Bacillus natto with reduced viscous material production capability and extracellular protease activity, and food product using the same

Abstract

To provide a Bacillus natto strain that can be easily used for food processing.SOLUTION: A Bacillus natto strain with reduced viscous material production capability and extracellular protease activity, and a food product and a food product production method using the strain.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a bacterium called natto having reduced mucilage-producing ability and reduced extracellular protease activity, and to a food product using the same. [Background technology]

[0002] Natto, a fermented food produced using natto bacteria, is widely consumed in Japan. However, much of the natto produced is distributed and consumed as is, without further processing. One reason for the limited variety of natto-based processed foods is the difficulty of handling natto's mucilage during food processing. The stringiness that characterizes natto is due to its highly viscous mucilage. However, natto's mucilage can cause problems during food processing, such as clogging production lines and making cleaning difficult. Another reason for the limited variety of natto-based processed foods is that unheated natto products continue to undergo secondary fermentation after production, leading to excessive protein degradation by proteases, which can lead to tyrosine precipitation and bitterness. In particular, in semi-liquid natto products such as gelées, and the crushed natto often used in such semi-liquid products, the enzyme reaction proceeds more rapidly, making bitter compounds more likely to be produced from the soy protein by the action of proteases.

[0003] Therefore, in recent years, a natto bacillus that produces less viscous substances and has weak stringiness has been developed (Patent Document 1).

[0004] On the other hand, with regard to protease activity, efforts have been made to develop natto bacteria with enhanced protease activity and improved fermentation ability. Patent Document 2 discloses that a natto bacterium has been obtained that has high protease activity and produces a large amount of mucilage.

[0005] No natto bacteria with reduced mucilage production and reduced protease activity have been reported to date. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Laid-Open No. 2015-208319 [Patent Document 2] Japanese Patent Application Publication No. 3-254677 Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a natto strain that is easy to use in food processing. [Means for solving the problem]

[0008] As a result of extensive research to solve the above problems, the present inventors succeeded in obtaining a natto strain with reduced mucilage-producing ability and reduced extracellular protease activity, and discovered that the natto strain can be used to produce a fermented product that is easy to process into food, thereby completing the present invention.

[0009] That is, the present invention includes the following. [1] A natto strain with reduced mucilage production and extracellular protease activity. [2] The natto strain according to [1] above, which is the Bacillus subtilis natto KA-145LV3 strain having the accession number NITE P-03977. [3] The natto strain according to [1] above, which is the Bacillus subtilis natto KA-145LV7 strain having the accession number NITE P-03978. [4] The natto strain according to [1] above, which is the Bacillus subtilis natto KKA-LV2 strain having the accession number NITE P-03979. [5] The natto strain according to [1] above, which is the Bacillus subtilis natto KKA-LV8 strain having the accession number NITE P-03980. [6] A food fermented product using the natto strain described in any one of [1] to [5] above. [7] The fermented food material described in [6] above, wherein the food material contains soybeans. [8] A food product containing the fermented food material described in [6] or [7] above. [9] The food product according to [8] above, wherein the fermented food material is natto.

[10] The food product according to [9] above, wherein the fermented food material is whole natto or crushed natto.

[11] The food product according to [9] or

[10] above, which is dried natto.

[12] A method for producing a food product, comprising the step of producing a fermented food material using the natto strain described in any one of [1] to [5] above.

[13] The method according to

[12] above, wherein the fermented food material is natto.

[14] The method according to

[12] or

[13] above, further comprising a step of seasoning and / or drying the produced fermented food material. [Effects of the Invention]

[0010] According to the present invention, it is possible to provide a natto strain having reduced mucilage-producing ability and reduced extracellular protease activity, and further, by using this natto strain, it is possible to produce a fermented product that is easy to process into food, and to efficiently produce foods using the fermented product. [Brief explanation of the drawings]

[0011] [Figure 1] Figure 1 is a graph showing the tyrosine concentration in ground natto produced using the obtained natto strains during storage at 5°C. A: KA-145 strain and its mutants, KA-145LV3 and KA-145LV7. B: Kamiamakusa strain and its mutants, KKA-LV2 and KKA-LV8. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention will be described in detail below. The present invention relates to a natto strain having reduced mucilage-producing ability and reduced extracellular protease activity, a fermented food material such as natto using the strain, and a food product and food production method using the fermented food material.

[0013] The "reduction in ability to produce a mucilage" of the natto strain according to the present invention refers to the property that, in natto produced using the natto strain, the amount of mucilage produced is reduced compared to when a general natto bacterium is used, and the stringiness of the natto is clearly weak or not visible when visually observed.

[0014] The "reduction in extracellular protease activity" of the natto strain according to the present invention refers to the property that the extracellular protease activity in natto produced using the natto strain is clearly reduced, typically to 30% or less, compared to when a general natto strain is used. The extracellular protease activity in natto can be measured, for example, by a protease activity measurement method using azocasein as a substrate, according to the procedure described in Example 1 below.

[0015] Common natto bacteria to be compared include, but are not limited to, Miyagino bacteria (Miyagino Natto Seizosho, Ltd., Japan), one of the three major natto bacteria in Japan, or other natto bacteria strains having the same ability to produce mucilage and extracellular protease activity as Miyagino bacteria (e.g., derivative strains of Miyagino bacteria, such as the KA-145 strain, KR-823 strain, and Kamiamakusa strain described below).

[0016] Examples of the natto strain according to the present invention that has reduced mucilage-producing ability and reduced extracellular protease activity include the natto strains KA-145LV3, KA-145LV7, KKA-LV2, and KKA-LV8.

[0017] The natto (Bacillus subtilis natto) KA-145LV3 strain was deposited on September 11, 2023, at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary (NPMD) (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan) under the accession number NITE P-03977.

[0018] The natto (Bacillus subtilis natto) strain KA-145LV7 was deposited on September 11, 2023, at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary (NPMD) (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan) under the accession number NITE P-03978.

[0019] The natto (Bacillus subtilis natto) KKA-LV2 strain was deposited on September 11, 2023, at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary (NPMD) (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan) under the accession number NITE P-03979.

[0020] The natto (Bacillus subtilis natto) KKA-LV8 strain was deposited on September 11, 2023, at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary (NPMD) (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan) under the accession number NITE P-03980.

[0021] In the present invention, foods can be produced by fermenting food materials using the natto strain of the present invention (the above-mentioned natto strain with reduced mucilage-producing ability and extracellular protease activity). More specifically, in the present invention, a fermented food material can be produced by fermenting food materials using the natto strain of the present invention, and foods can be produced using the fermented food material. The present invention provides a fermented food material produced using the natto strain of the present invention (a fermented food material using the natto strain of the present invention / a fermented food material fermented using the natto strain of the present invention), and foods containing the fermented food material.

[0022] The fermented food material using the natto strain according to the present invention is a fermented product produced by fermenting a food material with the natto strain according to the present invention. The fermented food material using the natto strain according to the present invention is not particularly limited, but is more preferably a fermented soybean product produced by fermenting soybeans with the natto strain, and is particularly preferably natto. The natto may be any type of natto, for example, whole natto or crushed natto.

[0023] The fermented food product using the natto strain according to the present invention is characterized by low viscosity and low protease activity, which makes protein degradation less likely to occur. The low viscosity of the fermented food product makes it relatively easy to process. Furthermore, the low protease activity of the fermented food product makes it less likely to undergo further fermentation, making it suitable for long-term transportation and storage. Therefore, the fermented food product using the natto strain according to the present invention is suitable for food processing.

[0024] Foods containing a food material fermented using the natto strain of the present invention may be any type of food produced using the fermented food material. In one embodiment, foods containing a food material fermented using the natto strain of the present invention may be foods produced by processing the fermented food material. Foods containing a food material fermented using the natto strain of the present invention may be produced by, for example, packaging, packaging, molding, drying, crushing, pulverizing, powdering, pasting, pressurizing, refrigerating, aging, freezing, lyophilizing (e.g., vacuum freeze-drying), and / or freeze-dried powdering the fermented food material. Foods containing a food material fermented using the natto strain of the present invention may be produced by adding other microorganisms and / or ingredients (e.g., salt) to the fermented food material and further fermenting and / or aging the fermented food material. Foods containing a food material fermented using the natto strain of the present invention may be produced by mixing two or more of the fermented food materials. Foods containing a food material fermented using the natto strain of the present invention may be produced by adding, mixing, embedding, spreading, topping, and / or coating the fermented food material with other food materials, seasonings, and / or food additives. Foods containing the fermented food product using the natto strain of the present invention may be those in which the fermented food product is enveloped in other food ingredients, seasonings, etc., or those in which the fermented food product is added to, mixed with, embedded in, coated on, and / or topped with other food ingredients, seasonings, etc. Foods containing the fermented food product using the natto strain of the present invention may be those in which the fermented food product is seasoned with a seasoning liquid, seasoning, spices, and / or herbs, etc., or those produced using a fermented food product seasoned in this manner. Foods containing the fermented food product using the natto strain of the present invention may be fried, baked, steamed, stir-fried, simmered, or cooked foods such as tempura. Foods containing the fermented food product using the natto strain of the present invention may include those in which the fermented food product has been subjected to any secondary or further food processing, including but not limited to those described above.

[0025] Foods containing a food material fermented using the natto strain according to the present invention may further contain, in addition to the fermented food material, other food materials, seasonings, etc. Foods containing a food material fermented using the natto strain according to the present invention may also contain food additives such as carriers, excipients, stabilizers, preservatives, antioxidants, anti-adherents, disintegrants, binders, humectants, thickeners, buffers, surfactants, suspending agents, solubilizers, emulsifiers, coating agents, flavorings, fragrances, colorants, etc.

[0026] A food product containing a food material fermented using the natto strain according to the present invention may have any overall form, such as a liquid, semi-liquid (jelly, paste, etc.), or solid (powder, granules, crushed material, or a material that retains the shape of a food material such as beans, etc.).

[0027] Foods containing a food ingredient fermented using the natto strain of the present invention may be any type of food, such as prepared dishes, confectioneries, seasonings, sauces, beverages, soups, furikake (rice toppings), breads, noodles, fish paste products, or frozen foods. In one embodiment, a food containing a food ingredient fermented using the natto strain of the present invention is a food that uses natto as the food ingredient fermented using the natto strain of the present invention or is a food made by processing natto, i.e., a natto product. In one embodiment, a food containing a food ingredient fermented using the natto strain of the present invention includes at least one of whole natto and ground natto as the food ingredient fermented using the natto strain of the present invention. In one embodiment, a food containing a food ingredient fermented using the natto strain of the present invention is dried natto. In one embodiment, dried natto includes dried whole natto or ground natto. In the present invention, "dried natto" refers to natto that has been dried (for example, but not limited to, hot air drying, cold air drying, vacuum drying, freeze drying, vacuum freeze drying, spray drying, sun drying, or the like), and may have been further subjected to other food processing such as seasoning, as necessary. In the production of dried natto, high viscosity can easily hinder processing steps such as seasoning and drying natto. Therefore, it is highly advantageous to produce dried natto using the natto strain of the present invention, which has reduced mucilage-producing ability and extracellular protease activity. In another embodiment, a food product containing a food ingredient fermented using the natto strain of the present invention is jelly natto (sometimes referred to as "natto jelly"). In the present invention, "jelly natto" refers to natto mixed with jelly. Jelly natto is often used as, but is not limited to, nursing care food and hospital food. In the present invention, "jelly" refers to an edible liquid such as dashi, broth, soup, sauce, juice, fruit juice, vegetable juice, or seasoning liquid, solidified into a gel or thickened by adding a thickening stabilizer (gelling agent / coagulant, thickener, etc.) such as gelatin, agar, carrageenan, or pectin. In one embodiment, jelly natto contains crushed natto in a jelly. The fermented food product using the natto strain of the present invention has low viscosity and, due to its low protease activity, is less susceptible to protein degradation and therefore is highly useful, particularly for use in nursing care foods, hospital foods, etc.

[0028] Foods containing a food material fermented using the natto strain of the present invention may be functional foods such as foods for specified health uses, foods with functional claims, foods with nutrient function claims, and supplements. In the present invention, functional foods encompass all health foods, such as health foods to which a health claim based on the food standards of Codex Alimentarius (the Joint FAO / WHO Food Standards Commission) is applied. Foods containing a food material fermented using the natto strain of the present invention may also be nursing care foods, hospital foods, etc.

[0029] In a preferred embodiment, in a food fermented product using the natto strain of the present invention and a food containing the same, the increase in tyrosine concentration during storage is suppressed compared to when common natto bacteria (e.g., Miyagino bacteria, or the KA-145 strain or Kamiamakusa strain owned by Marumiya Co., Ltd.) are used. For example, at a temperature of 5°C or below, the increase in tyrosine concentration is preferably suppressed compared to when common natto bacteria are used for a storage period of 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 8 days or more, or 10 days or more, or at least up to 12 days, 14 days (2 weeks), or 15 days. In one embodiment, the suppression of the increase in tyrosine concentration is indicated by, but is not limited to, a decrease of 10% or more, preferably 20% or more, and more preferably 30% or more, of the tyrosine concentration compared to when common natto bacteria are used. As the tyrosine concentration increases, tyrosine becomes more likely to precipitate, resulting in a deterioration in texture. Therefore, the effect of suppressing the increase in tyrosine concentration by using the natto strain of the present invention is very useful in food production.

[0030] The present invention also provides a method for producing a food product, which includes the step of producing a fermented food material using the natto strain of the present invention. In the food production method of the present invention, a food material is fermented using the natto strain of the present invention, thereby producing a fermented food material.

[0031] The food material to be fermented using the natto strain of the present invention may be any food material (fermentation substrate) that can be fermented by the natto strain. The food material to be fermented may be, for example, an edible seed such as a bean, but is preferably soybean. The food material to be fermented may be a heated food material (for example, steamed, boiled, simmered, roasted, steam-heated, infrared-heated or direct-flame-heated, electromagnetic induction-heated or electrostatic induction-heated, etc.), for example, heated soybeans such as steamed soybeans, but is not limited thereto. The food material to be fermented may include at least one food material that is preferred for fermentation by the natto strain, such as soybeans.

[0032] In one embodiment, a food production method according to the present invention comprises the step of producing natto using the natto strain according to the present invention. More specifically, such a food production method may comprise the step of fermenting soybeans or a food material containing soybeans using the natto strain according to the present invention, thereby producing natto.

[0033] The food production method of the present invention may be a method for producing the above-mentioned food using the fermented food material produced as described above. In addition to the step of producing a fermented food material using the natto strain of the present invention, the food production method of the present invention may further include a step of processing the fermented food material obtained by the step using any method. The food production method of the present invention may further include a step of, for example, packaging, packaging, shaping, drying, crushing, pulverizing, powdering, pasting, pressing, refrigerating, aging, freezing, freeze-drying, and / or freeze-dried powdering the fermented food material. The food production method of the present invention may further include a step of adding other microorganisms and / or ingredients (e.g., salt) to the fermented food material and further fermenting and / or maturing. The food production method of the present invention may further include a step of mixing two or more of the fermented food materials. The food production method of the present invention may further include a step of adding, mixing, embedding, spreading, topping, and / or coating other food materials, seasonings, and / or food additives to the fermented food material. The food production method of the present invention may further include a step of enveloping the fermented food material with other food materials, seasonings, etc., or a step of adding, mixing, embedding, coating, and / or topping the fermented food material with other food materials, seasonings, etc. (e.g., jelly). In one embodiment, the food production method of the present invention may further include, for example, a step of seasoning and / or drying the fermented food material. The fermented food material may be seasoned using a seasoning liquid, seasonings, spices, herbs, etc. The fermented food material may be dried by hot air drying, cold air drying, vacuum drying, freeze drying, vacuum freeze drying, spray drying, sun drying, etc.

[0034] In the food production method according to the present invention, the fermented food material (e.g., natto) produced using the natto strain according to the present invention can be processed relatively easily, thereby improving the efficiency of the food production process. The food production method according to the present invention is advantageous in that by using the natto strain according to the present invention in food production, it is possible to produce foods that are comparable to conventional foods produced using general natto bacteria while realizing an efficient food production process.

[0035] The present invention also provides use of the natto strain according to the present invention in the production of a fermented food material (e.g., natto) using the natto strain or a food product containing the same, which exhibits low viscosity and low protease activity. [Example]

[0036] The present invention will be described in more detail below using examples, although the technical scope of the present invention is not limited to these examples.

[0037] [Example 1] Screening of natto strains Bacillus subtilis natto strains KA-145, KR-823, and Kamiamakusa (all owned by Marumiya Co., Ltd.) were cultured overnight in LB medium. 4 2-fold dilution or 10 5 After 2-fold dilution, 0.1 mL of the diluted solution was spread onto a GSP agar medium and cultured at 37°C overnight.

[0038] After cultivation, colonies that had decreased in the amount of mucilage produced and had become less sticky were visually selected as spontaneous mutants. The selected colonies were then harvested and further cultivated in LB medium.

[0039] The extracellular protease activity of cultures derived from selected colonies was measured using a protease activity assay with azocasein as a substrate. Specifically, 0.1 mL of culture supernatant separated from the culture by centrifugation (10,000 rpm, 5 min) was mixed with 0.5 mL of assay buffer (100 mM Tris-HCl (pH 8.0) + 0.5% azocasein) and incubated at 37°C for 30 min. The reaction was then stopped by adding 1.2 mL of 5% trichloroacetic acid. The reaction solution was centrifuged (10,000 rpm, 5 min), and the supernatant was collected. 1 mL of the resulting supernatant was mixed with 50 μL of sodium hydroxide solution, and the absorbance at 440 nm (A440 nm) was measured. The absorbance at 440 nm of the original (pre-mutation parent strains) KA-145, KR-823, and Kamiamakusa strains was also measured in the same manner. As a negative control, water was used instead of the culture supernatant, and the absorbance at 440 nm was measured in the same manner. The relative protease activity was calculated using the following formula:

[0040] Relative protease activity (%) = [measurement value (A440nm) of each strain culture - measurement value (A440nm) of negative control] / [measurement value (A440nm) of original strain - measurement value (A440nm) of negative control] × 100

[0041] The results are shown in Table 1. Strains with relative protease activity values ​​of 20% or less or 10% or less compared to the original strain were selected. Among the spontaneous mutants with reduced mucilage production derived from the KR-823 strain or the Kamiamakusa strain, most strains exhibited relative protease activity values ​​exceeding 100%, indicating increased protease activity compared to the original strain. In contrast, among the spontaneous mutants with reduced mucilage production derived from the KA-145 strain, most strains exhibited relative protease activity values ​​of 10% or less, and the remaining strains exhibited relative protease activity values ​​of 20% or less, indicating a significant decrease in protease activity compared to the original strain.

[0042] [Table 1]

[0043] Two mutant strains derived from the KA-145 strain (KA145-G3 and KA145-G7) and two mutant strains derived from the Kamiamakusa strain (Kamiamakusa-G2 and Kamiamakusa-G8), which showed more stable reductions in mucilage production and protease activity, were selected as natto strains with reduced mucilage production and extracellular protease activity. These pure culture strains were given new names and deposited at the National Institute of Technology and Evaluation's Patent Microorganisms Depositary (NPMD) (Table 2).

[0044] [Table 2]

[0045] Furthermore, when the absorbance at 440 nm (A440nm) of Miyagino strain (Miyagino Natto Seizosho, Ltd., Japan), a commonly used commercially available natto starter strain, was measured using the same method as above, the measured value was approximately 1. Therefore, the original strains KA-145, KR-823, and Kamiamakusa strain are considered to have protease activity at the same level as Miyagino strain.

[0046] [Example 2] Characterization of the deposited strain of Bacillus subtilis natto The four natto strains obtained and deposited in Example 1 were evaluated for their characteristics.

[0047] First, the natto strains were cultured in liquid medium to check for spore formation. The KA-145LV3, KA-145LV7, KKA-LV2, and KKA-LV8 strains showed good spore formation, comparable to that of their parent strains.

[0048] Furthermore, natto was produced using these four natto strains. Domestic soybeans were washed with water, then soaked in water for about 14 to 15 hours, drained, and then autoclaved at 121°C for about 20 minutes to prepare steamed soybeans. A spore suspension (1.0 × 10) of natto bacteria (Bacillus subtilis natto) KA-145LV3, KA-145LV7, KKA-LV2, or KKA-LV8 strain was added to the natto soybean extract. 5cfu / mL, diluted 6000-fold) was sprayed onto the steamed soybeans at a 2% concentration, mixed thoroughly, and then 40 g of the solution was weighed and placed into natto production containers (made of polystyrene). The openings of the containers were covered with a polyethylene film with small holes, the lids were closed, and the natto was fermented at 38°C for approximately 18 hours, and then aged in a refrigerator at 5°C or below for at least 24 hours to produce natto.

[0049] A sensory evaluation was conducted on the produced natto. The top and bottom surfaces of the natto in the container were inspected for appearance, such as color. The top surface was inspected after removing the polyethylene film. For the bottom surface, the container was rubbed from the outside to remove the natto from the container, then the container was turned upside down and the natto was placed on the lid of the container, and the appearance (color, etc.) of the bottom surface of the natto was inspected. Furthermore, for stickiness and stringiness, the natto was stirred 20 times with chopsticks, and then one-half to one-third of the natto in the container was picked up with the chopsticks and lifted about 40 cm to observe. The natto's aroma, bean hardness, and taste were also inspected.

[0050] The specific evaluation items were the following eight items, and each item was rated on a 5-point scale. 1) Bacteria coverage The white cotton-like substance that covers the surface of natto is a film of natto bacteria called "bacterial covering." If the beans are evenly and uniformly covered with natto bacteria (bacterial covering) and there are no plain beans (beans not covered with natto bacteria) or spotted covering, the bean is rated "good" (maximum of 5 points), while if the bacterial covering is extremely thin, such as when the natto bacteria covering is spotty or plain beans are found here and there, the bean is rated "bad" (minimum of 1 point). 2) Lytic state If the covering bacterial layer (bacterial film) is not found to have dissolved, it is rated as "good" (maximum of 5 points), and if the bacterial layer has dissolved and is found to be sticky, it is rated as "bad" (minimum of 1 point). 3) Cracks and crushing If there is very little or no cracking, crushing, or peeling of the beans, the bean is rated as "good" (maximum of 5 points), and if there is a lot of cracking, crushing, or peeling of the beans, the bean is rated as "bad" (minimum of 1 point). 4) Color If the beans are brown to light brown in color and have a vivid color, they are rated as "good" (maximum of 5 points), and if the beans are dark brown to black in color, they are rated as "bad" (minimum of 1 point). 5) Fragrance If the product has a good sweet smell and is judged to have an appropriate aroma for natto in terms of ammonia smell, burnt smell, sour smell, and unpleasant smells, it is rated as "good" (maximum of 5 points), and if the product has an aroma that is inappropriate for natto in terms of sweet smell, ammonia smell, burnt smell, sour smell, and unpleasant smells, it is rated as "bad" (minimum of 1 point). 6) Hardness If the beans have a smooth texture they are rated "good" (maximum of 5 points) and if they have a rough texture they are rated "bad" (minimum of 1 point). 7) Taste Judging from the viewpoints of umami, bitterness, sweetness, and off-flavors, if the taste is appropriate for natto, it is rated as "good" (maximum of 5 points), and if the taste is inappropriate for natto, it is rated as "bad" (minimum of 1 point). 8) Stringiness If the natto is very sticky and stringy when stirred, it is rated as "good" (maximum of 5 points), and if it is not very sticky and stringy, it is rated as "bad" (minimum of 1 point).

[0051] For comparison, sensory evaluation was also carried out on natto produced using Miyagino bacteria, a commonly used commercially available natto starter strain. The results of the sensory evaluation are shown in Table 3.

[0052] [Table 3]

[0053] All four deposited natto strains were shown to be non-sticky. Strains KA-145LV3 and KA-145LV7, which have lower extracellular protease activity, did not exhibit bitterness.

[0054] [Example 3] Production of dried natto using Bacillus subtilis natto with reduced mucilage production ability and reduced extracellular protease activity Domestically produced soybeans were washed with water, soaked in water for 16 to 20 hours, drained, and then steamed at a pressure of 98 to 147 kPa for 30 to 40 minutes (start to finish) to prepare steamed soybeans. A spore suspension (1 × 10) of the deposited Bacillus subtilis natto strain KA-145LV3, KA-145LV7, KKA-LV2, or KKA-LV8 was added. 5 cfu / mL, diluted 6000 times) was sprayed at approximately 2% of 2 kg of steamed soybeans and mixed well. The steamed soybeans were then spread on a plastic strainer lined with a polyethylene sheet with small holes and wrapped in the sheet. Fermentation was carried out at approximately 40°C for approximately 18 hours, and then the soybeans were aged in a refrigerator at 5°C or below for 1 to 5 days to produce natto.

[0055] The produced natto was seasoned by adding salt and monosodium glutamate, cooled in a refrigerator for 3-4 days, and then dried with hot air. The dried natto was stored at a low temperature of 15°C or below for at least one month. After aging for a certain period of time, the dried natto was seasoned by adding a seasoning liquid containing chili pepper extract, monosodium glutamate, and sodium 5'-ribonucleotide, and then coated with wheat flour and dried again. During this seasoning, the natto was easy to mix, and there was little stickiness or adhesion, making it easy to work with.

[0056] The taste, flavor, texture, and color of the dried natto produced in this manner were compared with commercially available dried natto (Korumame; Busoan Co., Ltd.) and subjected to a sensory evaluation. The results of the sensory evaluation of the dried natto produced using the KA-145LV3 strain are shown in Table 4. The dried natto produced using the KA-145LV7 strain, the KKA-LV2 strain, and the KKA-LV8 strain each showed similar sensory evaluation results to those shown in Table 4.

[0057] [Table 4]

[0058] The dried natto produced using the deposited natto strains with reduced mucilage production ability and extracellular protease activity showed sensory evaluation results (Table 4) that were comparable to commercially available dried natto.

[0059] Ordinary natto is very sticky, and when seasoned, the stickiness increases even more, making it difficult to mix. Furthermore, after drying, the sticky substance in the natto causes the beans to adhere tightly to each other. Therefore, when producing dried natto, it is necessary to loosen each grain of natto that has adhered after drying, which requires a great deal of labor. In contrast, when producing dried natto using the deposited natto strain, stirring during seasoning is easy, and the beans in the dried natto are less likely to adhere to each other, significantly improving the efficiency of dried natto production. It is highly advantageous to be able to produce dried natto that exhibits the same sensory evaluation as conventional dried natto while streamlining the production process.

[0060] [Example 4] Measurement of tyrosine concentration in natto The tyrosine concentration in natto produced using the Bacillus subtilis natto strains KA-145LV3, KA-145LV7, KKA-LV2, and KKA-LV8, as well as their parent strains, the KA-145 strain and the Kamiamakusa strain, was measured.

[0061] Specifically, ground natto was divided into small polyethylene pouches and frozen. The frozen natto pouches were then stored (refrigerated) at 5°C for 1 to 15 days, and one pouch was removed at 1, 2, 3, 4, 5, 8, 10, 12, and 15 days after the start of storage at 5°C. Five grams of natto was removed from each pouch, mixed with 5 mL of sterilized water, shaken for 5 minutes, and then centrifuged at 8000 rpm for 10 minutes. The tyrosine concentration in the supernatant after centrifugation was measured using an amino acid analyzer JLC-500 / V2 (manufactured by JEOL Ltd.).

[0062] The results are shown in Figure 1. The mutant strains KA-145LV3 and KA-145LV7 exhibited lower tyrosine concentrations than their parent strain, KA-145, for approximately two weeks after storage at 5°C. Similarly, the mutant strains KKA-LV2 and KKA-LV8 exhibited lower tyrosine concentrations than their parent strain, Kamiamakusa, for more than two weeks after storage at 5°C.

[0063] Tyrosine is produced by the decomposition of proteins by the action of proteases during the fermentation process of natto, and as the tyrosine concentration increases, tyrosine tends to precipitate. Since tyrosine precipitation deteriorates the texture, natto bacteria that can reduce the tyrosine concentration can be suitably used for the production of natto.

Claims

1. A natto strain with reduced mucilage production ability and extracellular protease activity.

2. The natto strain according to claim 1, which is a Bacillus subtilis natto KA-145LV3 strain having the accession number NITE P-03977.

3. The natto strain according to claim 1, which is a Bacillus subtilis natto KA-145LV7 strain having the accession number NITE P-03978.

4. The natto strain according to claim 1, which is a Bacillus subtilis natto KKA-LV2 strain having the accession number NITE P-03979.

5. The natto strain according to claim 1, which is a Bacillus subtilis natto KKA-LV8 strain having the accession number NITE P-03980.

6. A food fermented product using the natto strain according to any one of claims 1 to 5.

7. The fermented food material of claim 6 , wherein the food material comprises soybeans.

8. A food product comprising the fermented food material according to claim 6.

9. The food product according to claim 8 , wherein the fermented food material is natto.

10. The food product according to claim 9, wherein the fermented food material is whole natto or crushed natto.

11. The food product according to claim 9, which is dried natto.

12. A method for producing a food product, comprising a step of producing a fermented food material using the natto strain according to any one of claims 1 to 5.

13. 13. The method of claim 12, wherein the fermented food material is natto.

14. The method of claim 12, further comprising the step of seasoning and / or drying the food material fermentate produced.

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