Preparation method of soy sauce containing active biological enzyme

Abstract

This invention discloses a method for preparing soy sauce containing active bio-enzymes. The method utilizes a strain of Aspergillus oryzae that produces a high yield of complex bio-enzymes. Aspergillus oryzae This strain, deposited on March 27, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42541, possesses the dual advantages of high enzyme production and high salt tolerance. In soy sauce production, no exogenous enzymes are added throughout the process. It employs segmented temperature-controlled koji making, low-temperature long-cycle high-salt dilute-state fermentation at 15–25℃, followed by gradual temperature gradient sterilization at 60–75℃, retaining over 85% of the natural enzyme activity. The resulting product exhibits neutral protease ≥600 U / 100mL, amylase ≥900 U / 100mL, and amino acid nitrogen ≥1.2 g / 100mL, making it suitable for industrial mass production.

Description

Technical Field

[0001] This invention relates to the field of soy sauce brewing technology, specifically a method for preparing soy sauce containing active biological enzymes. Background Technology

[0002] High-salt, liquid-state soy sauce is a mainstream category of high-end soy sauce in my country, characterized by its rich flavor, intense aroma, and abundant nutrients. Its quality hinges on the activity of the koji-making enzymes and the efficiency of fermentation conversion. Traditional high-salt, liquid-state soy sauce brewing processes commonly use commercially available Aspergillus oryzae strains for koji-making. This means that the composition and activity level of the enzyme system secreted by Aspergillus oryzae during the koji-making stage directly determine the degradation efficiency of raw material proteins, the amount of amino acid nitrogen generated, and the final flavor quality of the product during subsequent fermentation.

[0003] Currently, domestic soy sauce producers generally use Aspergillus oryzae strain Hu Niang 3.042 or its derivatives as the koji-making strain. However, this strain has limited enzyme production capacity, resulting in low overall enzyme activity in the koji. This leads to incomplete decomposition of proteins, starches, and cellulose in the raw materials, reducing the amino acid conversion rate of soy sauce and easily causing raw material waste and a thin flavor. While existing technologies have reported the screening of strains that can produce high levels of complex enzymes, these strains, although producing high levels of complex enzymes, are not salt-tolerant and cannot ferment in high-concentration saline solutions (18%–21%), or adapt to weakly acidic fermentation systems. They can only be used for ordinary fermentation and cannot be used in the production of high-salt, thin-state soy sauce.

[0004] To address the issue of insufficient enzyme activity, most companies in the industry currently enhance fermentation efficiency by adding exogenous industrial enzyme preparations such as proteases and amylases. For example, fermenting a mixture of Aspergillus oryzae strains with significantly different enzyme systems yields koji with significantly higher neutral and acidic protease activities than single-strain fermentation, and also improves the amino acid nitrogen and reducing sugar content of the finished soy sauce. Other studies have screened Aspergillus niger and Aspergillus oryzae from traditional soy sauce mash for koji production, resulting in a significant increase in neutral protease, saccharifying enzyme, and acidic protease activities. However, multi-strain koji production faces challenges in actual production, such as significant differences in strain growth characteristics and difficulty in coordinating koji-making conditions. Furthermore, the introduction of exogenous strains may increase the uncertainty of the fermentation process.

[0005] Meanwhile, traditional soy sauce sterilization generally adopts high-temperature instantaneous sterilization process (132-150℃, 2-8 s). Although this process can completely kill miscellaneous bacteria, it will cause a large number of active biological enzymes of natural metabolism of koji to be deactivated. The finished soy sauce has almost no active enzymes left. This not only results in the loss of the natural active nutrients of soy sauce, but also leads to poor stability of soy sauce in the later stage, easy separation, and rapid flavor decay.

[0006] In summary, given the technical shortcomings of existing technologies, such as weak enzyme production capacity of strains, the need for exogenous enzyme preparations, severe enzyme activity loss after sterilization, no active biological enzymes remaining in the finished product, and unstable product quality, there is an urgent need to develop a method for preparing high-salt dilute soy sauce that does not require exogenous enzyme addition, has high enzyme activity retention, and excellent flavor. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing soy sauce containing active bio-enzymes. This method involves screening, isolation, domestication, and cultivation to ultimately select and breed a strain of *Aspergillus oryzae* with high salt tolerance and high enzyme production. Aspergillus oryzae The strain QHJ003 not only exhibits high salt tolerance but also produces high levels of enzymes under high-salt conditions, making it suitable for use in the preparation of high-salt, diluted soy sauce. Further optimization of the koji-making and fermentation process parameters, combined with mild sterilization and low-temperature storage techniques, allows the finished soy sauce to maintain high levels of natural biological enzyme activity while simultaneously increasing the content of amino acid nitrogen and flavor compounds, all without the addition of any exogenous enzyme preparations.

[0008] To achieve the above-mentioned objectives, the specific technical solution of this invention is as follows: This invention first protects a strain of Aspergillus oryzae ( Aspergillus oryzae The strain was deposited on March 27, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42541. The address of the depository is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0009] The strains described above are strains that have undergone mutagenesis and domestication. Specifically, a strain of Aspergillus oryzae was first isolated from high-salt dilute soy sauce mash, and then mutagenesis was induced by ambient pressure room temperature plasma (ARTP) and / or ultraviolet mutagenesis, combined with domestication culture on high-salt (6%–18% NaCl) plates. A mutant strain with high production of protease and amylase and high enzyme activity retention rate under high-salt acid conditions was obtained and preserved.

[0010] More specifically, ARTP mutagenesis (10 SLM, 100 W, 50–80 s) was used to obtain mutant strains with increased enzyme activity by more than 30% after initial screening by casein clear zone and secondary screening by shake-flask fermentation. Then, after UV mutagenesis (15 W, 20 cm distance, 60–120 s) and directional acclimatization by gradually increasing NaCl concentration (6%→9%→12%→15%→18%), the target strain with good growth and stable enzyme production performance in high-salt environment was finally screened.

[0011] Due to the above properties, the aforementioned Aspergillus oryzae ( Aspergillus oryzae It is used in high-yield compound bio-enzyme products in high-salt environments.

[0012] Furthermore, in the aforementioned application, the product is high-salt liquid soy sauce (e.g., used in the koji-making step of the high-salt liquid soy sauce production process).

[0013] A method for preparing soy sauce containing active bio-enzymes, comprising the following steps: s1. Aspergillus oryzae with accession number CGMCC No. 42541 ( Aspergillus oryzae Inoculate onto slant culture medium, activate and culture at a constant temperature to prepare seed strains; s2. Pretreatment of raw materials for koji making: Defatted soybeans and wheat are selected as the main raw materials. After mixing them in a certain proportion, the raw materials are soaked, cooked and cooled to room temperature to obtain sterile raw materials for koji making. s3, High Enzyme Activity Koji Making: The seed culture obtained in s1 is evenly inoculated into the aseptic koji making raw material obtained in s2. The segmented temperature-controlled koji making process is adopted, and the entire process is carried out under constant temperature and humidity. The koji is turned at regular intervals. After the koji matures, high enzyme activity koji is obtained. s4, High-salt dilute state fermentation: Add sterile brine to the high-enzyme-active koji obtained in s3, mix evenly to prepare soy sauce mash; control the fermentation environment temperature and salinity, carry out constant temperature and sealed fermentation, and obtain soy sauce mash after fermentation is mature; no exogenous enzyme preparations are added throughout the process, and the raw material decomposition and flavor transformation are completed by relying on the natural biological enzymes of the koji itself. s5. Precise sterilization and enzyme preservation: After pressing, filtering and clarifying, the soy sauce mash obtained in s4 is sterilized using a gradient temperature mild sterilization process to retain the active biological enzymes in the finished product to the greatest extent, resulting in sterilized soy sauce concentrate. s6. Finished product bottling: The sterilized soy sauce concentrate obtained in s5 is cooled, inspected, and aseptically bottled to obtain (high enzyme activity) soy sauce finished product containing active biological enzymes (high salt dilute state).

[0014] In a preferred embodiment of this application, in step s2 of the method for preparing soy sauce containing active bio-enzymes, the mass ratio of defatted soybeans to wheat is 6-7:3-4; the soaking temperature is 25-30°C, and the soaking time is 4-6 h; the cooking is carried out under normal pressure, the cooking temperature is 100-105°C, the cooking time is 30-40 min, and the moisture content in the aseptic koji-making raw materials is controlled at 48-52 wt%.

[0015] In a preferred embodiment of this application, in step s3 of the method for preparing soy sauce containing active bio-enzymes, the inoculation amount of the seed strain is 0.3-0.5% of the total mass of the koji-making raw materials; the segmented temperature-controlled koji-making process is as follows: the initial culture temperature is 28-30℃ for 12 hours; the middle culture temperature is 30-32℃ for 12 hours; the later culture temperature is 26-28℃ for 12 hours; the relative humidity of the entire culture environment is 90-95%; the koji is turned twice, at 12 hours and 24 hours of koji-making, respectively, for a total koji-making time of 35-45 hours.

[0016] In this invention, the segmented temperature-controlled koji-making process is optimized to adapt to the growth and metabolic patterns of high-enzyme-producing strains, maximizing the activation of the strains' enzyme production capacity and ensuring stable and high enzyme activity in the koji.

[0017] As a preferred embodiment of this application, in step s3 of the method for preparing soy sauce containing active biological enzymes, the high-enzyme-active koji obtained has a neutral protease activity ≥1800 U / g, an amylase activity ≥2500 U / g, and a cellulase activity ≥800 U / g.

[0018] In a preferred embodiment of this application, in step s4 of the method for preparing soy sauce containing active bio-enzymes, the mass concentration of the sterile brine is 18-20%; the mass ratio of the starter culture to the sterile brine is 1:2.5-3; the fermentation temperature is kept constant at 15-25℃ throughout the process; the closed fermentation cycle is 180-380 days; and the mixture is stirred once every 15 days during the fermentation process, with each stirring lasting 10-15 minutes.

[0019] In this invention, the temperature, salinity, and fermentation cycle of high-salt dilute fermentation are precisely controlled, and the catalytic reaction conditions of natural enzyme systems are adapted to improve the hydrolysis efficiency of raw materials and the accumulation of flavor substances.

[0020] As a preferred embodiment of this application, in step s5 of the method for preparing soy sauce containing active bio-enzymes, the gradient heating and sterilization process specifically comprises: first heating to 60–65°C and holding for 15–20 min; then heating to 70–75°C and holding for 10–15 min; and finally cooling to 55–60°C for constant-temperature filtration and sterilization. Compared to traditional high-temperature instantaneous sterilization, this process can retain more than 85% of the natural active bio-enzymes in the finished product.

[0021] In this invention, a gradient temperature rise and mild sterilization process is used to replace the traditional high temperature sterilization process. Under the premise of ensuring that the product meets the sterility standards and the shelf life is qualified, the natural active biological enzymes in the finished product are preserved to the greatest extent.

[0022] The above-described method for preparing soy sauce containing active bio-enzymes does not involve the addition of any artificially synthesized enzymes, flavor enhancers, or preservatives throughout the entire process. It relies solely on the natural metabolic bio-enzymes of the bacterial strain to complete the hydrolysis of raw materials, flavor generation, and quality optimization.

[0023] This invention also protects a soy sauce containing active bio-enzymes prepared according to any one of the above methods.

[0024] Preferably, the soy sauce is a high-salt, dilute-state fermented soy sauce; under normal temperature storage conditions, the neutral protease activity is ≥600 U / 100mL, the amylase activity is ≥900 U / 100mL, the amino acid nitrogen content is ≥1.2 g / 100mL, and the enzyme activity retention rate is high, with stable and long-lasting flavor.

[0025] Compared with existing technologies, the present invention has the following advantages: 1. The Aspergillus oryzae strain preserved in this invention has the dual advantages of high enzyme production and high salt tolerance. When the koji material is mature, the neutral protease activity is ≥1800 U / g, the amylase activity is ≥2500 U / g, and the cellulase activity is ≥800 U / g. The enzyme production capacity is more than 30% higher than that of ordinary strains, and the enzyme activity retention rate is extremely high in high-salt and weakly acidic fermentation systems.

[0026] 2. This invention optimizes the segmented temperature-controlled koji-making process, precisely adapts to the metabolic rhythm of the strains, resulting in high koji-making efficiency and uniform and stable enzyme activity in the koji. It innovatively adopts a low-temperature constant-temperature fermentation process of 15-25℃, which significantly slows down the rate of enzyme thermal inactivation. This increases the residual enzyme activity rate in the fermented mash from less than 10% in the traditional process to more than 25% at the end of fermentation, prolonging the enzyme's action time and increasing the production of amino acid nitrogen and reducing sugar in soy sauce.

[0027] 3. This invention employs a gradient temperature mild sterilization process, which involves primary sterilization at 60-65℃, deep inactivation at 70-75℃, and low-temperature fine filtration at 55-60℃ to thoroughly kill Escherichia coli, molds, yeasts, and heat-resistant spores, meeting the national food microbiological safety standards for soy sauce products. Furthermore, it can stably retain over 85% of natural active proteases, amylases, and cellulases while ensuring long-term shelf-life stability.

[0028] 4. The high-salt dilute soy sauce product prepared by this invention has a neutral protease activity ≥600 U / 100mL, an amylase activity ≥900U / 100mL, and an amino acid nitrogen content ≥1.2 g / 100mL. It has high natural biological activity, high nutritional quality, and a rich and full-bodied soy sauce aroma. It does not separate or become cloudy during long-term storage at room temperature and has excellent flavor stability.

[0029] 5. The process parameters of this invention are precise and controllable, and are suitable for industrial mass production. It breaks through the bottlenecks of traditional processes from the source of the strain, koji making, fermentation and sterilization. The product has significant differentiation advantages and has extremely high industrial application and market promotion value. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the illustrative embodiments and descriptions of the present invention are only used to explain the present invention and are not intended to limit the present invention.

[0031] The following details an embodiment of the method for preparing soy sauce containing active bio-enzymes according to the present invention. However, unnecessary detailed descriptions may be omitted. For example, detailed descriptions of well-known matters and repeated descriptions may be omitted. This is to avoid making the following description unnecessarily lengthy and to facilitate understanding by those skilled in the art.

[0032] The "scope" disclosed in this invention is defined in the form of a lower limit and an upper limit. A given scope is defined by selecting a lower limit and an upper limit, which define the boundaries of the specific scope. The scope defined in this way can include or exclude end values, and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a scope.

[0033] Unless otherwise specified, all embodiments and optional embodiments of the present invention can be combined with each other to form new technical solutions.

[0034] Unless otherwise specified, all technical features and optional technical features of this invention can be combined to form new technical solutions.

[0035] Unless otherwise specified, the terms "comprising" and "including" as used in this invention can be open-ended or closed-ended. For example, "comprising" and "including" can mean that other substances not listed may also be included, or that only the listed substances may be included.

[0036] Unless otherwise specified, all steps of the present invention may be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), indicating that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, the mention that the method may also include step (c) indicates that step (c) may be added to the method in any order. For example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.

[0037] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0038] It should be noted that, unless otherwise specified, the experimental methods used in the embodiments are conventional methods. Unless otherwise specified, the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0039] A strain of Aspergillus oryzae that produces a high amount of complex biological enzymes ( Aspergillus oryzae This strain was deposited on March 27, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42541. The address of the depository is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0040] I. Isolation and Screening of Strains 1. Original strain We selected Aspergillus oryzae strain M3, which was collected, isolated, screened, and preserved in the laboratory in 2025 from our soy sauce brewing workshop for use in soy sauce brewing.

[0041] II. ARTP Mutagenesis 2.1 Preparation of spore suspension Aspergillus oryzae M3 strain was inoculated onto PDA slant agar and cultured at 30°C for 5–7 days until spores were fully mature. The slant was washed with sterile physiological saline (containing 0.05% Tween-80), and mycelium was removed by filtration through sterile defatted cotton. The spore suspension was collected. The spores were counted using a hemocytometer, and the spore concentration was adjusted to 1 × 10⁻⁶. 6 ~1×10 7 per mL.

[0042] 2.2 ARTP mutagenesis treatment Take 10 μL of spore suspension and spread it evenly on the surface of a sterile metal slide, then place it on the sample stage of the ARTP mutagenesis breeding instrument. The mutagenesis conditions are set as follows: power 100 W, gas flow rate 10 SLM (standard liters / minute). Four time gradient treatments are set: 50 s, 60 s, 70 s, and 80 s. Three parallel samples are prepared for each time gradient.

[0043] 2.3 Drawing the mortality rate curve After treatment at each time gradient, slides were quickly placed in EP tubes containing 1 mL of sterile physiological saline and eluted by shaking. The eluent was appropriately diluted and spread onto PDA plates, incubated at 30°C in the dark for 48–72 h, and the number of surviving colonies was counted. Using untreated spore suspension as a control (survival rate considered 100%), the lethality rate at each treatment time was calculated. The lethality rate calculation formula is as follows: Lethality rate (%) = (1 - number of surviving spores after mutagenesis / total number of spores before mutagenesis) × 100% The optimal mutagenesis conditions were selected based on a treatment time with a lethality rate between 90% and 95%. Measurements showed that a treatment time of 70 seconds resulted in a lethality rate of approximately 92%, which was determined to be the optimal treatment time for ARTP mutagenesis in this experiment.

[0044] 2.4 Initial screening of mutant strains—casein clear zone method The spore suspension treated under optimal mutagenesis conditions was appropriately diluted and spread onto casein selection medium plates, then incubated at 30°C in the dark for 48–72 h. Single colonies with good growth and clear zones (HC value ≥ 3.0) were selected and transferred to PDA slants for preservation. More than 50 candidate mutant strains with significantly higher HC values ​​than the original strain were obtained through screening.

[0045] 2.5 Rescreening of mutant strains – Validation by shake-flask fermentation The candidate mutants obtained from the initial screening were subjected to shake-flask fermentation (bran medium, 30℃, 72 h), and the activities of neutral protease and amylase were measured. Mutants with enzyme activities more than 30% higher than the original strain M3 were screened out. The dominant strain with the highest enzyme activity was selected and named Aspergillus oryzae M3-1 (i.e., the first-generation dominant strain obtained by ARTP mutagenesis screening), which will be used as the original strain for the next round of UV mutagenesis.

[0046] III. Ultraviolet Mutagenesis of M3-1 3.1 Preparation of spore suspension Aspergillus oryzae M3-1 was inoculated onto PDA slant and cultured at 30℃ for 5–7 days until full conidia were produced. A spore suspension was prepared according to method 2.1, and the spore concentration was adjusted to 1×10⁻⁶. 6 ~1×10 7 per mL.

[0047] 3.2 Ultraviolet Mutagenesis Treatment Place 5 mL of spore suspension in a sterile petri dish (9 cm in diameter) and place a sterile magnetic stirrer inside. Position the petri dish 20–30 cm vertically from a 15 W UV lamp. Open the petri dish lid and simultaneously turn on the magnetic stirrer (ensuring uniform irradiation of the spores) to begin UV irradiation. Set three irradiation time gradients: 60 s, 90 s, and 120 s. Prepare three parallel samples for each time gradient. All operations were performed in a dark room. Immediately after irradiation, cover the petri dish and wrap it with aluminum foil to prevent photoreactivation.

[0048] 3.3 Drawing the mortality rate curve The spore suspensions irradiated at each time gradient were appropriately diluted, spread on PDA plates, and incubated at 30°C in the dark for 48–72 h. The number of surviving colonies was counted, and the lethality rate was calculated. The irradiation time with a lethality rate between 90% and 95% was selected as the optimal mutagenesis condition. It was determined that an irradiation time of 90 s resulted in a lethality rate of approximately 92%, which was determined to be the optimal treatment time for UV mutagenesis in this experiment.

[0049] 3.4 Initial screening of mutant strains—casein clear zone method The spore suspension treated with the optimal irradiation time was appropriately diluted and spread onto casein selection medium plates (containing 1.5% NaCl for preliminary screening of salt-tolerant mutants) and incubated at 30°C in the dark for 48–72 h. Single colonies with excellent morphology, clear transparent zones, and HC values ​​≥3.5 were selected and transferred for preservation. Approximately 89 candidate mutant strains were obtained through screening.

[0050] 3.5 Secondary screening of mutant strains—shake flask fermentation and preliminary evaluation of salt tolerance Candidate mutant strains obtained from the initial screening were subjected to shake-flask fermentation, with 3% NaCl added to the culture medium to preliminarily assess the salt tolerance and enzyme production capacity of the strains. After incubation at 30℃ and 120 rpm for 72 h, the activities of neutral protease, amylase, and cellulase in the fermentation broth were measured. Using HC value and enzyme activity as comprehensive indicators, superior mutant strains with an F value (comprehensive score) greater than 2.5% were screened, resulting in a total of 12 dominant mutant strains. The strain with the best overall performance was named *Aspergillus oryzae* M3-2.

[0051] IV. Directed acclimatization under high salinity gradient (salt stress screening) 4.1 Gradient High-Salt Plate Acclimation The dominant strain M3-2 obtained by UV mutagenesis screening was used to prepare a spore suspension according to method 2.1, and the spore concentration was adjusted to 1×10⁻⁶. 6 ~1×10 7 spores / mL. The spore suspension was spread onto solid culture plates containing different NaCl concentration gradients: 6%, 9%, 12%, 15%, and 18% (w / v). Three parallel plates were prepared for each concentration gradient, and the plates were incubated at 30°C.

[0052] Acclimation process: M3-2 was sequentially subcultured on 6%→9%→12%→15%→18% NaCl plates. Each concentration was subcultured 3–5 times, with each subculture lasting 5–7 days. After each acclimation, colony growth and the size of the clear zone were observed. Colonies with good growth and clear zones were selected and transferred to the next higher concentration acclimation plate.

[0053] 4.2 Evaluation of enzyme production performance under high salt stress After each stage of acclimatization, the best-growing single colonies were selected for shake-flask fermentation (with the same NaCl concentration in the culture medium as the acclimatization stage), and cultured at 30℃ and 120 rpm for 72 h. The activities of neutral protease, acidic protease, and amylase were then measured. The enzyme production capacity and enzyme activity retention rate of the strains under high-salt environments of 15% NaCl and 18% NaCl were evaluated in particular.

[0054] 4.3 Determination of the final target strain After complete gradient acclimatization, strains that could still grow normally on 18% NaCl plates, with clear transparent zones, and exhibiting the highest retention rates of neutral and acidic protease activities under 15% NaCl conditions were selected. This strain was the final target strain selected and preserved in this study, named *Aspergillus oryzae* QHJ003, and its taxonomic name is [not specified]. Aspergillus oryzae It was deposited on March 27, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42541. The address of the depository is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0055] Example 1 A method for preparing soy sauce containing active bio-enzymes includes the following steps: s1. Activation of high enzyme-producing strain: The Aspergillus oryzae strain QHJ003 of CGMCC No. 42541 preserved in the specific embodiment was inoculated into PDA slant medium and activated at 30℃ for 4 days to obtain a plump, contaminant-free seed strain rich in yellow-green spores for later use.

[0056] s2. Pretreatment of raw materials for koji making: Select defatted soybeans and dehulled wheat, mix them evenly at a mass ratio of 6.5:3.5; soak in water at 28℃ for 5 hours, stirring once every 1 hour, and drain the free water after soaking; cook at 102℃ under normal pressure for 35 minutes to achieve moderate denaturation of protein and complete gelatinization of starch; after cooking, air cool and spread out to 30℃, control the moisture content of the raw materials to 50%, and obtain uniform and sterile raw materials for koji making.

[0057] s3. Segmented temperature-controlled high-enzyme-activity koji making: The activated QHJ003 seed culture is evenly mixed into the koji-making raw materials at an inoculation rate of 0.4% of the total raw material mass and placed in a constant temperature and humidity koji-making disc for cultivation, with a relative humidity of 92% throughout the process; a three-stage precise koji-making process is adopted: the early stage is cultivated at 29℃ for 12 h, the middle stage at 31℃ for 12 h, and the late stage at 27℃ for 12 h; the koji is turned once at 12 h and 24 h respectively, with a total koji-making time of 36 h; after the koji-making is completed, the koji is tested: neutral protease activity 1893 U / g, amylase activity 2657 U / g, cellulase activity 833 U / g, the koji is loose, the spores are full, and there is no moldy or off-flavor.

[0058] s4. Low-temperature, long-cycle, high-salt, dilute-state fermentation: Prepare a 19% food-grade sterile brine solution. Mix the mature, highly active koji with the sterile brine solution at a mass ratio of 1:2.8 and stir at low speed for 12 minutes until completely homogeneous and free of dry material lumps to prepare homogeneous fermented sauce mash. Transfer the fermented sauce mash to a sealed fermentation tank and ferment at a constant temperature of 22℃ for 280 days. Stir and turn the mash every 15 days during the fermentation process, with each stirring session lasting 12 minutes to break up the surface crust, promote the exchange of substances in the system, and ensure uniform fermentation. No exogenous enzyme preparations, preservatives, or artificial flavor enhancers are added throughout the entire process.

[0059] s5. Gradient Temperature and Mild Enzyme Preservation Sterilization: The fermented and matured soy sauce mash is extracted by spiral pressing, filtered through a plate and frame filter to remove solid residues, and allowed to stand for 24 hours to clarify naturally, removing bottom sediment impurities to obtain clarified soy sauce concentrate. A graded mild enzyme preservation sterilization process is adopted: the first stage is held at 62℃ for 18 minutes to kill common bacteria such as yeast, mold, and E. coli; the second stage is held at 72℃ for 12 minutes to thoroughly kill heat-resistant spores and meet food aseptic safety standards; the third stage is cooled to 58℃ for constant temperature precision filtration sterilization to avoid enzyme denaturation and inactivation caused by high temperature, and to retain the activity of natural biological enzymes to the greatest extent.

[0060] s6. Cooling and Filling: The sterilized and filtered soy sauce concentrate is naturally and slowly cooled to room temperature. All physical, chemical, microbiological, and sensory indicators are tested. After passing the tests, the concentrate is aseptically filled, capped, and labeled in a Class 10,000 cleanroom to produce a high-activity, high-flavor, and high-stability high-salt diluted soy sauce product containing active biological enzymes.

[0061] Example 2 A method for preparing soy sauce containing active bio-enzymes includes the following steps: s1. Activation of high enzyme-producing strains: The preserved CGMCC No. 42541 Aspergillus oryzae QHJ003 strain was inoculated into PDA slant medium and activated at 30℃ for 4 days to obtain plump, contaminant-free seed strains rich in yellow-green spores for later use.

[0062] s2. Pretreatment of raw materials for koji making: Select defatted soybeans and dehulled wheat, mix them evenly in a mass ratio of 6:4, soak them in water at 25℃ for 6 hours, stirring once every 1 hour, and drain the free water after soaking; cook at 100℃ and normal pressure for 40 minutes to achieve moderate denaturation of protein and complete gelatinization of starch; after cooking, air cool and spread out to 28℃, and control the moisture content of the raw materials to 48% to obtain uniform and sterile raw materials for koji making.

[0063] s3. Segmented temperature-controlled high-enzyme-activity koji making: The activated QHJ003 seed culture is evenly mixed into the koji-making raw materials at an inoculation rate of 0.3% of the total raw material mass and placed in a constant temperature and humidity koji-making disc for cultivation, with a relative humidity of 90% throughout the process; a three-stage precise koji-making process is adopted: the early stage is cultivated at 28℃ for 12 h, the middle stage at 30℃ for 12 h, and the late stage at 26℃ for 12 h; the koji is turned once at 12 h and 24 h respectively, with a total koji-making time of 40 h; after the koji-making is completed, the koji is tested and found to have: neutral protease activity of 1822 U / g, amylase activity of 2586 U / g, and cellulase activity of 814 U / g. The koji is loose, with full spores and no moldy or off-flavors.

[0064] s4. Low-temperature, long-cycle, high-salt, dilute-state fermentation: Prepare a food-grade sterile brine with a mass concentration of 18%. Mix the mature, highly active koji with the sterile brine at a mass ratio of 1:2.5 and stir at low speed for 12 minutes until completely homogeneous and free of dry material lumps to prepare homogeneous fermented mash. Transfer the fermented mash to a sealed fermentation tank and ferment at a constant temperature of 20℃ for 380 days. Stir and turn the mash every 15 days during fermentation, with each stirring session lasting 10 minutes. This breaks up the surface crust, promotes the exchange of substances in the system, and ensures uniform fermentation. No exogenous enzymes, preservatives, or artificial flavor enhancers are added throughout the entire process.

[0065] S5. Gradient Temperature and Mild Enzyme Preservation Sterilization: The fermented and matured soy sauce mash is extracted by spiral pressing, filtered through a plate and frame filter to remove solid residues, and allowed to stand for 24 hours to clarify naturally, removing bottom sediment impurities to obtain clarified soy sauce concentrate. A graded mild enzyme preservation sterilization process is adopted: the first stage is held at 60℃ for 20 minutes to kill common bacteria such as yeast, mold, and E. coli; the second stage is held at 70℃ for 15 minutes to thoroughly kill heat-resistant spores and meet food aseptic safety standards; the third stage is cooled to 55℃ for constant temperature precision filtration sterilization to avoid enzyme denaturation and inactivation caused by high temperature and to retain the activity of natural biological enzymes to the greatest extent.

[0066] s6. Cooling and Filling: The sterilized and filtered soy sauce concentrate is naturally and slowly cooled to room temperature. All physical, chemical, microbiological, and sensory indicators are tested. After passing the tests, the concentrate is aseptically filled, capped, and labeled in a Class 10,000 cleanroom to produce a high-activity, high-flavor, and high-stability high-salt diluted soy sauce product containing active biological enzymes.

[0067] Example 3 A method for preparing soy sauce containing active bio-enzymes includes the following steps: s1. Activation of high enzyme-producing strains: The preserved CGMCC No. 42541 Aspergillus oryzae QHJ003 strain was inoculated into PDA slant medium and activated at 30℃ for 4 days to obtain plump, contaminant-free seed strains rich in yellow-green spores for later use.

[0068] s2. Pretreatment of raw materials for koji making: Select defatted soybeans and dehulled wheat, mix them evenly at a mass ratio of 7:3, soak them in water at 30℃ for 4 hours, stirring once every 1 hour, and drain the free water after soaking; cook at 105℃ under normal pressure for 30 minutes to achieve moderate denaturation of protein and complete gelatinization of starch; after cooking, air cool and spread out to 32℃, and control the moisture content of the raw materials to 52% to obtain uniform and sterile raw materials for koji making.

[0069] s3. Segmented temperature-controlled high-enzyme-activity koji making: The activated QHJ003 seed culture was evenly mixed into the koji-making raw materials at an inoculation rate of 0.5% of the total raw material mass and placed in a constant temperature and humidity koji-making disc for cultivation, with a relative humidity of 95% throughout the process; a three-stage precise koji-making process was used: the early stage was cultivated at 30℃ for 12 h, the middle stage at 32℃ for 12 h, and the late stage at 28℃ for 12 h; the koji was turned once at 12 h and 24 h respectively, with a total koji-making time of 45 h; after the koji-making was completed, the koji was tested and found to have a neutral protease activity of 1915 U / g, an amylase activity of 2717 U / g, and a cellulase activity of 846 U / g. The koji was loose, with full spores and no moldy or off-flavors.

[0070] s4. Low-temperature, long-cycle, high-salt, dilute-state fermentation: Prepare a 20% (w / w) food-grade sterile brine. Mix the mature, highly active koji with the sterile brine at a mass ratio of 1:3 and stir at low speed for 12 minutes until completely homogeneous and free of dry material lumps to prepare homogeneous fermented mash. Place the fermented mash into a sealed fermentation tank and ferment at a constant temperature of 25℃ for 180 days. Stir and turn the mash every 15 days during fermentation, with each stirring session lasting 15 minutes. This breaks up the surface crust, promotes the exchange of substances in the system, and ensures uniform fermentation. No exogenous enzymes, preservatives, or artificial flavor enhancers are added throughout the entire process.

[0071] S5. Gradient Temperature and Mild Enzyme Preservation Sterilization: The fermented and matured soy sauce mash is extracted by spiral pressing, filtered through a plate and frame filter to remove solid residues, and allowed to stand for 24 hours to clarify naturally, removing bottom sediment impurities to obtain clarified soy sauce concentrate. A graded mild enzyme preservation sterilization process is adopted: the first stage is held at 65℃ for 15 minutes to kill common bacteria such as yeast, mold, and E. coli; the second stage is held at 75℃ for 10 minutes to thoroughly kill heat-resistant spores and meet food aseptic safety standards; the third stage is cooled to 60℃ for constant temperature filtration and sterilization to avoid enzyme denaturation and inactivation caused by high temperature, and to retain the activity of natural biological enzymes to the greatest extent.

[0072] s6. Cooling and Filling: The sterilized and filtered soy sauce concentrate is naturally and slowly cooled to room temperature. All physical, chemical, microbiological, and sensory indicators are tested. After passing the tests, the concentrate is aseptically filled, capped, and labeled in a Class 10,000 cleanroom to produce a high-activity, high-flavor, and high-stability high-salt diluted soy sauce product containing active biological enzymes.

[0073] Comparative Example 1 (Traditional commercial strains + conventional processes + high-temperature sterilization) Commercially available Aspergillus oryzae strains were used, and a conventional koji-making process of 48–72 hours was employed. Exogenous compound enzyme preparations were added during the fermentation process, and the fermentation cycle was 100 days. After fermentation, high-temperature instantaneous sterilization was carried out at 121°C for 15 minutes. The remaining raw material ratios and pretreatment methods were the same as in Example 1.

[0074] Comparative Example 2 (Common bacterial strain + Invention process + High-temperature sterilization) Referring to the koji-making and fermentation process in the published patent CN 121647370 A "A method for improving the quality of high-salt thin-state soy sauce and high-salt thin-state soy sauce", the original strain M3 in this application (i.e. without ARTP, ultraviolet mutagenesis and high-salt domestication) is used. The koji-making and low-temperature long-cycle fermentation process of Example 1 of this invention is adopted. After fermentation, traditional high-temperature instantaneous sterilization at 121℃ is used without the addition of exogenous enzymes.

[0075] Comparative Example 3 (QHJ003 strain of this invention + high-temperature sterilization) The preserved Aspergillus oryzae strain QHJ003 (CGMCC No. 42541) and the complete koji-making and low-temperature fermentation process of this invention were used. After fermentation, the traditional 121°C high-temperature instantaneous sterilization process in this field was replaced. All other parameters were completely consistent with those in Example 1.

[0076] Performance test results 1. Enzyme activity in koji production: In Examples 1-3 of this invention, strain QHJ003 was used for koji production. The neutral protease activity was ≥1800 U / g, amylase activity was ≥2500 U / g, and cellulase activity was ≥800 U / g, which is more than 30% higher than that of ordinary commercial strains. The overall enzyme activity of koji produced by Comparative Examples 1 and 2 was significantly lower, and there was no advantage in high enzyme production.

[0077] Table 1. Comparison of comprehensive enzyme activity between the examples and the comparative examples.

[0078] 2. Enzyme activity residue performance during fermentation: The present invention adopts a low temperature long-cycle fermentation process of 15-25℃, which effectively slows down the thermal inactivation of enzymes. After fermentation, the enzyme activity residue rate of the fermented mash is stably maintained at more than 25%. In contrast, the enzyme activity residue rate of traditional high temperature short-cycle fermentation is less than 10%, the enzyme action time is greatly shortened, and the raw material hydrolysis is insufficient.

[0079] Table 2 Comparison of enzyme activity residue in fermented mash of the Example and Comparative Examples

[0080] 3. Retention performance of finished product active enzymes: Examples 1-3 of this invention use a gradient mild sterilization process, and the retention rate of natural protease, amylase and cellulase in the finished product can reach more than 85%; Comparative Examples 1, 2 and 3 use traditional high-temperature sterilization, and the enzyme proteins are almost completely denatured and inactivated, with a finished product enzyme activity retention rate of less than 4%, and they basically lose their biological activity.

[0081] Table 3 Comparison of enzyme activity between the examples and the comparative examples

[0082] 4. Nutritional and flavor indicators: The amino acid nitrogen content of the finished product in the embodiment of this invention is ≥1.28 g / 100mL, which is significantly higher than that of the comparative examples; the product has a pure and rich soy sauce aroma, a mellow and smooth taste, no raw or bitter taste, and a full flavor profile; the comparative products have lower amino acid nitrogen content, a thin taste, a light soy sauce aroma, and some samples have a burnt bitter taste.

[0083] Table 4 Comparison of amino acid nitrogen content and sensory evaluation scores between the examples and comparative examples

[0084] 5. Storage stability: The finished product of this invention remains clear and bright, without layering, turbidity or sediment, and its flavor decays very slowly after 12 months of storage at room temperature; the comparative sample showed slight sedimentation, loss of luster, and a weakening of flavor after 6 months of storage, indicating poor storage stability.

[0085] Table 5 Comparison of the storage conditions of finished products in the examples and comparative examples

[0086] The embodiments described above merely illustrate specific implementation methods of this application, and while the descriptions are detailed, they should not be construed as limiting the scope of protection of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the technical solution of this application, and these modifications and improvements all fall within the scope of protection of this application.

[0087] This background section is provided to generally present the context of the invention. The work of the currently named inventors, the work to the extent described in this background section, and aspects of this section that did not constitute prior art at the time of application are neither expressly nor impliedly acknowledged as prior art to the invention.

Claims

1. A strain of Aspergillus oryzae that produces a high amount of complex biological enzymes ( Aspergillus oryzae The strain is characterized by: This strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on March 27, 2026, with accession number CGMCC No. 42541.

2. The Aspergillus oryzae with high production of complex biological enzymes as described in claim 1 ( Aspergillus oryzae Application of strains in the preparation of high-salt dilute-state fermented soy sauce.

3. A method for preparing soy sauce containing active bio-enzymes, characterized in that, Includes the following steps: s1. The Aspergillus oryzae (a high-yield complex bioenzyme) described in claim 1 Aspergillus oryzae The strain was inoculated into slant culture medium and activated at a constant temperature to obtain seed strains; s2. Pretreatment of raw materials for koji making: Defatted soybeans and wheat are selected as the main raw materials. After mixing them, they are soaked, steamed, and cooled to room temperature to obtain sterile raw materials for koji making. s3, High Enzyme Activity Koji Making: The seed culture obtained in s1 is evenly inoculated into the aseptic koji making raw material obtained in s2. The segmented temperature-controlled koji making process is adopted, and the entire process is carried out under constant temperature and humidity. The koji is turned at regular intervals. After the koji matures, high enzyme activity koji is obtained. s4, high-salt dilute state fermentation: add sterile brine to the high-enzyme-active koji obtained in s3, mix evenly and start preparing sauce mash. Control the fermentation environment temperature and salinity, carry out constant temperature and closed fermentation, and do not add any exogenous enzyme preparations throughout the process. Rely on the natural biological enzymes in the koji to complete the decomposition of raw materials and flavor transformation. After fermentation is mature, sauce mash is obtained. s5, Precise Sterilization and Enzyme Preservation: After the fermentation mash obtained in s4 is pressed, filtered and clarified, it is sterilized using a gradient temperature mild sterilization process to retain the active biological enzymes in the finished product to the greatest extent. s6. Finished Product Bottling: The sterilized soy sauce concentrate from s5 is cooled, inspected, and aseptically filled to obtain a high-enzyme-active, high-salt, diluted soy sauce finished product containing active biological enzymes.

4. The method for preparing soy sauce containing active bio-enzymes according to claim 3, characterized in that: In s1, Aspergillus oryzae, which produces high levels of complex bioenzymes ( Aspergillus oryzae The strain exhibits neutral protease activity ≥1800 U / g, amylase activity ≥2500 U / g, and cellulase activity ≥800 U / g in mature koji.

5. A method for preparing soy sauce containing active bio-enzymes according to claim 3, characterized in that: In s2, the mass ratio of defatted soybeans to wheat is 6-7:3-4; the soaking temperature is 25-30℃, and the soaking time is 4-6 h; the cooking is carried out under normal pressure, the cooking temperature is 100-105℃, the cooking time is 30-40 min, and the moisture content of the raw materials after cooking is controlled at 48-52 wt%.

6. A method for preparing soy sauce containing active bio-enzymes according to claim 3, characterized in that: In s3, the inoculation amount of the seed strain is 0.3-0.5% of the total mass of the raw materials for koji making; the segmented temperature-controlled koji making process is as follows: the initial culture temperature is 28-30℃ for 12 h, the middle culture temperature is 30-32℃ for 12 h, and the later culture temperature is 26-28℃ for 12 h; the relative humidity of the culture environment is 90-95%; the koji is turned twice at 12 h and 24 h respectively, and the total koji making time is 35-45 h to obtain mature koji with high enzyme activity.

7. A method for preparing soy sauce containing active bio-enzymes according to claim 3, characterized in that: In s4, the mass concentration of the sterile brine is 18-20%; the mass ratio of the starter culture to the sterile brine is 1:2.5-3; the fermentation temperature is constant at 15-25℃ throughout the process; the closed fermentation cycle is 180-380 days; and the mixture is stirred once every 15 days during the fermentation process, with each stirring lasting 10-15 minutes.

8. A method for preparing soy sauce containing active bio-enzymes according to claim 3, characterized in that, In s5, the gradient heating and sterilization process specifically involves: first heating to 60-65°C and holding for 15-20 minutes, then heating to 70-75°C and holding for 10-15 minutes, and finally cooling to 55-60°C for constant temperature filtration and sterilization.

9. Soy sauce containing active bio-enzymes prepared by the method according to any one of claims 3-8.

10. The soy sauce containing active bio-enzymes according to claim 9, characterized in that: The soy sauce is a high-salt, dilute-state fermented soy sauce. Under normal temperature storage conditions, the neutral protease activity is ≥600 U / 100mL, the amylase activity is ≥900 U / 100mL, the amino acid nitrogen content is ≥1.2 g / 100mL, and the enzyme activity retention rate is high, with stable and long-lasting flavor.

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