Aspergillus oryzae at23 and use thereof
By screening for Aspergillus oryzae strain AT23, which produces high levels of protease and low levels of ammonia byproduct, and combining it with a high-salt, low-temperature fermentation process, the problem of high ammonia content in soy sauce brewing was solved, thus enhancing the aroma and flavor of the soy sauce.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- FOSHAN HAITIAN GAOMING FLAVORING & FOOD
- Filing Date
- 2025-08-15
- Publication Date
- 2026-07-10
AI Technical Summary
Existing Aspergillus strains, during the soy sauce brewing process, produce high levels of protease while also exhibiting high levels of the byproduct ammonia (ammonium salt form), leading to a reduction in the content of effective substances and affecting the aroma and flavor of the soy sauce.
A strain of Aspergillus oryzae AT23 with high protease production and low ammonia byproduct was screened out. A strain with high glutaminase activity was screened out by ARTP mutagenesis. Combined with a high-salt dilute state fermentation process, the yield of glutamic acid was increased and the ammonia content was reduced.
During the soy sauce brewing process, Aspergillus oryzae AT23 increases glutamic acid production, promotes the utilization rate of raw material protein, enhances the aroma and flavor of soy sauce, and reduces the ammonia content of by-products.
Smart Images
Figure CN121022606B_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of brewing soy sauce technology, and in particular to Aspergillus oryzae AT23 and its applications. Background Technology
[0002] Chinese invention patent application CN202411190034.1 describes a strain of Aspergillus oryzae ZA304 and its application. The Aspergillus oryzae can produce protease in large quantities, which can improve the utilization rate of raw materials. However, not all protein hydrolysates can be effectively used, such as the generation of additional ammonium salts, which leads to a decrease in the effective utilization rate.
[0003] Chinese invention patent application CN202411334479.2 describes a high-protease-producing Aspergillus oryzae strain SICC3.428 and its uses. The Aspergillus oryzae can produce high levels of protease, which improves the aroma of fermentation products. As amino acids are the main source of protein hydrolysis aroma, they also face the problem of excessive downward reaction, which reduces the content of the final effective substances, such as the formation of additional ammonium salts.
[0004] Chinese invention patent application CN202410096103.6 describes a high-yield Aspergillus oryzae, its fermentation broth, its preparation method and application. The Aspergillus oryzae in the fermentation broth has a relatively high level of enzymes such as protease and amylase that degrade macromolecular nutrients, but it does not address the maintenance of hydrolysates to prevent further decomposition.
[0005] To address the shortcomings of the aforementioned technologies, a high-protease-producing Aspergillus oryzae was screened out while simultaneously enhancing the activity of the corresponding glutaminase. This increased the fermentation yield of protein hydrolysates (especially glutamic acid) while reducing the yield of the byproduct ammonia (mainly in the form of ammonium salts). Summary of the Invention
[0006] Based on this, one or more embodiments of this application provide Aspergillus oryzae AT23 and its applications. The technical solutions include the following:
[0007] One or more embodiments of this application provide an Aspergillus oryzae AT23, with accession number GDMCC No: 66246.
[0008] One or more embodiments of this application provide a method for preparing koji, the method comprising the steps of inoculating the Aspergillus oryzae AT23 into a koji-making culture medium and culturing it.
[0009] In some embodiments of this application, the koji-making culture medium comprises legume raw materials and wheat raw materials.
[0010] In some embodiments of this application, the legume raw material includes soybeans.
[0011] In some embodiments of this application, the wheat-based raw material includes flour.
[0012] In some embodiments of this application, the mass ratio of the legume raw material to the wheat raw material is (6~8):(2~4).
[0013] In some embodiments of this application, the culture temperature is 25°C to 34°C, and the culture time is 36h to 60h.
[0014] One or more embodiments of this application provide a melody prepared by the method described above.
[0015] One or more embodiments of this application provide a method for preparing soy sauce, the method comprising:
[0016] The method described above is used to prepare the melody; and,
[0017] Soy sauce is prepared using the koji.
[0018] In some embodiments of this application, the method uses high-salt dilute-state fermentation to prepare soy sauce.
[0019] In some embodiments of this application, the high-salt dilute state fermentation time is 50 days to 70 days.
[0020] In some embodiments of this application, the temperature of the high-salt dilute fermentation is 25°C to 34°C.
[0021] One or more embodiments of this application provide a soy sauce prepared by the method described.
[0022] Compared with traditional technologies, this application has the following advantages:
[0023] This application provides a *Aspergillus oryzae* AT23 strain that produces high levels of protease and glutamic acid, while exhibiting a reduced content of the byproduct ammonia (in the form of ammonium salts). In the soy sauce brewing process, using the *Aspergillus oryzae* AT23 strain selected in this application can maintain a high level of glutamic acid production, which helps promote the utilization of raw material proteins during the koji-making process, thereby enhancing aroma and flavor during fermentation. Attached Figure Description
[0024] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0025] Figure 1This is an operation flowchart of an embodiment of this application.
[0026] Figure 2 This is a plate morphology diagram of the target mutagen strain AT23 implemented in this application.
[0027] Figure 3 This is a microscopic morphological image of the target mutagen strain AT23 in an embodiment of this application.
[0028] The Aspergillus oryzae AT23 strain provided in this application, taxonomically named Aspergillus oryzae, was deposited on April 30, 2025, at the Guangdong Microbial Culture Collection Center, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, with accession number GDMCC No: 66246. This strain was received and registered by the collection center on April 30, 2025, and was confirmed as a viable strain by the collection center on the same day. Detailed Implementation
[0029] The present application will be further described in detail below with reference to the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.
[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for descriptive purposes only and is not intended to be limiting of the application.
[0031] the term
[0032] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:
[0033] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").
[0034] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.
[0035] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.
[0036] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.
[0037] In this document, terms such as “preferred,” “better,” “more suitable,” and “ideal” are merely used to describe implementation methods or examples that achieve better results, and should be understood not to limit the scope of protection of this application.
[0038] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.
[0039] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.
[0040] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.
[0041] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.
[0042] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.
[0043] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.
[0044] In this application, %(w / w) and wt% both represent weight percentage, %(v / v) refers to volume percentage, and %(w / v) refers to mass-volume percentage.
[0045] All references to this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the purpose and / or technical solution of this application, all cited references are incorporated herein by reference in their entirety and for all purposes. When references are cited in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. Examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.
[0046] In a first aspect of this application, an Aspergillus oryzae AT23 is provided, with accession number GDMCC No: 66246.
[0047] A second aspect of this application provides a method for preparing koji (fermented rice), the method comprising the step of inoculating Aspergillus oryzae AT23 into a koji-making culture medium and culturing it.
[0048] In the process of making koji using Aspergillus oryzae according to the embodiments of this application, this application does not particularly limit the raw materials for koji making. In some examples of this application, the koji-making culture medium includes legume raw materials and wheat raw materials. The legume raw materials in this application include, but are not limited to, soybeans. The wheat raw materials in this application include, but are not limited to, wheat flour. This application does not particularly limit the amount of legume raw materials and wheat raw materials. The appropriate amount can be used according to the koji-making requirements. In some examples of this application, the mass ratio of the legume raw materials to the wheat raw materials is (6~8):(2~4), for example, 6:2, 6:3, 6:4, 7:2, 7:3, 7:4, 8:2, 8:3, 8:4.
[0049] In the process of making koji using Aspergillus oryzae according to the embodiments of this application, there are no special limitations on the cultivation conditions. Suitable koji-making conditions can be used. In some examples of this application, the cultivation temperature is 25℃~34℃ (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34℃), and the cultivation time is 36h~60h (e.g., 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60h).
[0050] A third aspect of the embodiments of this application provides a melody prepared by the method described in the second aspect.
[0051] A fourth aspect of this application provides a method for preparing soy sauce, the method comprising:
[0052] The method described above is used to prepare the melody; and,
[0053] Soy sauce is prepared using the koji.
[0054] This application does not impose particular limitations on the preparation process of soy sauce, such as using high-salt liquid-state fermentation, or low-salt solid-state fermentation. In some examples of this application, the method uses high-salt liquid-state fermentation to prepare soy sauce. This application does not impose particular limitations on the conditions for high-salt liquid-state fermentation; it can be carried out under suitable conditions. In some examples of this application, the high-salt liquid-state fermentation time is 50 days to 70 days, for example, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, or 70 days. In some examples of this application, the high-salt liquid-state fermentation temperature is 25℃ to 34℃, for example, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34℃.
[0055] A fifth aspect of this application provides a soy sauce prepared by the method described above.
[0056] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.
[0057] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.
[0058] Example 1
[0059] This embodiment uses Aspergillus oryzae AS3.951 (Hu Niang 3.042) as the starting strain, employs high-throughput ARTP (atmospheric pressure room temperature plasma source) mutagenesis, superimposed with high-salt dilute-state fermentation, to screen for target mutagenic strains, such as... Figure 1 As shown. The solution is as follows:
[0060] 1. Inoculation of Aspergillus oryzae AS3.951 and preparation of spore suspension
[0061] (1) Take an appropriate amount of Aspergillus oryzae AS3.951 (Hu Niang 3.042) spores and spread them on malt extract agar plates and incubate at 30℃ for 5 days.
[0062] (2) After the culture is completed, rinse the cultured spores with sterile physiological saline and transfer them to centrifuge tubes at 4°C and 8000 rpm.
[0063] (3) Discard the supernatant and repeat twice, then resuspend in 5 mL of sterile physiological saline and dilute to 10. 7 Quantity / mL, ready for use.
[0064] 2 ARTP mutagenesis
[0065] The suspension was coated onto an iron sheet for ARTP mutagenesis. The mutagenesis time was 240 s, the power was 120 W, and the gas flow rate was 10 SLM. After mutagenesis, the iron sheet was placed in an EP tube containing 1 mL of physiological saline and incubated at 30 °C and 150 rpm for 2 h.
[0066] 3. Screening of target strains
[0067] (1) Screening of high glutamate-producing strains
[0068] Mutagenized Aspergillus oryzae spores and a glutamate fluorescence sensor were encapsulated in droplets. After 48 hours, the fluorescence of each droplet was measured and sorted, with a sorting speed of approximately 100 droplets per second.
[0069] (2) Screening of high protease-producing strains
[0070] Green fluorescent protein was injected during the microdroplet screening process. The mutant with stronger protease activity degraded GFP faster. Based on this, mutants with weaker fluorescence intensity were sorted and subjected to proteolysis zone testing.
[0071] (3) Screening of strains with low byproduct transformation
[0072] The purified mutant strains were used for high-salt dilute-state fermentation, and the strains with the highest glutamic acid content and the lowest ammonium salt content in the system after fermentation were selected.
[0073] 4. Application of mutagenic strains
[0074] 4.1 Preparation of koji using mutagenic strains
[0075] (1) Select whole, dried soybeans and add twice the amount of pure water. Soak them in a refrigerator at 4°C for 12 hours. Filter the water through a sieve and weigh them. After draining, wrap them in gauze and sterilize them at 121°C for 15 minutes. Cooked soybeans are brown, with intact coatings. They are elastic when lightly squeezed and become a paste when pressed. Let them cool before use.
[0076] (2) Weigh out flour at a mass ratio of 7:3 for the dried soybeans and flour, and make a final concentration of 10. 7 Inoculate the spore suspension at a rate of 1 spore / g (calculated based on the total weight of dried soybeans and flour to determine the final concentration) onto the inner wall of the flour container. After the spore suspension dries, scrape off the spores with a spatula and mix them evenly with the flour. The flour will turn slightly green after mixing.
[0077] (3) Mix the cooked soybeans from step (1) with the flour from step (2) until each soybean is fully coated with flour. After spreading evenly, take 50g and transfer it to a 500mL fermentation bottle. Seal the bottle with gauze and place it in a constant temperature and humidity incubator at 32℃ and 85%RH for 2 days.
[0078] 4.2 High-salt dilute-state fermentation of mutagenic strains
[0079] (1) Prepare a sterile saline solution with a salt concentration of 20wt%, take 50g and add it to the fermentation bottle and continue to culture at 30℃ for 60 days to prepare soy sauce.
[0080] (2) Compare the fermentation results and select strains with excellent performance.
[0081] 5. Comparison of target mutagenic strain and starting strain
[0082] 5.1 Comparison of casein hydrolysis zone size between the target mutagenized strain and the starting strain
[0083] The mutant strain and the original strain AS3.951 were inoculated into casein agar medium (Huankai Microbiology, catalog number 028370) and cultured at 32°C for 3 days. The colony diameter and the inner and outer diameters of the hydrolysis zone were then compared.
[0084] Table 1
[0085]
[0086] 5.2. Comparison of sporulation capacity between the target mutagenized strain and the starting strain
[0087] (1) The mutant strain and the starting strain AS3.951 were inoculated into malt extract agar medium (Huankai Microbiology, catalog number 021110) and cultured at 32°C. After 4 days, the surface spores were washed off with sterile physiological saline (containing 0.5% Tween 80) and stored in Erlenmeyer flasks. An appropriate amount was taken into a hemocytometer and the spore suspension concentration was calculated using the five-point method.
[0088] (2) Take equal amounts of the mutagenized strain and the starting strain AS3.951 and prepare the koji according to the description in "4.1 Preparation of koji by mutagenized strain". After the culture is completed, break up the koji material and mix it thoroughly with sterile physiological saline (containing 0.5% Tween 80). Take an appropriate amount into a hemocytometer and calculate the spore suspension concentration using the five-point method.
[0089] Table 2
[0090]
[0091] 5.3 Comparison of neutral protease activity between the target mutagenized strain and the starting strain in koji preparation
[0092] Take equal amounts of the mutagenized strain and the original strain AS3.951 and prepare koji according to the instructions in section “4.1 Koji Preparation with Mutagenized Strains”. Take the koji material and test the neutral protease activity.
[0093] Table 3
[0094]
[0095] in:
[0096] The method for determining the activity of neutral proteases is described in SB / T 10317-1999, "Protein Activity Determination Method".
[0097] Moisture determination method: Take 20g of material in a tray, place it in a 37℃ oven and dry for 24h, then calculate the moisture content of the koji material by 1-(weight of material after drying / weight of material before drying).
[0098] 5.4 Comparison of fermentation levels between the target mutagenized strain and the starting strain
[0099] Take the mutagenized strain and the original strain AS3.951, and follow the instructions in "4 Application of Mutagenized Strains" for koji making and high-salt dilute state fermentation. Take the fermentation products for testing.
[0100] Table 4
[0101]
[0102] in:
[0103] The method for determining total nitrogen content is specified in GB / T 18186-2000 Brewed Soy Sauce;
[0104] The method for determining amino nitrogen content is specified in GB / T 18186-2000 Brewed Soy Sauce;
[0105] The method for determining glutamic acid content is specified in GB 5009.124-2016 National Food Safety Standard - Determination of Amino Acids in Food.
[0106] The method for determining ammonium salt content is specified in GB 5009.234-2016 National Food Safety Standard - Determination of Ammonium Salts in Food.
[0107] 5.5 Plate morphology of the target mutagen strain AT23
[0108] Czapek's medium culture: AT23 colonies were cultured on Czapek's medium at 30°C until they filled a 9cm diameter culture dish. The texture was velvety to thick flocculent, with many conidial structures. The color was initially light yellow, later turning light brownish-yellow. There was no exudate. The reverse side of the colony was colorless or light brownish-yellow.
[0109] Malt extract culture medium: AT23 colonies were cultured on malt extract medium at 30°C until they filled a 9cm diameter petri dish. The colonies had a velvety to thick flocculent texture and were yellowish-green. The reverse side of the colonies was colorless or pale yellow.
[0110] Cultivation on Saccharomyces chapensis medium: AT23 colonies were cultured on Saccharomyces chapensis medium at 30°C until they filled a 9cm diameter culture dish. The texture was velvety to thick flocculent, with radial grooves, and was light yellowish-brown. The reverse side of the colonies was brownish-brown.
[0111] like Figure 2 As shown.
[0112] Comparative analysis showed that AT23 and the starting strain did not differ significantly in plate morphology during the culture process.
[0113] 5.6 Microscopic morphology of the target mutagenized strain AT23
[0114] The conidiophores are initially spherical, later becoming radial, with a diameter of (40-)80-250(-300)μm. The conidiophores arise from the substrate or aerial hyphae. The conidiophore stems are (200-)500-3000(-3500)μm or longer, with a diameter of (5.5-)8-20μm. Those arising from aerial hyphae are shorter. The apical sac is nearly spherical or flask-shaped, (12.8-)20-45(-50)μm, and fertile on the entire or three-quarters surface. The sporulation structure is single or double-layered. The conidia are spherical or nearly spherical, (3.6-)5-8.6(-9.6)μm.
[0115] like Figure 3 As shown, Figure 3 The top image shows a conidiophore, and the bottom image shows a conidium.
[0116] In summary, the embodiments of this application utilize ARTP mutagenesis technology to screen for Aspergillus oryzae with high protease and high glutamate production, while reducing the content of the byproduct ammonia (existing in the form of ammonium salts). Using the mutagenic strains screened in this application can maintain a high level of glutamate production, which helps to improve the utilization rate of raw material protein during the koji-making process in soy sauce brewing, thereby enhancing aroma and flavor during fermentation.
[0117] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.
[0118] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification and drawings can be used to interpret the content of the claims.
Claims
1. A type of Aspergillus oryzae AT23, characterized in that, This strain was deposited on April 30, 2025, at the Guangdong Microbial Culture Collection Center, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, with accession number GDMCC No: 66246.
2. A method for preparing koji, characterized in that, The method includes the step of inoculating Aspergillus oryzae AT23 as described in claim 1 into a koji-making culture medium and culturing it. The koji-making culture medium contains legume raw materials and wheat raw materials; The legume raw materials include soybeans; The wheat-based raw materials include flour; The mass ratio of the legume raw material to the wheat raw material is (6~8):(2~4).
3. The method for preparing koji according to claim 2, characterized in that, The incubation temperature is 25℃~34℃, and the incubation time is 36h~60h.
4. The melody prepared by the method according to any one of claims 2 to 3.
5. A method for preparing soy sauce, characterized in that, The method includes: The melody is prepared by the method according to any one of claims 2 to 3; and, Soy sauce is prepared using the aforementioned koji. The method uses high-salt dilute-state fermentation to prepare soy sauce; The high-salt dilute state fermentation time is 50-70 days; The temperature for the high-salt dilute state fermentation is 25℃~34℃.