Soy sauce with reduced heat-resistant protease activity

Abstract

To solve the problem of deterioration in quality in terms of color, taste and aroma components in a conventionally known method for producing soy sauce with low thermostable protease activity, and to obtain soy sauce of good quality despite having low thermostable protease activity. [Solution] By producing soy sauce using the koji mold mutant strain obtained by the method of the present invention, it is possible to obtain soy sauce with new properties that have not previously existed, such as sufficiently low heat-resistant protease activity and no deterioration in quality, such as taste and aroma components.

Description

[Technical Field]

[0001] The present invention relates to soy sauce with reduced thermostable protease activity, a method for producing the same, and a method for producing koji mold with low thermostable protease activity that can be used to produce soy sauce with reduced thermostable protease activity. [Background technology]

[0002] Soy sauce is used in a variety of processed foods due to its excellent flavor and versatility, but when used in the exceptional cases of fish paste products or boiled eggs, it can impair the texture and reduce the commercial value. This is thought to be due to the heat-resistant proteases produced during the soy sauce manufacturing process by Aspergillus oryzae and Aspergillus sojae, which are koji molds used in soy sauce brewing, remaining and acting in the finished soy sauce (Patent Document 1, Patent Document 2).

[0003] Conventionally, a method for removing heat-resistant proteases by membrane filtration using an ultrafiltration membrane or a reverse osmosis membrane has been known as a method for obtaining soy sauce with low heat-resistant protease activity (Patent Document 1, Patent Document 2).

[0004] To remove heat-resistant proteases by membrane filtration, it is necessary to remove fractions with a molecular weight cutoff of greater than 10,000 (Patent Document 2). However, since some components in soy sauce with a molecular weight greater than 10,000 have a significant effect on the taste (Non-Patent Document 1), such filtration may result in a significant deterioration in quality.

[0005] In addition, a method for obtaining soy sauce with low thermostable protease activity is known in which thermostable proteases are autolyzed by prolonged heat treatment (Patent Document 2), but excessive heating is not preferred because it significantly affects the aroma components and color of soy sauce. For example, it is known that the lower the content of 2-acetylpyrrole, one of the aroma components that increases with heating, the better the sensory quality (Non-Patent Document 2).

[0006] That is, although there have been methods for obtaining soy sauce with low thermostable protease activity, all of these methods inevitably result in deterioration of the quality of the soy sauce. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Application Publication No. 04-016162 [Patent Document 2] Japanese Patent Application Publication No. 02-131554 [Non-patent literature]

[0008] [Non-Patent Document 1] Imamura et al., Journal of the Brewing Society of Japan, 110(5), 318-326, 2015 [Non-patent document 2] Kikuchi et al., Fukushima Prefectural High-Tech Plaza Research Report 2023, 2023 Summary of the Invention [Problem to be solved by the invention]

[0009] Therefore, an object of the present invention is to solve the problem of deterioration in quality in terms of color, taste, and aroma components that occurs in conventionally known methods for producing soy sauce with low thermostable protease activity, and to obtain soy sauce of good quality despite having low thermostable protease activity. [Means for solving the problem]

[0010] One ideal method for producing soy sauce that does not have heat-resistant protease activity would be to select a mutant strain of koji mold that does not secrete the heat-resistant protease responsible for the soy sauce and use it to produce the soy sauce.

[0011] However, it has not been clear how many genes contributing to thermostable protease activity are present in the koji mold genome used in soy sauce brewing. Therefore, if multiple genes contributing to thermostable protease activity are present in the koji mold genome, it is expected that a huge number of strains would need to be examined to obtain a mutant with low thermostable protease activity by mutant selection. However, the actual situation, including the number of strains required for screening, has not been fully investigated.

[0012] Furthermore, a known method for measuring heat-stable protease activity involves reacting an enzyme solution with a substrate and then quantifying the liberated amino acids using ninhydrin (Patent Documents 1 and 2). However, because the free amino acids contained in the enzyme solution must be removed in advance by dialysis or other methods, this method of measuring heat-stable protease activity and obtaining strains with low activity does not have a high throughput and is therefore not applicable to examining many bacterial strains.

[0013] For these reasons, there have been no known cases in which methods for obtaining koji mold strains with reduced thermostable protease activity by selecting mutant strains or the like have been investigated, nor have there been any known cases in which mutant strains with reduced thermostable protease activity have actually been obtained.

[0014] Therefore, the present inventors have conducted extensive research and have found that (Step 1) growing koji mold on a solid medium and extracting a crude enzyme solution from the culture; (Step 2) heating the obtained crude enzyme solution to inactivate heat-resistant proteases; (Step 3) reacting the heated crude enzyme extract with an antibacterial enzyme substrate; (Step 4) culturing a microorganism whose growth is inhibited by the antibacterial activity derived from the enzyme substrate in a medium containing the reaction solution, and detecting a change in the growth of the microorganism; (Step 5) A step of selecting koji mold strains that exhibit poor growth of the microorganism as candidate mutant strains with reduced thermostable protease activity. It has been revealed that the method comprising the steps of: (1) makes it possible to easily evaluate the heat-resistant protease activity of koji mold based on the growth of the microorganism; and (2) makes it possible to efficiently obtain koji mold mutant strains that do not secrete heat-resistant proteases.

[0015] The present inventors have found that by producing soy sauce using the koji mold mutant strain obtained by the above method, it is possible to obtain soy sauce with new properties that have not previously existed, namely, sufficiently low heat-resistant protease activity and no deterioration in quality, such as taste and aroma components, and have thus completed the present invention. [Effects of the Invention]

[0016] The soy sauce of the present invention has a sufficiently low heat-resistant protease activity of 1.5 nkat / mL or less, and has excellent properties in terms of suitability for processing and cooking, such as not causing deterioration of ingredients when used in processing fish paste products, boiled eggs, etc. Furthermore, the soy sauce contains 1% or more by dry weight of a fraction with a molecular weight of 13,000 or more, which affects the taste of soy sauce, and the content of 2-acetylpyrrole, a component known to be formed by prolonged heating of soy sauce and to reduce sensory quality, is 5 ppm or less. Therefore, this soy sauce has novel properties that do not suffer from any deterioration in quality, as seen in soy sauce with reduced heat-resistant protease activity obtained by conventional manufacturing methods.

[0017] Furthermore, the soy sauce production method of the present invention can produce soy sauce having the above-mentioned properties by a conventional soy sauce production method, except for using a koji mold strain having a thermostable protease activity of 8 nkat / g or less for koji production. In other words, it is possible to produce soy sauce having the desired properties by a conventional method without using processes that require equipment and energy, such as membrane treatment or prolonged heating.

[0018] Furthermore, the method of the present invention for selecting koji mold with low thermostable protease activity has excellent throughput and enables efficient discrimination of the protease activity of a large number of koji mold strains, thereby making it possible to select koji mold strains with low thermostable protease activity that have never been obtained before. [Brief explanation of the drawings]

[0019] [Figure 1] FIG. 1 shows the heat-resistant protease activity of the parent strain of koji mold and the mutant strain of koji mold obtained from the parent strain by the method of the present invention. [Figure 2] FIG. 2 shows the maximum load generated when a probe of a texture analyzer was inserted into a piece of kamaboko immersed in the soy sauce of the present invention and the control liquid, until the probe was completely penetrated. [Figure 3] FIG. 3 shows the maximum load generated when a probe of a texture analyzer was inserted into kamaboko pieces after the pieces were soaked in the soy sauce of the present invention and commercially available soy sauce, until the probe completely penetrated the pieces. DETAILED DESCRIPTION OF THE INVENTION

[0020] <1> soy sauce The soy sauce of the present invention is (1) The heat-resistant protease activity is 1.5 nkat / mL or less, (2) Contains a fraction with a molecular weight of 13,000 or more at a dry weight ratio of 1% or more, and (3) The content of 2-acetylpyrrole is 5 ppm or less. It is characterized by:

[0021] The soy sauce of the present invention is not particularly limited as long as it is soy sauce produced by a conventional method, but is preferably pasteurized soy sauce that includes a pasteurization step in the production process to eliminate the activity of various enzymes such as proteases, including but not limited to heat-resistant proteases. Examples of types of soy sauce include dark soy sauce, light soy sauce, tamari soy sauce, and re-brewed soy sauce.

[0022] (1) Heat-stable protease activity The soy sauce of the present invention has a heat-resistant protease activity of 1.5 nkat / mL or less, which may be 1.4 nkat / mL or less, 1.2 nkat / mL or less, 1.1 nkat / mL or less, 1.0 nkat / mL or less, 0.9 nkat / mL or less, or 0.8 nkat / mL or less, or may have no heat-resistant protease activity at all.

[0023] In the present invention, the heat-resistant protease activity in a sample refers to a value calculated by the following method. In particular, when the target sample is soy sauce, it refers to the protease activity per mL of soy sauce.

[0024] (Method for measuring heat-resistant protease activity) The sample is placed in a 13,000 molecular weight dialysis membrane and dialyzed against 0.1 M sodium phosphate buffer (pH 7.0), then transferred to a 1.5 mL tube and heated in boiling water for 10 minutes. The heated solution is used as the crude enzyme solution, and 1% protamine sulfate is added as a substrate. The mixture is incubated at 30°C for 30 minutes, and the amino acids liberated in the reaction solution are measured by the ninhydrin method.

[0025] The enzyme unit is calculated as 1 enzyme unit = 1 kat (1 kat), which is the amount of enzyme that produces ninhydrin color-reactive substance equivalent to 1 mole of L-arginine from the substrate per second using the above measurement method, and is expressed as enzyme activity (kat / mL).

[0026] (2) Fraction with a molecular weight of 13,000 or more Next, the soy sauce of the present invention contains 1% or more by dry weight of a fraction having a molecular weight of 13,000 or more. The content of the fraction having a molecular weight of 13,000 or more by dry weight may be 1.3% or more, 1.5% or more, 1.7% or more, 2.0% or more, 2.3% or more, 2.5% or more, 2.7% or more, or 3.0% or more.

[0027] The content of fractions having a molecular weight of 13,000 or more can be measured by the following steps (1) and (2).

[0028] (Step 1) Molecular weight fractionation 10 mL of the soy sauce sample is dialyzed overnight at 4°C in deionized water using a cellulose dialysis membrane with a molecular weight of 13,000 to remove low molecular weight fractions.

[0029] (Step 2) Measuring the dry weight The soy sauce sample from which the low molecular weight fraction has been removed by molecular weight fractionation is freeze-dried and the dry weight is measured. The soy sauce sample before molecular weight fractionation is also freeze-dried and the dry weight is measured. The dry weight ratio of the fraction with a molecular weight of 13,000 or more is calculated from the dry weight of the soy sauce sample before molecular weight fractionation and the dry weight after molecular weight fractionation.

[0030] (3) 2-acetylpyrrole content Furthermore, the soy sauce of the present invention has a 2-acetylpyrrole content of 5.0 ppm or less, which may be 3.5 ppm or less, 3.0 ppm or less, 2.5 ppm or less, 2.0 ppm or less, 1.5 ppm or less, or 1.0 ppm or less.

[0031] The amount of 2-acetylpyrrole can be determined by the following method.

[0032] (Quantitative method for acetylpyrrole) 6 mL of soy sauce sample is mixed with 3 mL of dichloromethane as an organic solvent to extract the aroma components into the solvent. The solvent layer is then separated by centrifugation and subjected to GC-MS analysis.

[0033] (GC-MS analysis conditions) Quantitative analysis using GC-MS is performed by the standard addition method in SIM mode under the following analytical conditions: Measurement device: 7890B-5977B MSD (Agilent Technologies) Column: DB-WAX (length 60 m, diameter 0.25 mm, film thickness 0.25 μm) (Agilent Technologies) Injection volume: 3μL Temperature conditions: 40°C (10 min) → Heat up to 230°C at 5°C / min → Hold for 12 min Carrier: High-purity helium, 161.34kPa Transfer temperature: 250℃ Ion source temperature: 230℃ Ionization method: EI Target ion: m / z 94 Qualifier ion: m / z 109

[0034] <2> Soy sauce production method The soy sauce of the present invention may be produced by any method as long as the soy sauce to be produced satisfies the following conditions: (1) the heat-stable protease activity is 1.5 nkat / mL or less, (2) the fraction with a molecular weight of 13,000 or more is contained in an amount of 1% or more by dry weight, and (3) the 2-acetylpyrrole content is 5 ppm or less.

[0035] The present invention also provides a new method for producing soy sauce. Specifically, the present invention relates to a method for producing soy sauce that (1) has a thermostable protease activity of 1.5 nkat / mL or less, (2) contains a fraction with a molecular weight of 13,000 or more at 1% or more by dry weight, and (3) has a 2-acetylpyrrole content of 5 ppm or less, by using a koji mold strain with a thermostable protease activity of 8 nkat / g or less in the koji production process.

[0036] In the soy sauce production method of the present invention, soy sauce may be produced by a conventional method, except that a koji mold strain having a heat-resistant protease activity of 8 nkat / g or less is used in the koji production step. For example, the production method of dark soy sauce may include the following steps 1 to 4.

[0037] (Step 1) Steamed soybeans and roasted and crushed wheat are mixed, and seed koji (containing koji mold) is added and cultured for 1 to 5 days to obtain koji (koji production process). (Step 2) The resulting koji is mixed with salt water and fermented and aged for 3 to 24 months to obtain the moromi. (Step 3) The matured moromi is pressed to separate the solid and liquid, and the liquid portion (raw soy sauce) is obtained. (Step 4) The raw soy sauce is heated (pasteurized) at 68-130°C for less than 3 hours (but not including 0 hours) to obtain dark soy sauce.

[0038] To obtain the soy sauce of the present invention, a koji mold strain having a heat-resistant protease activity of 8 nkat / g or less can be used as the koji mold contained in the seed koji in step 1. The koji mold used may have a heat-resistant protease activity of 8 nkat / g or less, 6 nkat / g or less, 4 nkat / g or less, or 2 nkat / g or less. A koji mold strain having such properties is, for example, <3> It can be obtained by the method described in the paragraph.

[0039] The koji mold strain used in the soy sauce production method of the present invention may have a heat-resistant protease activity of 8 nkat / g or less. However, it is preferable that the koji mold strain also has sufficient non-heat-resistant protease activity in order to adequately decompose proteins derived from raw materials. Specifically, when activity is measured according to the total protease activity measurement method described in "Soy Sauce Testing Methods" edited by the Japan Soy Sauce Research Institute, it is preferable that the total protease activity of the koji mold strain retains 60%, 70%, or 80% or more of that of the parent strain.

[0040] The soy sauce of the present invention is such that the final soy sauce is <1> As long as the conditions described in the paragraph are met, the soy sauce may be a mixture of the soy sauce obtained by the above-mentioned soy sauce production method and soy sauce produced by other production methods, water, salt, edible alcohol, etc.

[0041] The type of koji mold to be used may be any strain that can be used for soy sauce brewing, such as Aspergillus sojae (soy sauce koji mold) or Aspergillus oryzae (yellow koji mold).

[0042] In the present invention, the heat-resistant protease activity in a sample refers to the protease activity per gram of the solid culture when the sample is a solid culture of koji mold. <1> The measurement can be performed by the measurement method described in the item 1., and the sample for measurement can be prepared by the following method.

[0043] (Sample preparation method) 7 ml of water is added to 5 g of defatted soybeans, and 5 g of crushed wheat is mixed thoroughly. After that, the solid medium is autoclaved at 121°C for 40 minutes, and then koji mold is seeded on the medium and cultured for 4 days at 30°C. 50 ml of deionized water is added to the resulting solid culture, and the mixture is stirred well. After leaving to stand overnight at 5°C, the mixture is filtered through a filter paper to prepare a measurement sample.

[0044] In the soy sauce manufacturing method of the present invention, it is preferable that the method does not include a step of filtering the brewed soy sauce through an ultrafiltration membrane, a reverse osmosis membrane, or the like, or a step of heating the brewed soy sauce for a long period of time, specifically, for 3 hours or more, 4 hours or more, 5 hours or more, or 6 hours or more, in order to prevent deterioration of the quality of the soy sauce.

[0045] <3> How to obtain koji mold The present invention provides a method for obtaining koji mold having low thermostable protease activity. (Step 1) growing koji mold on a solid medium and extracting a crude enzyme solution from the culture; (Step 2) heating the obtained crude enzyme solution to inactivate heat-resistant proteases; (Step 3) reacting the heated crude enzyme extract with an antibacterial enzyme substrate; (Step 4) culturing a microorganism whose growth is inhibited by the antibacterial activity derived from the enzyme substrate in a medium containing the reaction solution, and detecting a change in the growth of the microorganism; (Step 5) A step of selecting koji mold strains that exhibit poor microbial growth as candidate mutant strains with reduced thermostable protease activity. The present invention relates to a method for obtaining a koji mold having low thermostable protease activity, which comprises:

[0046] As used herein, "koji mold" refers to a filamentous fungus that is a microorganism classified in the genus Aspergillus and has a proven track record of being used in the production of fermented foods and beverages such as sake, shochu, miso, soy sauce, mirin, amazake, etc. Therefore, koji mold in this specification can include Aspergillus oryzae (yellow koji mold), Aspergillus sojae (soy sauce koji mold), Aspergillus luchuensis (black koji mold), and Aspergillus luchuensis mut. kawachii (white koji mold), with A. oryzae or A. sojae being preferred.

[0047] (Step 1) Growing koji mold on a solid medium and extracting a crude enzyme solution from the culture It is known that koji mold does not produce sufficient amounts of proteases, including heat-stable proteases, unless it is grown on a solid medium. Therefore, in step 1, the koji mold (parent strain) to be selected is grown on a solid medium, and a crude enzyme solution is extracted from the resulting culture.

[0048] There are no particular limitations on the koji mold strain to be used for selection; it may be selected from naturally occurring koji mold strains, commercially available koji mold strains, or any koji mold strain that has been subjected to genetic mutation treatment.

[0049] When a strain that has been subjected to genetic mutation treatment is subjected to selection, the genetic mutation treatment method may be any known method, including physical mutation treatment using ultraviolet light or radiation, or chemical mutation treatment using N-methyl-N'-nitro-N-nitrosoguanidine (NTG) or ethyl methanesulfonate (EMS).

[0050] There are no particular limitations on the raw materials for the solid medium, but raw materials used in actual brewing are preferred because they allow for accurate evaluation of the characteristics when applied to actual brewing. Specific examples include beans, rice, wheat, and potatoes, with soybeans, rice, and wheat being more preferred. One type of raw material may be used, or multiple types may be used.

[0051] To grow koji mold on a solid medium, for example, a parent strain of koji mold is seeded on the medium and cultured for 24 to 240 hours under conditions of a temperature of 20 to 40°C and a humidity of 85 to 100%.

[0052] Next, a crude enzyme solution is extracted from the solid culture by, for example, ultrasonic treatment in a heated, very dilute surfactant for 1 to 10 minutes.

[0053] (Step 2) Heating the crude enzyme solution to inactivate the heat-resistant protease Step 2 is a step of removing the activity of heat-resistant proteases from the crude enzyme extract obtained in step 1. An example of a method for removing the activity of heat-resistant proteases is a method of heating at a temperature of 70 to 130°C for a heating time of less than 3 hours (but not including 0 minutes).

[0054] (Step 3) A step of reacting the heated crude enzyme extract with an enzyme substrate having antibacterial properties (Step 4) A step of culturing a microorganism whose growth is inhibited by the antibacterial activity derived from the enzyme substrate in a medium containing the reaction solution, and detecting a change in the growth of the microorganism. (Step 5) A step of selecting koji mold strains that exhibit poor microbial growth as candidate mutant strains with reduced thermostable protease activity. Step 3 is a step of reacting a crude enzyme extract from which the activity of heat-insensitive proteases has been removed by heating with a heat-stable protease substrate having antibacterial activity, and step 4 includes a step of mixing a reaction solution obtained by reacting the heat-treated crude enzyme extract with the substrate having antibacterial activity with microorganisms, culturing the microorganisms in the reaction solution, and detecting changes in the growth of the microorganisms.

[0055] In steps 3 and 4, the degradation of the antibacterial substrate by the heat-resistant protease is detected by the growth of a separately mixed microorganism.

[0056] In step 5, strains showing poor growth based on the detection of microbial growth in step 4 are selected as candidates for strains with reduced thermostable protease activity.

[0057] That is, if the koji mold strain to be evaluated has heat-resistant protease activity and the crude enzyme extract after heating also has heat-resistant protease activity, the antibacterial substrate will be decomposed in step 3, allowing the microorganisms separately mixed in the culture step in step 4 to grow sufficiently. On the other hand, if the koji mold strain to be evaluated does not have or has reduced heat-resistant protease activity, the crude enzyme extract will also not have heat-resistant protease activity, and the antibacterial substrate will remain without being decomposed, resulting in reduced microbial growth due to the antibacterial effect.

[0058] Therefore, by selecting strains that do not grow well, it is possible to obtain koji mold strains that have no or reduced thermostable protease activity.

[0059] In the method of the present invention, any combination of substrate and microorganism having antibacterial properties that can be used in the above-mentioned mechanism can be used.

[0060] As the substrate having antibacterial activity, a protein or polypeptide having antibacterial activity can be used, and specific examples of such types include lysozyme, protamine, histone, etc. When protamine is used, it is preferable to use protamines extracted from fish milt, such as milt, milt protein, milt protein extract, and milt hydrolysate. Among these, salmine, a protamine derived from salmon, is more preferable.

[0061] The conditions for reacting the heat-treated crude enzyme extract with protamine include, for example, a temperature of 15 to 40°C for about 0.5 to 15 days.

[0062] The microorganisms used for detection can be those whose growth is inhibited by a protein substrate having antibacterial activity and whose growth level can be easily detected. The type of microorganism is not particularly limited, but suitable examples include microorganisms that produce organic acids to change the pH of the culture solution, and microorganisms that express fluorescent proteins or luminescent proteins. Examples of microorganisms that change the pH through growth include butyric acid bacteria, acetic acid bacteria, and lactic acid bacteria. As lactic acid bacteria, lactic acid bacteria of the genus Latilactobacillus are more preferred.

[0063] When culturing the crude enzyme extract and the microorganism, a medium suitable for culturing the microorganism may be further added. After mixing the crude enzyme extract and the microorganism, the culture may be carried out under culture conditions depending on the type of microorganism, for example, at a temperature of 15 to 40°C for 3 to 72 hours.

[0064] After the cultivation, changes in the growth of the microorganisms are detected. The detection method may be selected according to the properties of the microorganisms used. For example, if microorganisms expressing fluorescent proteins or luminescent proteins are used, the intensity of fluorescence or luminescence may be measured.

[0065] Furthermore, when a microorganism that changes the pH, such as lactic acid bacteria, is used as the microorganism, an example method is to add a pH indicator such as bromocresol purple to the culture medium and visually detect the pH change after cultivation using the pH indicator.

[0066] After detection, koji mold strains that result in poor growth of the microorganism are selected as candidates for mutant strains with reduced thermostable protease activity.

[0067] When a microorganism expressing a fluorescent protein or a luminescent protein is used as the microorganism, if the koji mold strain has heat-stable protease activity, the protein substrate with antibacterial activity, such as salmine, is degraded by the heat-stable protease, resulting in the loss of antibacterial activity. Therefore, the microorganism can grow sufficiently, and fluorescence and luminescence can be detected sufficiently. On the other hand, if the koji mold strain has reduced heat-stable protease activity, the protein substrate with antibacterial activity is not degraded, so the antibacterial activity is maintained, the microorganism cannot grow, and fluorescence and luminescence are not observed or are significantly reduced. Therefore, by measuring and detecting the intensity of fluorescence and luminescence, candidate strains with reduced heat-stable protease activity can be easily selected.

[0068] When a microorganism that changes the pH, such as lactic acid bacteria, is used as the microorganism, if the koji mold strain has heat-resistant protease activity, the heat-resistant protease will decompose antibacterial protein substrates, such as salmine, resulting in a loss of antibacterial activity. Therefore, the lactic acid bacteria can grow sufficiently, and the pH of the culture medium changes to an acidic state due to the lactic acid produced. On the other hand, if the koji mold strain has reduced heat-resistant protease activity, the antibacterial protein substrates will not be decomposed, maintaining the antibacterial activity, preventing the lactic acid bacteria from growing, and the pH will not change. By detecting the presence or absence of this pH change based on the color change of a pH indicator, such as bromocresol purple, candidate strains with reduced heat-resistant protease activity can be easily selected.

[0069] After selecting candidate strains with reduced thermostable protease activity through steps 4 and 5 above, the thermostable protease activity of the selected strains can be measured by the method described above to obtain koji molds with reduced thermostable protease activity. [Example]

[0070] The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[0071] Example 1: Selection of koji mold with low thermostable protease activity using protamine We investigated whether it is possible to select koji mold with low heat-resistant protease activity using an experimental system using protamine.

[0072] (gene mutation treatment) A koji mold strain (Aspergillus oryzae, hereafter referred to as the "parent strain") owned by Yamasa Shoyu Co., Ltd. was irradiated with UV light to induce genetic mutations. Specifically, koji mold spores suspended in a 0.05% Tween solution were exposed to UV light with gentle stirring to achieve a survival rate of approximately 0.1-1.0%. The spores were then inoculated onto malt medium (Malt Extract Broth (Becton Dickinson) 1.5%, Triton X-100 0.25%, agar 1.5%, pH 6.0) and cultured at 30°C for 4-5 days to allow colony formation.

[0073] (solid culture) Equal amounts of defatted soybeans and wheat were mixed and powdered in a mixer. Approximately 0.1 g of the powder was then placed in a 96-well deep well plate, allowed to absorb 50 μl of deionized water, and sterilized in an autoclave at 121°C for 40 minutes to prepare the medium. After the mutation treatment, the koji mold colonies were scraped off with a sterilized cotton swab, inoculated onto solid medium, and cultured at 30°C and 98% relative humidity for 3 to 4 days.

[0074] (Preparation of replica plates) After solid culture, the mutant koji mold was replicated onto malt medium using a sterilized bamboo skewer from the deep-well plate.

[0075] (Extraction of crude enzyme extract) After the solid culture, 200 μl of a heated 0.005% Tween solution was added to the deep well plate, which was then sonicated for 5 minutes and then allowed to stand at 4° C. overnight.

[0076] (Reaction of crude enzyme extract with substrate) 25 μL of the crude enzyme extract was transferred to a sterilized PCR tube and heated in a thermal cycler at 99°C for 15 minutes. After heating, 25 μL of 0.1% protamine sulfate (salmon-derived) solution was added, and the mixture was allowed to react at 30°C for 1 week.

[0077] (detection) To the reaction mixture, 200 μL of 4x BCP medium (6.25% glucose, 0.625% bacteriologic meat extract, 0.625% polypeptone, 0.625% yeast extract, 0.125% mercaptoacetic acid, 0.0125% bromocresol purple, pH 8.0) was added. Furthermore, 20 mL of culture medium of Latilactobacillus sakei NBRC 15893 strain, which had been cultured overnight at 30°C in MRS medium, was collected by centrifugation, washed thoroughly with sterile water, and finally suspended in 40 mL of sterile water. 10 μL of this suspension was added to the reaction mixture, mixed well, and cultured overnight at 30°C.

[0078] (Isolation of strains) After cultivation, the reaction solution was selected to remain blue in color, and the strains replicated from the solid medium used to prepare the crude enzyme extract added to the reaction solution were selected as strains with low heat-resistant protease activity.

[0079] (Confirmation of heat-resistant protease activity) Approximately 20,000 UV-induced mutagenesis strains were screened, and four strains with blue reaction mixtures were selected. These were then inoculated onto solid medium prepared by adding 5 g of defatted soybeans, 7 mL of water, and 5 g of crushed wheat, followed by thorough mixing. The mixture was then autoclaved at 121°C for 40 minutes and cultured at 30°C for 4 days. Each culture was mixed with 50 mL of deionized water, stirred thoroughly, and incubated overnight at 5°C. The filtrate was then placed in a 13,000 molecular weight dialysis membrane and dialyzed against 0.1 M sodium phosphate buffer (pH 7.0). The filtrate was then transferred to a 1.5 mL tube and heated in boiling water for 10 minutes. The heated mixture was used as the crude enzyme solution, and 1% protamine sulfate was added as a substrate. The mixture was incubated at 30°C for 30 minutes. The released amino acids in the reaction mixture were measured by the ninhydrin method.

[0080] As a result, it was confirmed that all four strains had reduced heat-resistant protease activity compared to the parent strain (Figure 1). Among them, strains No. 11, No. 133, and No. 200 had heat-resistant protease activity of 8 nkat / g or less, and several koji mold strains with low heat-resistant protease activity were obtained.

[0081] That is, it was revealed that the method for selecting koji molds with low thermostable protease activity using protamine described in this example has excellent throughput and is capable of discriminating the activities of a large number of strains.

[0082] (Example 2) Soy sauce brewing using the obtained koji mold strain and evaluation of the properties of the brewed soy sauce To verify the characteristics of the koji mold strain obtained by the method described in Example 1, soy sauce brewing was carried out.

[0083] (Soy sauce brewing) Equal amounts of defatted soybeans and cracked wheat were mixed, and then the parent strain and one of the obtained koji mold strains, No. 200, were inoculated to produce soy sauce koji. The resulting soy sauce koji was then brewed with 1.6 times the amount of 23.7% salt water and aged for 5 months. After aging, the moromi was pressed to produce raw soy sauce, which was then heated at 123°C in a plate heater to obtain soy sauce.

[0084] (Measurement of heat-resistant protease activity) The heat-stable protease activity of soy sauce brewed using the Aspergillus oryzae No. 200 strain and soy sauce brewed using the parent strain was measured. 10 ml of sample soy sauce was placed in a dialysis membrane with a molecular weight cutoff of 13,000 and dialyzed against 0.1 M sodium phosphate buffer (pH 7.0). 1 ml of 1% protamine sulfate was added as a substrate to 0.2 ml of the dialyzed sample, and the mixture was incubated at 30°C for 30 minutes. The amino acids liberated in the reaction solution were measured by the ninhydrin method.

[0085] As a result, the heat-stable protease activity was 2.94 nkat / mL in the soy sauce brewed with the parent strain and 0.75 nkat / mL in the soy sauce brewed with the koji mold No. 200 strain.

[0086] (Verification of suitability for soy sauce processing) To confirm the processing suitability of the soy sauces obtained, kamaboko cut into 2 cm widths was soaked in each soy sauce and left to stand at 5°C for 4 days. As a control, a product was soaked in 17% salt water.

[0087] The hardness was evaluated using a texture analyzer (TA.XT2i, Eiko Seiki Co., Ltd.). Specifically, a cylindrical probe with a diameter of 5 mm was inserted into the side of the kamaboko piece at a speed of 2 mm / s, and the evaluation was carried out by comparing the maximum load (gf) generated until the probe was completely penetrated.

[0088] The results are shown in Figure 2. Kamaboko pickled in soy sauce made with strain No. 200 maintained the same hardness as kamaboko pickled in 17% salt water, while the parent strain lost significantly more hardness, clearly demonstrating different physical properties.

[0089] Furthermore, to evaluate the suitability for processing into boiled eggs, egg whites were heated and coagulated to prepare egg white pieces for immersion tests, each measuring 3 cm in length, 3 cm in width, and 0.5 cm in thickness. The egg white pieces were then immersed in three-fold diluted versions of the brewed soy sauce and left to stand at 5°C for 18 hours.

[0090] The evaluation was carried out by seven well-trained panelists using a two-point preference method. Each egg white fragment was presented as a blind sample, and they were asked to choose which texture they preferred as the boiled egg white, maintaining its original texture.

[0091] As a result, all seven panelists chose the white meat pieces soaked in soy sauce made with No. 200 as preferable, and judged them to be significantly preferable to those soaked in soy sauce made with the parent strain.

[0092] The results of this example confirmed that soy sauce brewed using the koji mold strain obtained by the method described in Example 1 is suitable for processing into fish paste products such as kamaboko and boiled eggs.

[0093] (Example 3) Comparison of High Molecular Weight Fraction Content The soy sauce brewed by the method described in Example 2 was compared in terms of the content of the high molecular weight fraction with that of soy sauce from which the high molecular weight fraction containing the heat-resistant protease had been removed using membrane filtration.

[0094] (Test sample) The samples were soy sauce brewed using the parent strain according to the method described in Example 2, soy sauce brewed using the No. 200 strain, two commercially available dark soy sauces (soy sauce A and soy sauce B), and as a control, soy sauce A was fractionated at a molecular weight of 10,000 to remove the high molecular weight fraction (hereinafter referred to as the control product), for a total of five samples. The control sample was fractionated using 10000NWML of Amicon Ultra (Merck).

[0095] (molecular weight fractionation by dialysis) 10 ml of each soy sauce sample was dialyzed overnight at 4°C in deionized water using a cellulose dialysis membrane with a molecular weight of 13,000 to remove low molecular weight fractions.

[0096] (Measurement of dry weight) Each soy sauce sample from which the low molecular weight fraction had been removed was freeze-dried and its dry weight was measured. In addition, each soy sauce sample before molecular weight fractionation was similarly freeze-dried and its dry weight was measured.

[0097] The difference between the dry weight of each soy sauce sample before and after molecular weight fractionation was calculated, and this was taken as the dry weight of components with a molecular weight of 13,000 or more. The results are shown in Table 1 as a percentage of the dry weight of each soy sauce sample before molecular weight fractionation.

[0098] [Table 1]

[0099] The control product, in which the high molecular weight fraction with a molecular weight of 10,000 was fractionated and removed by membrane filtration, contained almost no fractions with a molecular weight of 13,000 or more. On the other hand, the soy sauce brewed using the No. 200 strain contained high molecular weight fractions at the same level as the soy sauce brewed with the parent strain and the two commercially available soy sauces.

[0100] In other words, it was demonstrated that by using strain No. 200, it is possible to brew soy sauce containing the same amount of high molecular weight fractions that affect taste as regular soy sauce.

[0101] (Example 4) Comparison of 2-acetylpyrrole content The soy sauce brewed by the method described in Example 2 was compared in terms of 2-acetylpyrrole content with that of soy sauce in which the thermostable protease had been inactivated by heating.

[0102] (Heating soy sauce samples and activity evaluation) One commercially available dark soy sauce (soy sauce A) and two light soy sauces (soy sauces C and D) were prepared, and samples were prepared by heating each of these soy sauces in a 70°C water bath for 6.5 hours, 14.5 hours, and 21 hours. The activity of the heat-resistant protease in each soy sauce sample was confirmed by immersing kamaboko cut into 2 cm widths in each soy sauce, leaving it to stand at 5°C for 4 days, and then measuring the load using a texture analyzer, in the same manner as described in Example 2 (Verification of suitability for soy sauce processing).

[0103] The results are shown in Figure 3. To reach a hardness of 475 gf, equivalent to that of soy sauce brewed with 17% salt water and strain No. 200, soy sauce A required 14.5 hours of heating, and soy sauces C and D required 21 hours of heating.

[0104] (Quantitative determination of 2-acetylpyrrole content) Using the method described in Example 2, a total of 14 samples were quantified using GC-MS: soy sauce brewed using the parent strain, soy sauces A, C, and D (unheated samples) brewed using the No. 200 strain, and samples obtained by heating soy sauces A, C, and D in a 70°C water bath for 6.5 hours, 14.5 hours, and 21 hours, respectively.

[0105] (Method for extracting aroma components from soy sauce samples) 6 ml of each soy sauce sample was mixed with 3 ml of dichloromethane as an organic solvent to extract the aroma components into the solvent. The solvent layer was then separated by centrifugation and subjected to GC-MS analysis.

[0106] (GC-MS analysis conditions) Quantitative analysis using GC-MS was carried out by the standard addition method in SIM mode under the following analytical conditions. Measurement device: 7890B-5977B MSD (Agilent Technologies) Column: DB-WAX (length 60 m, diameter 0.25 mm, film thickness 0.25 μm) (Agilent Technologies) Injection volume: 3μl Temperature conditions: 40°C (10 min) → Heat up to 230°C at 5°C / min → Hold for 12 min Carrier: High-purity helium, 161.34kPa Transfer temperature: 250℃ Ion source temperature: 230℃ Ionization method: EI Target ion: m / z 94 Qualifier ion: m / z 109

[0107] The results are shown in Table 2. Heating soy sauces A, C, and D significantly increased the amount of 2-acetylpyrrole. On the other hand, the amount of 2-acetylpyrrole in soy sauce brewed with strain No. 200 was the same as or lower than that of the parent strain and the unheated samples of soy sauces A, C, and D.

[0108] [Table 2]

[0109] These results indicate that conventional soy sauce requires prolonged heating to reduce thermostable protease activity, which inevitably results in the development of off-flavors such as 2-acetylpyrrole. However, by using strain No. 200, soy sauce with low thermostable protease activity can be obtained without being affected by changes in aroma components due to heating.

Claims

1. Soy sauce that satisfies the following conditions (1) to (3). (1) The heat-resistant protease activity is 1.5 nkat / mL or less; (2) Contains a fraction with a molecular weight of 13,000 or more at a dry weight ratio of 1% or more, and (3) The content of 2-acetylpyrrole is 5 ppm or less.

2. The soy sauce according to claim 1, wherein the soy sauce is pasteurized soy sauce.

3. A method for producing soy sauce that satisfies the following conditions (1) to (3), characterized in that a koji mold strain having a heat-resistant protease activity of 8 nkat / g or less is used in the koji-making step. (1) The heat-resistant protease activity is 1.5 nkat / mL or less; (2) Contains a fraction with a molecular weight of 13,000 or more at a dry weight ratio of 1% or more, and (3) The content of 2-acetylpyrrole is 5 ppm or less.

4. A method for obtaining a koji mold strain having reduced thermostable protease activity, comprising the following steps (1) to (5): (Step 1) growing a koji mold strain on a solid medium and extracting a crude enzyme solution from the culture; (Step 2) heating the obtained crude enzyme solution to inactivate heat-resistant proteases; (Step 3) reacting the heated crude enzyme extract with an antibacterial enzyme substrate; (Step 4) culturing a microorganism whose growth is inhibited by the antibacterial activity derived from the enzyme substrate in a medium containing the reaction solution, and detecting a change in the growth of the microorganism; (Step 5) A step of selecting koji mold strains that result in poor growth of the microorganism as candidate mutant strains with reduced thermostable protease activity.

5. 5. The method for obtaining a koji mold mutant strain according to claim 4, wherein the koji mold mutant strain has a thermostable protease activity of 8 nkat / g or less.

6. A method for producing soy sauce, comprising the steps of obtaining a mutant strain of koji mold by the method according to claim 4 or 5, and using the obtained mutant strain in a koji-making step.

Images