and 0.15% by mass). In the non-added group, an antifoaming agent was added 18 hours after culture, and it was confirmed that it continued to be added appropriately depending on foaming.

[0036] Example 7: Taste verification in industrial fermentors 1. Cultivation in a jar fermenter The experiment was carried out using 3 L of synthetic liquid medium with the same composition as that used in Examples 1 to 5. The medium was cultured for 48 hours at 30°C with a maximum stirring speed of 600 rpm. After 48 hours of culture, 30 mL of an enzyme solution containing papain (Nagase ChemteX) was added. The enzyme solution was adjusted to a concentration of 0.1% by mass of enzyme relative to the medium. After the culture was completed, the enzyme reaction was carried out for 1 hour at 30°C.

[0037] 2. Sensory evaluation The cultures were subjected to sensory evaluation by four trained sensory evaluators. The results shown are for off-taste evaluation by four experts and for odor evaluation by three experts. A group without enzyme addition and a group without enzyme addition but with washed bacterial cells were used as controls. Off-taste and odor were evaluated on a 5-point scale from 1 to 5. The control group without enzyme addition was placed in the mouth, and the taste and odor were assigned a score of 5. The other enzyme-treated and washed groups were evaluated based on the following evaluation criteria in Table 7 for taste and Table 8 for odor. Evaluation criteria were used: a score of 4 or less was considered to indicate that the enzyme had reduced the off-taste or odor; and a score of 3 or less was considered to indicate that the off-taste or odor no longer interfered with eating. [Table 7] [Table 8] The results are shown in Tables 9 and 10 below: [Table 9] [Table 10] It was thought that the off-flavor and odor of cultured products could also be reduced by treating them with enzymes after cultivation. [Industrial Applicability]

[0038] The filamentous fungal cells produced by the production method of the present invention can be used as a food ingredient or can be added to food, and thus have industrial applicability. Because of its high protein content, it can also be used as a solution to future food problems. Furthermore, the method for reducing off-taste and / or off-odor of the present invention can suppress the off-taste and / or off-odor of filamentous fungal cells, thereby reducing the water washing step and being industrially useful.

Claims

1. Cultivating a filamentous fungus in a liquid medium containing a protease; and a step of recovering the grown bacterial cells and removing the liquid medium to recover the bacterial cells; A method for producing edible filamentous fungal cells, comprising:

2. The method according to claim 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. 2. 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 of claim 1, wherein the protease is a protease of EC.3.4.11 to EC.3.4.

25.

5. The method of claim 1 , wherein the protease has either endopeptidase or exopeptidase activity, or has both endopeptidase and exopeptidase activity, or includes both endopeptidase and exopeptidase activity.

6. 2. The method according to claim 1, wherein the protease is one or a combination of two or more selected from the group consisting of papain, pancreatin, trypsin, chymotrypsin, pepsin, cathepsin, elastase, subtilisin, streptoglycin, bromelain, zingibain, ficain, and actinidin.

7. The method according to claim 1, which does not include a washing step with water after the culture.

8. The method according to claim 1 , wherein the protease is added after the liquid medium has been subjected to a sterilization treatment.

9. The method according to claim 1, wherein the protease is contained in an amount of 0.001 to 10% by mass.

10. The method according to claim 1, wherein the protease has an activity temperature of 20°C to 50°C.

11. The method according to claim 1, wherein the protease has an activity of 10,000 U / g or more at the active temperature.

12. The method according to claim 1 , wherein the protease reduces the off-flavor of edible filamentous fungal cells.

13. The method of claim 1 , wherein the protease inhibits foam formation during incubation.

14. An edible filamentous fungus produced by the method according to any one of claims 1 to 13.

15. A food composition or food comprising the edible bacterial cells of claim 14.

16. A method for reducing off-taste and / or off-odor in a filamentous fungal culture, comprising culturing the filamentous fungus in a liquid medium containing a protease.

17. A method for defoaming a filamentous fungal culture, comprising culturing the filamentous fungus in a liquid medium containing a protease.

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