Preparation method of sodium-reduced zero-sugar soy sauce

Abstract

The present application relates to a kind of preparation methods of sodium-reduced zero-sugar soy sauce, belong to the technical field of condiment preparation.The preparation method of sodium-reduced zero-sugar soy sauce of the present application includes the following steps:S1 first fermentation: after the first fermentation of the koji material and brine into fermentation tank, then add composite enzyme preparation 1 and ferment, prepare sauce mash;S2 second fermentation: after the second fermentation of adding into sauce mash and then connecting into composite strain, ferment;S3 third fermentation: after the third fermentation of adding into sauce mash prepared in step S2 and enzyme mash and composite enzyme preparation 2, ferment;S4 press sterilization, prepare sodium-reduced zero-sugar soy sauce.The total cycle of three-stage fermentation of the present application is controlled in 4-5 months, compared with the traditional high-salt fermentation process of more than 180 days, the fermentation cycle is greatly shortened.The present application uses intermittent aeration and gradient feeding process to reduce energy consumption and production cost, meet the needs of industrial cost reduction and efficiency improvement.

Description

Technical Field

[0001] This invention relates to the field of condiment preparation technology, and in particular to a method for preparing a sodium-reduced and sugar-free soy sauce. Background Technology

[0002] With increasing consumer health awareness and the promotion of the national "three reductions" policy (reducing salt or sodium, sugar, and oil), the demand for zero-sugar and reduced-sodium healthy condiments continues to grow in the market. Traditional brewed soy sauce produces various sugars, such as glucose, fructose, and maltose, through microbial decomposition during fermentation. Its total sugar content is typically between 3-8g / 100mL, which is insufficient to meet the national standard GB 28050's requirement for "0 sugar" claims (total sugar ≤0.5g / 100mL). Simultaneously, to meet the needs of preservation and fermentation, traditional soy sauce generally has a high salt content of 15-18g / 100mL and a sodium content of 6000-7200mg / 100ml. Long-term excessive intake can easily increase health risks such as hypertension and cardiovascular disease. Therefore, sodium reduction has become a key direction for the upgrading of the soy sauce industry.

[0003] Existing patented technologies have attempted to reduce the sugar content of soy sauce from multiple dimensions. For example, the invention patent (application number: 202110286587.7) introduces a compound enzyme preparation (such as amylase) in the raw material pretreatment stage for pre-saccharification intervention to reduce fermentable sugar sources; after fermentation, it is heated (maintained at 40-50°C for 8-24 hours) and uses encapsulated bacterial agents to promote the metabolism of residual sugars; the invention patent (patent application number CN202311271938.2) mainly consumes sugars by preparing wheat mash and inoculating it with *Saccharomyces cerevisiae* CZ1 during fermentation; the invention patent (patent application number CN202411959717.9) produces sugar-free soy sauce by adding gluten powder, using compound bacteria and enzymatically hydrolyzed mash prepared with added enzymes for mixed fermentation, and adding *Rhodotorula glutinis* for secondary fermentation after fermentation maturity; in addition, the preparation process in the invention patent (patent application number CN202210633096.X) is to prepare soy sauce without adding exogenous sugars, but does not measure or process the sugar content of the fermented soy sauce itself.

[0004] The above-mentioned invention patents all provide technical solutions for the preparation of low-sugar or zero-sugar soy sauce, but some shortcomings still exist: First, over-reliance on specific microorganisms or enzyme preparations may lead to limited synthesis pathways of flavor substances, resulting in insufficient umami and richness. Furthermore, the biosafety of some strains is not a conventional fermentation strain for soy sauce and needs further investigation. Second, sodium reduction can easily lead to decreased antibacterial ability, unstable fermentation, weak flavor, and insufficient umami, making it difficult to balance the low-sodium environment with fermentation safety. Third, some processes require additional temperature control or extended fermentation cycles, increasing energy consumption and production costs. Fourth, the "zero-sugar" target often comes at the cost of sacrificing amino acid nitrogen content or color stability, affecting the overall quality of the soy sauce. In summary, this invention provides a method for preparing sodium-reduced, zero-sugar soy sauce that simultaneously achieves the sodium reduction target while meeting the zero-sugar requirement (total sugar ≤ 0.5g / 100mL), and the resulting soy sauce possesses excellent sensory and flavor qualities, enabling industrial application. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing sodium-reduced and sugar-free soy sauce.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a method for preparing a sodium-reduced, sugar-free soy sauce, comprising the following steps: S1 First Fermentation: After fermenting the koji and brine in a fermentation tank, compound enzyme preparation 1 is added for further fermentation to obtain soy sauce mash; S2 Second Fermentation: First, add wine-producing mash to the sauce mash obtained in step S1 for fermentation, and then inoculate with compound bacteria for further fermentation; S3 Third fermentation: Add enzyme-producing mash and compound enzyme preparation 2 to the mash obtained in step S2 and ferment simultaneously; S4 involves pressing and sterilizing the mash obtained in step S3 to produce reduced-sodium, zero-sugar soy sauce. Among them, the complex enzyme preparation 1 in S1 is composed of glucose isomerase and mannanase, with a mass ratio of (5-1):1; The composite strain in S2 consists of Hansenula anomala, Pichia pastoris and Pichia chinensis in a mass ratio of (7-2):3:3. The complex enzyme preparation 2 in S3 is composed of glucose oxidase and acidic protease in a mass ratio of 1:(1-4).

[0007] This invention produces a sodium-reduced, sugar-free soy sauce by adding compound enzymes 1 and 2 and compound bacterial strains in stages.

[0008] This invention involves the two-stage addition of compound enzyme preparations 1 and 2 to avoid enzyme inhibition and precisely match the needs of each fermentation stage. At the end of the first fermentation, the glucose isomerase and mannanase compound enzyme preparation 1 is added. Mannanase decomposes polysaccharides in the raw materials, releasing starch and oligosaccharides, while glucose isomerase converts glucose into fructose, which is easily utilized by microorganisms, accelerating monosaccharide consumption. The two enzymes work synergistically to target the decomposition of polysaccharides and optimize monosaccharide metabolic pathways, providing readily available substrates for microorganisms. At the beginning of the third fermentation, the glucose oxidase and acidic protease compound enzyme preparation 2 is added. Glucose oxidase directly oxidizes reducing sugars to gluconic acid, while acidic protease decomposes proteins to generate amino acids, promoting microbial proliferation and enhancing the Maillard reaction to consume sugar sources.

[0009] In the second stage, a combination of *Hansenula anomala*, *Saccharomyces cerevisiae*, and *Pichia pastoris* is added to form a stepwise metabolic chain of "disaccharides—monosaccharides—residual sugars." *Saccharomyces cerevisiae* can decompose disaccharides such as maltose and sucrose and secrete a small amount of saccharifying enzymes to assist in the hydrolysis of dextrin. *Hansenula anomala* primarily focuses on monosaccharide metabolism and generates flavor compounds, while *Pichia pastoris* further consumes residual monosaccharides and some oligosaccharides. This eliminates the need for large amounts of additional enzymes, resulting in higher sugar reduction efficiency. *Hansenula anomala* generates aromatic substances such as ethyl acetate, while *Pichia pastoris* produces lactic acid-based flavor compounds, solving the problem of thin umami and insufficient richness in traditional low-sodium, zero-sugar soy sauce, thus creating a unique flavor profile. Compared to the traditional *Saccharomyces rouxii* + *Saccharomyces globosa* combination, this combination has a significant advantage in the types of sugars metabolized. The traditional combination can only preferentially metabolize monosaccharides such as glucose and fructose and a small amount of maltose, with weak ability to decompose disaccharides and oligosaccharides. It requires a large amount of external enzymes to hydrolyze polysaccharides into monosaccharides before metabolism. In contrast, the yeast combination of this invention enhances sugar metabolism, covering a wider range of sugar types.

[0010] This invention adds alcohol-producing mash and enzyme-producing mash to the fermentation system. The alcohol-producing mash provides suitable ethanol to inhibit unwanted microorganisms, balances the low-sodium environment with fermentation stability, and provides carbon and nitrogen nutrient substrates to promote the rapid proliferation of complex yeasts and enhance the efficiency of stepwise sugar metabolism. The enzyme-producing mash continuously provides endogenous enzyme systems such as saccharifying enzymes, proteases, and lactases, which work synergistically with exogenous enzymes to achieve full-cycle sugar reduction.

[0011] Furthermore, after in-depth research and experimental verification, this invention has also discovered that through the synergistic effect of two carefully selected and matched sets of compound enzymes and compound microbial strains, combined with the participation of endogenous enzymes in the enzyme-producing mash, the sodium content in soy sauce can be effectively reduced. This unique process allows the zero-sugar soy sauce produced by this invention to significantly reduce its sodium content to 4400-4800 mg / 100 ml, thereby further improving the overall quality of the soy sauce and greatly enhancing and optimizing its health benefits and taste.

[0012] Furthermore, in S1, the mass ratio of glucose isomerase to mannanase is 3:1; in S2, the mass ratio of *Hansenula anomala*, *Cytomyces clausti*, and *Pichia pastoris* is 4:3:3; and in S3, the mass ratio of glucose oxidase to acidic protease is 1:2.

[0013] Through optimization, it was found that when the mass ratio of each component in the above-mentioned compound enzyme preparation and compound microbial agent is as described above, the sugar reduction and salt reduction effects of the fermentation system can be significantly improved.

[0014] In a preferred embodiment of the first aspect, the amount of compound enzyme preparation 1 added in step S1 is 0.05-0.1% based on the total mass of the koji and brine; the amount of compound bacteria added in step S2 is 0.2-0.6% based on the mass of the fermented mash without added yeast; and the amount of compound enzyme preparation 2 added in step S3 is 0.03-0.08% based on the total mass of the fermented mash in step S2.

[0015] In a preferred embodiment of the first aspect, the total amount of mash added in step S2 is 0.1-0.3 times the mass of the sauce mash obtained in step S1, and the amount of enzyme mash added in step S3 is 0.3-0.6 times the total mass of the sauce mash obtained in step S2.

[0016] As a preferred embodiment of the first aspect, in step S1, the koji material and brine are put into a fermentation tank and fermented at 15-20°C for 20-25 days, and then compound enzyme preparation 1 is added and the temperature is raised to 23-27°C for 20-25 days of fermentation.

[0017] In this stage, the invention employs variable-temperature fermentation. First, a temperature of 15-20℃ is used to promote the targeted metabolism of sugar sources by microorganisms. Then, in the later stages of fermentation, the temperature of the fermented mash is raised to 23-27℃, which is the optimal reaction temperature for the compound enzyme preparation, maximizing enzyme activity. Mannanase breaks down polysaccharides in the raw materials, releasing starch and oligosaccharides, while glucose isomerase converts glucose into fructose, which is easily utilized by microorganisms, accelerating the consumption of monosaccharides. Experiments have shown that, compared to isothermal or constant-temperature fermentation (Example 5), this variable-temperature fermentation stage improves the sugar-reducing effect of the fermentation system.

[0018] Through comparative experiments (Example 6), this invention found that when the compound enzyme preparation, koji, and brine are fermented simultaneously in step S1, it affects the sugar decomposition capacity of the fermentation system, leading to an increase in the sugar content of the soy sauce. Therefore, this invention adopts a method of first fermenting the koji and brine, followed by fermentation of the compound enzyme preparation 1, to further enhance the sugar-reducing capacity of the fermentation system.

[0019] Furthermore, in step S1, the mass ratio of the koji material to the brine is 1:(2.0-2.2).

[0020] Further, the preparation method of the koji material in S1 is as follows: after mixing roasted wheat and soybeans, inoculate with Aspergillus oryzae, the koji-making temperature is 28-32℃, and the koji-making time is 44-48h; the mass ratio of roasted wheat to soybeans is (1-2):(8-9); the inoculation amount of Aspergillus oryzae is 0.3-0.6% by mass (based on the mass of materials).

[0021] Furthermore, the soybeans are subjected to microwave and steaming treatment, specifically as follows: after screening and washing, the soybeans are microwaved at 300-500W power for 5-10 minutes; the treated soybeans are soaked in warm water for 30-60 minutes, put into a steaming pot, and steamed at 0.1-0.2MPa pressure for 30-40℃ for 30 minutes, and then cooled to 30-40℃ before being taken out.

[0022] Through comparative experiments (Example 4), this invention found that microwave treatment can destroy the cell wall structure of soybeans, accelerate the subsequent decomposition of starch and protein, and reduce the production of residual sugar.

[0023] Furthermore, the wheat undergoes roasting and grinding processes, specifically: the screened wheat is roasted using a roasting machine, ground, and then passed through a 30-mesh sieve, with the wheat flour content controlled at 15-30%.

[0024] Furthermore, the method for preparing the S2 mash is as follows: inoculating wheat flour slurry with brewing yeast for fermentation, with a fermentation pH of 4.0-5.0 and a fermentation temperature of 25-30℃, first carrying out aerobic fermentation and then carrying out anaerobic fermentation; the aeration rate for aerobic fermentation is 0.5-1.5 vvm, aerobic fermentation lasts for 3-5 days, and anaerobic fermentation lasts for 7-10 days.

[0025] As a preferred embodiment of the first aspect, the specific method for the second fermentation in step S2 includes the following steps: (1) Add the fermenting mash to the mash in step S1 in batches. Each addition is 0.03-0.1 times the mass of the mash without fermenting mash. The interval between each addition is 10 days, for a total of 30 days. The fermentation temperature is 23-30℃. (2) Inoculate the compound bacteria into the sauce mash of step (1) and ferment for 20-30 days at a temperature of 23-30℃.

[0026] This invention discovers that gradient replenishment of mash can avoid the inhibition of yeast activity by a single high alcohol concentration, and that the complex yeast combination can improve the efficiency of alcohol and ester flavor substance production, while enhancing sugar metabolism.

[0027] Furthermore, in step S2, the fermentation temperature of the compound microbial strain is 23-30℃, and the fermentation time is 20-30 days.

[0028] Furthermore, the preparation method of the S3 enzyme-producing mash is as follows: Rhizopus oryzae and yeast extract are added to wheat flour slurry for fermentation. The fermentation pH is 5.0-6.0, the temperature is 28-32℃, the aeration rate is 1-2 vvm, and the fermentation time is 3-7 days. The amount of Rhizopus oryzae added is 0.2-0.5% by mass (based on the mass of roasted wheat flour), and the amount of yeast extract added is 1.0% by mass (based on the mass of roasted wheat flour).

[0029] This invention, through comparative experiments (Comparative Example 14), found that *Rhizopus oryzae* can produce specific enzyme systems such as saccharifying enzymes, proteases, and lactases. Lactase does not directly degrade sugars, but indirectly enhances the hypoglycemic effect through a three-pronged mechanism: First, it catalyzes the conversion of pyruvate, an intermediate product of glucose metabolism, into lactate, consuming these intermediate products to promote the continuous decomposition of glucose. Simultaneously, lactate enhances the umami and richness of soy sauce. Second, the generated lactate lowers the pH of the fermented soy sauce mash, creating a weakly acidic environment that enhances the metabolic activity of *Hansenula polymorpha* and other strains on residual sugars, forming a dual hypoglycemic pathway of "enzymatic conversion + microbial metabolism." Third, it promptly consumes pyruvate, preventing its accumulation and inhibition of sugar decomposition, ensuring the smooth operation of the sugar metabolism pathway. The endogenous enzymes of *Rhizopus oryzae* also work synergistically with compound enzyme preparation 2 to efficiently decompose residual sugars.

[0030] As a preferred embodiment of the first aspect, the fermentation method in S3 is: intermittent aeration mode, with a 12-hour aeration followed by a 12-hour aeration stop cycle, a fermentation temperature of 28-30℃, and fermentation for 30-40 days until maturity.

[0031] Secondly, the present invention provides a sodium-reduced, sugar-free soy sauce prepared by the method of the first aspect, wherein the total sugar content of the soy sauce is ≤0.5g / 100mL and the sodium content of the soy sauce is 4400-4800mg / 100ml.

[0032] A sodium-reduced, sugar-free soy sauce was prepared using the method described above. The total sugar content was determined to be ≤0.5g / 100mL, and the sodium content was 4400-4800mg / 100ml. This meets the national standards for "0 sugar" and "reduced sodium" soy sauce and achieves the desired sodium reduction effect.

[0033] Thirdly, the present invention provides a compound enzyme preparation, wherein the compound enzyme preparation is composed of glucose isomerase and mannanase, and the mass ratio of glucose isomerase to mannanase is (5-1):1; or, the compound enzyme preparation is composed of glucose oxidase and acidic protease, and the mass ratio of glucose oxidase to acidic protease is 1:(1-4).

[0034] This invention discovers a synergistic effect between glucose isomerase and mannanase, as well as between glucose oxidase and acidic protease. Using these synergistic effects in the preparation of soy sauce can reduce the total sugar and sodium content in soy sauce.

[0035] Fourthly, the present invention provides a compound microbial agent, which is composed of *Hansenula anomala*, *Cytomyces clausti*, and *Pichia pastoris*, with the mass ratio of *Hansenula anomala*, *Cytomyces clausti*, and *Pichia pastoris* being (7-2):3:3.

[0036] This invention discovers a synergistic effect among *Hansenula anomala*, *Pichia pastoris*, and *Pichia giardi*, and their use in soy sauce preparation can reduce the total sugar and sodium content in soy sauce.

[0037] Fifthly, the present invention provides the application of the compound enzyme preparation described in the third aspect or the compound microbial agent described in the fourth aspect in the preparation of sodium-reduced and sugar-free soy sauce.

[0038] Compared with the prior art, the beneficial effects of the present invention are as follows: The strains used in this invention are safe and require no equipment modification: the Aspergillus oryzae, Hansenula anomala, Saccharomyces cerevisiae, Pichia pastoris, and Rhizopus oryzae used are all food-grade safe strains, posing no risk of introducing specific strains. The production equipment uses conventional soy sauce fermentation equipment, requiring no additional investment or modification, facilitating industrial-scale promotion.

[0039] Shorter cycle and lower energy consumption: The total fermentation cycle of the three-stage process is controlled within 4-5 months, which is significantly shorter than the traditional high-salt fermentation process of more than 180 days. Intermittent aeration and gradient feeding processes reduce energy consumption and production costs, meeting the industry's needs for cost reduction and efficiency improvement. Detailed Implementation

[0040] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0041] The microbial information used in the embodiments of this invention is as follows: Aspergillus oryzae ( Aspergillus oryzae ): Strain number CICC 2339, purchased from China Industrial Microbial Culture Collection Center (CICC).

[0042] brewing yeast ( Saccharomyces cerevisiae ): Strain number CICC 31759, purchased from China Industrial Microbial Culture Collection Center (CICC).

[0043] Abnormal Hansenula polymorpha ( Anomaly Hansenula ): Strain number CICC 31343, purchased from China Industrial Microbial Culture Collection Center (CICC).

[0044] Capsule-forming yeast ( Saccharomycopsis fibuligera ): Strain number CICC 33667, purchased from China Industrial Microbial Culture Collection Center (CICC).

[0045] Jiyemeng Pichia pastoris ( Pichia guilliermondii ): Strain number CICC 1232, purchased from China Industrial Microbial Culture Collection Center (CICC).

[0046] Rhizopus oryzae ( Rhizopus rice ): Strain number CICC 41567, purchased from China Industrial Microbial Culture Collection Center (CICC).

[0047] Saccharomyces cerevisiae ( Torulopsis candida ): Strain number CICC 1019, purchased from China Industrial Microbial Culture Collection Center (CICC).

[0048] Red yeast rice ( Rhodotorula mucilaginosa ): Strain number CICC 33371, purchased from China Industrial Microbial Culture Collection Center (CICC).

[0049] Aspergillus niger ( Aspergillus niger ): Strain number CICC 2041, purchased from China Industrial Microbial Culture Collection Center (CICC).

[0050] The enzyme preparations used in the embodiments of this invention are as follows: Glucose isomerase: enzyme activity 100,000-500,000 U / g, food grade, purchased from Shandong Longket Enzyme Preparation Co., Ltd.

[0051] Mannanase: enzyme activity 50,000-100,000 U / g, food grade, purchased from Jiangsu Yinong Biotechnology Co., Ltd.

[0052] Glucose oxidase: enzyme activity 10,000-100,000 U / g, food grade, purchased from Novozymes (China) Investment Co., Ltd.

[0053] Acidic protease: enzyme activity 50,000-100,000 U / g, food grade, purchased from Shandong Longket Enzyme Preparation Co., Ltd.

[0054] α-Amylase: Enzyme activity 100,000 U / g, food grade, purchased from Novozymes (China) Investment Co., Ltd.

[0055] Example 1 This embodiment provides a method for producing low-sodium, zero-sugar soy sauce, including the following steps: 1. Raw material processing: 1.1 Microwave and steaming treatment of soybeans: After screening and washing, the soybeans are microwaved for 8 minutes at 400W power. The treated soybeans are then soaked in warm water for 40 minutes, put into a steamer, steamed for 20 minutes at 0.15MPa pressure, and then cooled to 35℃ before being taken out.

[0056] 1.2 Roasting and grinding of wheat: The screened wheat is roasted by a wheat roasting machine, crushed and passed through a 30-mesh sieve, with the wheat flour content controlled at 20%. The crushed wheat is divided into three portions, which are used for the following processes: making koji, producing wine mash, and producing enzyme mash.

[0057] 2. First fermentation: 2.1 Composition: Roasted wheat and soybeans were mixed in a mass ratio of 1.5:8.5, and then 0.4% (by mass of materials) of Aspergillus oryzae was added. The koji-making temperature was 30℃ and the koji-making time was 45h to obtain the koji material.

[0058] 2.2 Preparation of fermented soybean paste: After adding the starter culture and brine in a fermentation tank at a mass ratio of 1:2.1, fermentation was carried out at 18°C ​​for 22 days. Then, 0.08% (by mass of total material) of compound enzyme preparation 1 was added, the fermentation temperature was raised to 25°C, and fermentation was carried out for 22 days (variable temperature fermentation) to obtain the sauce mash.

[0059] In this step, the compound enzyme preparation 1 is composed of glucose isomerase and mannanase in a mass ratio of 3:1.

[0060] 3. Second fermentation: 3.1 Preparation of wine mash: Roasted wheat flour is mixed with water to prepare a wheat flour slurry with a mass concentration of 20%. 0.4% (by mass of roasted wheat flour) of brewing yeast is added. Fermentation is carried out at pH 4.5 and a temperature of 28°C. The fermentation is first carried out with aeration for 4 days at a rate of 1.0 vvm to promote yeast proliferation. Then, it is sealed and anaerobic fermented for 8 days to produce mash.

[0061] 3.2 Add the wine-producing mash to the mash obtained in step 2.2: The fermenting mash was added to the mash obtained in step 2.2 in three separate additions, each time at an interval of 10 days, for a total of 30 days and a total addition of 0.18 times the original weight of the mash before the addition of the fermenting mash. Then, 0.4% (based on the weight of the mash before the addition of the fermenting mash) of compound bacteria was introduced for fermentation for 25 days, with the fermentation temperature maintained at 25°C.

[0062] In this step, the compound strain consists of *Hansenula anomala*, *Cytomyces clausti*, and *Pichia pastoris*, in a mass ratio of 4:3:3.

[0063] 4. Third fermentation: 4.1 Preparation of enzyme-producing mash: Roasted wheat flour was mixed with water to prepare a wheat flour slurry with a mass concentration of 18%. 0.3% (by mass of roasted wheat flour) of Rhizopus oryzae and 1.0% (by mass of roasted wheat flour) of yeast extract were added. Fermentation was carried out at pH 5.5, fermentation temperature of 30℃, and aeration rate of 1.5 vvm for 5 days to obtain enzyme mash.

[0064] 4.2 Add enzyme-producing mash to the mash obtained in step 3.2: Add 0.4 times (based on the mass of the mash in step 3.2) of the enzyme-producing mash to the mash obtained in step 3.2, and at the same time add 0.05% (based on the mass of the mash in step 3.2) of the compound enzyme preparation 2; at the same time, switch to intermittent aeration mode for fermentation and ferment for 35 days, and maintain the fermentation temperature at 29°C in this step.

[0065] In this step, the compound enzyme preparation 2 consists of glucose oxidase and acidic protease in a mass ratio of 1:2. The intermittent ventilation mode specifically involves a cycle of 12 hours of ventilation followed by 12 hours of ventilation stoppage.

[0066] 5. Pressing and sterilization: The fermented soy sauce mash from step 4.2 was pressed and filtered (diatomaceous earth filter, filtration pressure 0.25 MPa, filtration temperature 30℃), and then pasteurized (sterilization temperature 90℃, sterilization time 20min) to obtain sodium-reduced and sugar-free soy sauce.

[0067] Example 2 The difference between Example 2 and Example 1 lies in the parameters. The specific steps of Example 2 are as follows: 1. Raw material processing: 1.1 Microwave and steaming treatment of soybeans: After screening and washing, the soybeans are microwaved at 300W for 10 minutes. The treated soybeans are then soaked in warm water for 60 minutes, put into a steamer, steamed at 0.2MPa pressure for 10 minutes, and then cooled to 40℃ before being taken out.

[0068] 1.2 Roasting and grinding of wheat: The screened wheat is roasted by a wheat roasting machine, crushed and passed through a 30-mesh sieve, with the wheat flour content controlled at 15%. The crushed wheat is divided into three portions, which are used for the following processes: making koji, producing wine mash, and producing enzyme mash.

[0069] 2. First fermentation: 2.1 Composition: Roasted wheat and soybeans were mixed at a mass ratio of 1:8, and then 0.6% (by mass of materials) of Aspergillus oryzae was added. The koji-making temperature was 32℃ and the koji-making time was 44 hours.

[0070] 2.2 Preparation of fermented soybean paste: After adding the starter culture and brine to the fermentation tank at a mass ratio of 1:2.0, ferment at 15°C for 20 days. Then, add 0.05% (by mass of total material) of compound enzyme preparation 1, raise the fermentation temperature to 23°C, and ferment for another 20 days (variable temperature fermentation) to obtain the sauce mash.

[0071] In this step, the compound enzyme preparation 1 is composed of glucose isomerase and mannanase in a mass ratio of 5:1.

[0072] 3. Second fermentation: 3.1 Preparation of wine mash: Roasted wheat flour is mixed with water to prepare a wheat flour slurry with a mass concentration of 15%. 0.2% (by mass of roasted wheat flour) of brewing yeast is added. Fermentation is carried out at pH 4.0 and a temperature of 25°C. The fermentation is first carried out by aeration for 3 days with an aeration rate of 0.5 vvm to promote yeast proliferation. Then, it is sealed and anaerobic fermented for 7 days to produce mash.

[0073] 3.2 Add the wine-producing mash to the mash obtained in step 2.2: The fermenting mash obtained in step 2.2 was added in three separate additions, each time at an interval of 10 days, for a total of 30 days and a total addition of 0.12 times the original weight of the mash before the addition of the fermenting mash. Then, 0.2% (based on the original weight of the mash before the addition of the fermenting mash) of compound bacteria was introduced and fermented for 20 days, with the fermentation temperature maintained at 30°C.

[0074] In this step, the compound strain consists of *Hansenula anomala*, *Cytomyces clausti*, and *Pichia pastoris*, in a mass ratio of 7:3:3.

[0075] 4. Third fermentation: 4.1 Preparation of enzyme-producing mash: Roasted wheat flour was mixed with water to prepare a wheat flour slurry with a mass concentration of 20%. 0.2% (by mass of roasted wheat flour) of Rhizopus oryzae and 0.5% (by mass of roasted wheat flour) of yeast extract were added. Fermentation was carried out at pH 5.0, fermentation temperature 28℃, and aeration rate of 1 vvm for 3 days to obtain enzyme mash.

[0076] 4.2 Add enzyme-producing mash to the mash obtained in step 3.2: Add 0.3 times (based on the mass of the mash from step 3.2) of the enzyme-producing mash to the mash obtained in step 3.2, and simultaneously add 0.03% (based on the mass of the mash from step 3.2) of the compound enzyme preparation 2; at the same time, switch to intermittent aeration mode for fermentation and ferment for 30 days, maintaining the fermentation temperature at 30°C in this step.

[0077] In this step, the compound enzyme preparation consists of glucose oxidase and acidic protease in a mass ratio of 1:4. The intermittent ventilation mode is specifically a cycle of 12 hours of ventilation followed by 12 hours of ventilation stoppage.

[0078] 5. Pressing and sterilization: The fermented soy sauce mash from step 4.2 was pressed and filtered (diatomaceous earth filter, filtration pressure 0.2 MPa, filtration temperature 35℃), and then sterilized by pasteurization (sterilization temperature 85℃, sterilization time 30min) to obtain sodium-reduced and sugar-free soy sauce.

[0079] Example 3 The difference between Example 3 and Example 1 lies in the parameters. The specific steps of Example 3 are as follows: 1. Raw material processing: 1.1 Microwave and steaming treatment of soybeans: After screening and washing, the soybeans are microwaved for 5 minutes at 500W power. The treated soybeans are then soaked in warm water for 30 minutes, put into a steamer, and steamed for 40 minutes at 0.1MPa pressure. After cooling to 30℃, they are taken out.

[0080] 1.2 Roasting and grinding of wheat: The screened wheat is roasted by a wheat roasting machine, crushed and passed through a 30-mesh sieve, with the wheat flour content controlled at 30%. The crushed wheat is divided into three portions, which are used for the following processes: making koji, producing wine mash, and producing enzyme mash.

[0081] 2. First fermentation: 2.1 Composition: Roasted wheat and soybeans were mixed in a 2:9 mass ratio, and then 0.3% (by mass of materials) of Aspergillus oryzae was added. The koji-making temperature was 28℃ and the koji-making time was 48h.

[0082] 2.2 Preparation of fermented soybean paste: The raw materials and brine were added to the fermentation tank at a mass ratio of 1:2.2 and fermented at 20°C for 25 days. Then, 0.1% (by mass of total material) of compound enzyme preparation 1 was added, the fermentation temperature was raised to 27°C, and fermentation continued for 25 days (variable temperature fermentation) to obtain the sauce mash.

[0083] In this step, the compound enzyme preparation 1 is composed of glucose isomerase and mannanase in a mass ratio of 1:1.

[0084] 3. Second fermentation: 3.1 Preparation of wine mash: Roasted wheat flour is mixed with water to prepare a wheat flour slurry with a mass concentration of 30%. 0.5% (by mass of roasted wheat flour) of brewing yeast is added. Fermentation is carried out at pH 5.0 and a temperature of 30°C. The fermentation is first carried out with aeration for 5 days at a rate of 1.5 vvm to promote yeast proliferation. Then, it is sealed and anaerobic fermented for 10 days to produce mash.

[0085] 3.2 Add the wine-producing mash to the mash obtained in step 2.2: Add the fermenting mash obtained in step 2.2 in three separate additions, each time by an amount equal to 0.1 times the mass of the mash before adding the fermenting mash, with an interval of 10 days between each addition, for a total of 30 days and a total addition amount of 0.3 times the mass. Then, inoculate with 0.6% (based on the mass of the mash before adding the fermenting mash) of compound bacteria and ferment for 30 days, maintaining the fermentation temperature at 23°C.

[0086] In this step, the compound strain consists of *Hansenula anomala*, *Cytomyces cuspidata*, and *Pichia pastoris*, in a mass ratio of 2:3:3.

[0087] 4. Third fermentation: 4.1 Preparation of enzyme-producing mash: Roasted wheat flour was mixed with water to prepare a wheat flour slurry with a mass concentration of 15%. 0.5% (by mass of roasted wheat flour) of Rhizopus oryzae and 1.5% (by mass of roasted wheat flour) of yeast extract were added. Fermentation was carried out at pH 6.0, fermentation temperature 32℃, and aeration rate of 2 vvm for 7 days to obtain enzyme mash.

[0088] 4.2 Add enzyme-producing mash to the mash obtained in step 3.2: Add 0.6 times (based on the mass of the mash from step 3.2) of enzyme-producing mash to the mash obtained in step 3.2, and at the same time add 0.08% (based on the mass of the mash from step 3.2) of compound enzyme preparation 2; at the same time, switch to intermittent aeration mode for fermentation and ferment for 40 days, while maintaining the fermentation temperature at 28℃ in this step.

[0089] In this step, the compound enzyme preparation consists of glucose oxidase and acidic protease in a mass ratio of 1:1. The intermittent ventilation mode is specifically a cycle of 12 hours of ventilation followed by 12 hours of ventilation stoppage.

[0090] 5. Pressing and sterilization: The fermented soy sauce mash from step 4.2 was pressed and filtered (diatomaceous earth filter, filtration pressure 0.3 MPa, filtration temperature 25℃), and then sterilized by pasteurization (sterilization temperature 95℃, sterilization time 15min) to obtain sodium-reduced and sugar-free soy sauce.

[0091] Example 4 The only difference between Example 4 and Example 1 is that the soybeans are not microwaved; the remaining steps and parameters are the same as in Example 1, as detailed below: After screening and washing the soybeans, soak them in warm water for 40 minutes, put them into a steamer, steam them for 20 minutes under a pressure of 0.15MPa, and then remove them when the temperature drops to 35℃.

[0092] Example 5 The only difference between Example 5 and Example 1 is that step 2.2 uses constant temperature fermentation; the remaining steps and parameters are the same as in Example 1, as detailed below: 2.2 Preparation of fermented soybean paste: The raw materials and brine were added to the fermentation tank at a mass ratio of 1:2.1 and fermented at 25°C for 22 days. Then, 0.08% (by mass of total material) of compound enzyme preparation was added, and the fermentation temperature was maintained at 25°C for 22 days (constant temperature fermentation) to obtain the sauce mash.

[0093] Example 6 The only difference between Example 6 and Example 1 is that in step 2.2, the koji, brine, and compound enzyme preparation are fermented simultaneously. The remaining steps and parameters are the same as in Example 1, as detailed below: 2.2 Preparation of fermented soybean paste: The raw materials and brine were added to the fermentation tank at a mass ratio of 1:2.1, along with 0.08% (by mass of total materials) of compound enzyme preparation. The fermentation temperature was maintained at 18°C ​​for the first 22 days, and then increased to 25°C for 22 days (variable temperature fermentation) to produce the fermented sauce mash.

[0094] Example 7 The only difference between Example 7 and Example 1 is that the compound enzyme preparation is added later in step 4.2. The remaining steps and parameters are the same as in Example 1, as detailed below: 4.2 Add enzyme-producing mash to the mash obtained in step 3.2: Add 0.4 times the amount of the enzyme-producing mash to the mash obtained in step 3.2 and ferment for 20 days; then add 0.05% by weight of the compound enzyme preparation and ferment for 15 days.

[0095] Comparative Example 1 The only difference between Comparative Example 1 and Example 1 is that the compound microbial strain is missing in step 3.2, the second fermentation; the remaining steps and parameters are the same as in Example 1. Specifically: Add the fermented mash obtained in step 2.2 in three batches, each batch being 0.06 times the mass of the mash before adding the fermented mash, with an interval of 10 days between each batch, for a total of 30 days and a total addition amount of 0.18 times the mass of the mash. Then ferment for another 25 days.

[0096] Comparative Example 2 The only difference between Comparative Example 2 and Example 1 is that the compound enzyme preparation 2 is missing in step 4.2, the third fermentation; the remaining steps and parameters are the same as in Example 1. Specifically: Add 0.4 times the amount of enzyme-producing mash to the mash obtained in step 3.2, and use intermittent aeration mode for fermentation for 35 days.

[0097] Comparative Example 3 The only difference between Comparative Example 3 and Example 1 is that the order of step 3 (second fermentation) and step 4 (third fermentation) is reversed; the remaining steps and parameters are the same as in Example 1. Specifically: 3. Second fermentation: 3.1 Preparation of enzyme-producing mash: Roasted wheat flour was mixed with water to prepare a wheat flour slurry with a mass concentration of 18%. 0.3% by mass of Rhizopus oryzae and 1.0% by mass of yeast extract were added. Fermentation was carried out at pH 5.5, fermentation temperature of 30℃, and aeration rate of 1.5 vvm for 5 days to obtain enzyme mash.

[0098] 3.2 Add enzyme-producing mash to the mash obtained in step 2.2: Add 0.4 times the amount of the enzyme-producing mash to the mash obtained in step 2.2, and add 0.05% by weight of the compound enzyme preparation; at the same time, use intermittent aeration for fermentation for 35 days.

[0099] 4. Third fermentation: 4.1 Preparation of wine mash: Roasted wheat flour is mixed with water to prepare a wheat flour slurry with a mass concentration of 20%. 0.4% brewing yeast is added, and the fermentation is carried out at a pH of 4.5 and a temperature of 28°C. The fermentation is first carried out with aeration for 4 days at a rate of 1.0 vvm to promote yeast proliferation. Then, the fermentation is carried out in a sealed anaerobic environment for 8 days to produce mash.

[0100] 4.2 Add the wine-producing mash to the mash obtained in step 3.2: Add the fermenting mash to the mash obtained in step 3.2 in three separate additions, each time by an amount equal to 0.06 times the mass of the mash before adding the fermenting mash, with an interval of 10 days between each addition, for a total of 30 days and a total addition amount of 0.18 times the fermenting mash. Then, inoculate with 0.4% (by mass of the mash before adding the fermenting mash) of compound microbial strains and ferment for 25 days.

[0101] 5. Pressing and sterilization: The fermented soy sauce mash is pressed and filtered, and then pasteurized to produce reduced-sodium and zero-sugar soy sauce.

[0102] Comparative Example 4 The only difference between Comparative Example 4 and Example 1 is that no wine-producing mash is added in step 3.2 of the second fermentation process; the remaining steps and parameters are the same as in Example 1. Specifically: Add 0.4% by weight (based on the weight of the fermented sauce prepared in step 2.2) of the compound microbial strain to the fermented sauce mash obtained in step 2.2 and ferment for 55 days.

[0103] Comparative Example 5 The only difference between Comparative Example 5 and Example 1 is that enzyme-producing mash is not added in step 4.2 of the third fermentation process; the remaining steps and parameters are the same as in Example 1. Specifically: Add 0.05% by weight (based on the weight of the mash in step 3.2) of compound enzyme preparation 2 to the mash obtained in step 3.2; at the same time, switch to intermittent aeration mode for fermentation and ferment for 35 days.

[0104] Comparative Example 6 The only difference between Comparative Example 6 and Example 1 is that the enzyme preparation in step 2.2 contains only glucose isomerase and does not contain mannanase, and the total amount used is 0.08% (based on the total mass of the material). The other steps and parameters are the same as in Example 1.

[0105] Comparative Example 7 The only difference between Comparative Example 7 and Example 1 is that the enzyme preparation in step 2.2 contains only mannanase and does not contain glucose isomerase, and the total amount used is 0.08% (based on the total mass of the material). The other steps and parameters are the same as in Example 1.

[0106] Comparative Example 8 The only difference between Comparative Example 8 and Example 1 is that in step 2.2, the complex enzyme preparation is prepared by replacing glucose isomerase with acidic protease. The remaining steps and parameters are the same as in Example 1.

[0107] Comparative Example 9 The only difference between Comparative Example 9 and Example 1 is that the strain used in step 3.2 is only *Hansenula anomala*, without *Saccharomyces cerevisiae* and *Pichia pastoris*, and the inoculation amount is 0.4% (based on the weight of the mash before adding the mash). The other steps and parameters are the same as in Example 1.

[0108] Comparative Example 10 The only difference between Comparative Example 10 and Example 1 is that the strain used in step 3.2 is only *Saccharomyces cerevisiae*, without *Hansenula anomala* and *Pichia pastoris*, and the inoculation amount is 0.4% (based on the weight of the mash before adding the mash). The other steps and parameters are the same as in Example 1.

[0109] Comparative Example 11 The only difference between Comparative Example 11 and Example 1 is that the compound strain in step 3.2 contains only *Hansenula anomala* and *Saccharomyces cerevisiae*, without *Pichia pastoris*, and the inoculation amount is 0.4% (based on the mass of the mash before adding the mash). The mass ratio of *Hansenula anomala* to *Saccharomyces cerevisiae* is 1:1. The remaining steps and parameters are the same as in Example 1.

[0110] Comparative Example 12 The only difference between Comparative Example 12 and Example 1 is that in the compound strain of step 3.2, *Saccharomyces cerevisiae* is used instead of *Saccharomyces cerevisiae*. The other steps and parameters are the same as in Example 1.

[0111] Comparative Example 13 The only difference between Comparative Example 13 and Example 1 is that in the compound strain of step 3.2, Rhodotorula glutinis is used instead of Pichia pastoris. The other steps and parameters are the same as in Example 1.

[0112] Comparative Example 14 The only difference between Comparative Example 14 and Example 1 is that Aspergillus niger is used instead of Rhizopus oryzae in step 4.1, while the other steps and parameters are the same as in Example 1.

[0113] Comparative Example 15 The only difference between Comparative Example 15 and Example 1 is that the enzyme preparation in step 4.2 contains only glucose oxidase and does not contain acidic protease, and the total amount used is 0.05%. The remaining steps and parameters are the same as in Example 1.

[0114] Comparative Example 16 The only difference between Comparative Example 16 and Example 1 is that the enzyme preparation in step 4.2 consists only of acidic protease and does not contain glucose oxidase, and the total amount used is 0.05%. The remaining steps and parameters are the same as in Example 1.

[0115] Comparative Example 17 The only difference between Comparative Example 17 and Example 1 is that the enzyme preparation in step 4.2 is replaced with α-amylase instead of acidic protease; the remaining steps and parameters are the same as in Example 1.

[0116] Test case The amino acid content in soy sauce was determined according to GB 5009.235 "National Food Safety Standard - Determination of Amino Acid Nitrogen in Food"; the total sugar content was determined according to GB 5009.8 "National Food Safety Standard - Determination of Fructose, Glucose, Sucrose, Maltose and Lactose in Food"; and the sodium content was determined according to GB 5009.91 "National Food Safety Standard - Determination of Potassium and Sodium in Food". The content of ethyl acetate and guaiacol, characteristic aroma compounds of soy sauce, was determined by gas chromatography-mass spectrometry (GC-MS). The results of Examples 1-7 and Comparative Examples 1-17 are shown in Table 1 below. Table 1: Test results of sodium-reduced and sugar-free soy sauces from Examples 1-7 and Comparative Examples 1-17 As shown in Table 1, the sugar content of Examples 1-7 is all below ≤0.5g / 100mL, meeting the national standard of "0 sugar". Furthermore, the amino acid nitrogen content, ethyl acetate, and guaiacol content are relatively high, making the soy sauce of this invention not only a "0 sugar" soy sauce but also possessing a rich soy aroma and mellow flavor, with outstanding nutritional value. Meanwhile, the sodium content of Examples 1-7 is between 4400-4800mg / 100ml, lower than the comparative example, indicating a significant sodium reduction effect. The total fermentation time is also less than 180 days, demonstrating that the preparation process of this invention has advantages far exceeding conventional processes, and can also endow the product with a richer flavor profile and more stable quality performance.

[0117] Examples 1-7 show that Example 1 had the highest content of amino acid nitrogen, ethyl acetate, and guaiacol, and the lowest total sugar and sodium content, resulting in the best overall quality. Examples 2-7 were slightly less effective than Example 1, but still met the requirements for zero sugar and reduced sodium. This is because the process parameters in Examples 2 and 3 were adjusted, affecting the final soy sauce quality. In Example 4, because the soybeans were not microwave-treated, the soybean cell structure was not completely destroyed, and the starch and protein were not completely decomposed, leading to the production of residual sugar and a significant decrease in the content of ethyl acetate and guaiacol. Therefore, microwave treatment of soybeans can destroy the soybean cell wall structure, accelerate the subsequent decomposition of starch and protein, reduce the production of residual sugar, and increase the generation of soy sauce flavor substances. In Example 5, because the koji and brine in step 2.2 were fermented at a constant temperature, the microbial sugar metabolism was slow, resulting in slow sugar decomposition and an increased sugar content in the soy sauce, with insufficient accumulation of ethyl acetate and guaiacol. This indicates that variable-temperature fermentation promotes the targeted metabolism of sugar sources by microorganisms. In the later stages of fermentation, the temperature of the fermented mash rises to around 25°C, which matches the optimal reaction temperature of the two enzymes, maximizing enzyme activity and simultaneously enhancing sugar reduction and the synthesis of flavor compounds. In Example 6, the simultaneous fermentation of the starter culture, brine, and compound enzyme preparation led to mutual inhibition of the enzyme system, failing to maximize the effect of the enzyme preparation, resulting in increased sugar content and reduced flavor compound synthesis in the soy sauce. Therefore, adding a compound enzyme preparation of glucose isomerase and mannanase at the end of the first fermentation stage can enhance the synergistic effect of the two enzymes, targeting the decomposition of polysaccharides and optimizing monosaccharide metabolic pathways, providing readily available substrates for microorganisms. In Example 7, adding the enzyme-producing mash first and then the compound enzyme preparation during the third fermentation weakened the synergistic effect of endogenous and exogenous enzymes, resulting in insufficient accumulation of flavor compounds and incomplete removal of residual sugar. This indicates that simultaneously adding the enzyme-producing mash and the compound enzyme preparation is more conducive to deep sugar reduction and flavor enhancement.

[0118] Comparative Example 1 lacked a second fermentation with a compound microbial strain, thus missing the stepwise metabolic pathway of "disaccharide → monosaccharide → residual sugar," resulting in incomplete sugar metabolism. Consequently, the total sugar content of the soy sauce increased significantly, while ethyl acetate and guaiacol decreased substantially, leading to a weak soy sauce aroma. Comparative Example 2 lacked a third fermentation compound enzyme preparation, resulting in insufficient deep oxidation and removal of residual reducing sugars. It also failed to enhance the sugar reduction of the microbial cells through proteolysis, leading to an increase in total sugar and a significant decrease in flavor compounds. Comparative Example 3 reversed the order of the second and third fermentations, disrupting the synergistic sequence of "first yeast stepwise sugar reduction, then deep sugar reduction by compound enzymes," causing disordered carbon and nitrogen metabolism. Consequently, the effects of sugar reduction, sodium reduction, and flavor enhancement were significantly reduced. Comparative Example 4 lacked mash production, resulting in insufficient ethanol microenvironment regulation and carbon and nitrogen nutrient supply. The compound yeast proliferation was insufficient, and sugar metabolism efficiency decreased, leading to an increase in total sugar, a weak flavor, and a poorer sodium reduction effect. Comparative Example 5 lacked enzyme-producing mash, and lacked the supply of endogenous saccharifying enzymes, proteases, and lactases from Rhizopus oryzae. The enzyme-bacterial synergistic sugar-lowering chain was broken, resulting in an increase in total sugar, insufficient umami and soy sauce aroma, and a decrease in sodium reduction effect.

[0119] Comparative Examples 6-8 used only one enzyme or other enzymes for the first fermentation, which disrupted the synergistic mechanism of sugar release and sugar isomerization between glucose isomerase and mannanase. As a result, the supply of sugar metabolism substrates was insufficient and their utilization was inefficient, thus increasing the total sugar content of soy sauce and decreasing the content of ethyl acetate and guaiacol.

[0120] In Comparative Examples 9-13, only 1-2 strains or alternative strains were used for the second fermentation, which disrupted the synergistic effect of the complex microbial community, which includes the complementary division of labor, stepwise sugar reduction, and simultaneous aroma enhancement among *Hansenula polymorpha*, *Pichia pastoris*, and *Pichia chinensis*. As a result, sugar degradation was insufficient, the synthesis of soy sauce-flavored substances was inadequate, flavor layers were lacking, and the sodium reduction effect was weakened.

[0121] Comparative Example 14 used Aspergillus niger to replace Rhizopus oryzae in the preparation of enzyme-producing mash. The lack of the lactase-mediated pyruvate conversion pathway meant that glucose could not be continuously broken down by consuming intermediate products of sugar metabolism, resulting in the loss of deep hypoglycemic effect. As a result, total sugar increased, umami and soy sauce aroma decreased, and sodium reduction effect deteriorated.

[0122] Comparative Examples 15-17 used only one enzyme or other enzymes for the third fermentation, which disrupted the dual-effect sugar-reducing synergy between glucose oxidase and acidic protease, which directly oxidizes residual sugar and indirectly promotes bacterial consumption of sugar. As a result, the removal of residual sugar was incomplete, leading to an increase in total sugar, a decrease in flavor and nutritional indicators, and an insignificant sodium reduction effect.

[0123] Therefore, this invention utilizes the multi-dimensional synergistic effects of segmented compound enzymes, stepwise compound strains, wine-producing mash, and enzyme-producing mash to efficiently metabolize starch and sugars. Simultaneously, it relies on the antibacterial effect of ethanol in the wine-producing mash, the enhanced fermentation stability of the compound microbial community, and precise temperature control and intermittent aeration to ensure the operation of the sodium reduction system. This achieves stable fermentation under a sodium-reduced environment, resulting in a final product with a total sugar content ≤0.5g / 100mL and a sodium content consistently between 4400-4800mg / 100ml. This meets the national standard requirements for zero-sugar soy sauce and achieves a significant sodium reduction effect. Furthermore, it retains and enhances the core qualities of soy sauce, such as amino acid nitrogen, ethyl acetate, and guaiacol, achieving a unified balance of zero sugar, reduced sodium, rich soy sauce aroma, and mellow umami flavor, making it suitable for industrial production.

[0124] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for preparing a sodium-reduced, sugar-free soy sauce, characterized in that, Includes the following steps: S1 First Fermentation: After fermenting the koji and brine in a fermentation tank, compound enzyme preparation 1 is added for further fermentation to obtain soy sauce mash; S2 Second Fermentation: First, add wine-producing mash to the sauce mash obtained in step S1 for fermentation, and then inoculate with compound bacteria for further fermentation; S3 Third fermentation: Add enzyme-producing mash and compound enzyme preparation 2 to the mash obtained in step S2 and ferment simultaneously; S4 involves pressing and sterilizing the mash obtained in step S3 to produce reduced-sodium, zero-sugar soy sauce. Among them, the complex enzyme preparation 1 in S1 is composed of glucose isomerase and mannanase, with a mass ratio of (5-1):1; The composite strain in S2 consists of Hansenula anomala, Pichia pastoris and Pichia chinensis in a mass ratio of (7-2):3:

3. The complex enzyme preparation 2 in S3 is composed of glucose oxidase and acidic protease in a mass ratio of 1:(1-4).

2. The preparation method according to claim 1, characterized in that, In step S1, the amount of compound enzyme preparation 1 added is 0.05-0.1% based on the total mass of the starter culture and brine; in step S2, the amount of compound microbial strain added is 0.2-0.6% based on the mass of the fermented mash without added yeast; in step S3, the amount of compound enzyme preparation 2 added is 0.03-0.08% based on the total mass of the fermented mash in step S2.

3. The preparation method according to claim 1, characterized in that, In step S2, the total amount of fermented mash added is 0.1-0.3 times the mass of the fermented mash obtained in step S1, and in step S3, the amount of enzyme-producing mash added is 0.3-0.6 times the total mass of the fermented mash obtained in step S2.

4. The preparation method according to claim 1, characterized in that, In step S1, the starter culture and brine are added to a fermentation tank and fermented at 15-20℃ for 20-25 days. Then, compound enzyme preparation 1 is added and the temperature is raised to 23-27℃ for another 20-25 days of fermentation.

5. The preparation method according to claim 1, characterized in that, The specific method for the second fermentation in step S2 includes the following steps: (1) Add the fermenting mash to the mash in step S1 in batches. Each addition is 0.03-0.1 times the mass of the mash without fermenting mash. The interval between each addition is 10 days, for a total of 30 days. The fermentation temperature is 23-30℃. (2) Inoculate the compound bacteria into the sauce mash of step (1) and ferment for 20-30 days at a temperature of 23-30℃.

6. The preparation method according to claim 1, characterized in that, The fermentation method in S3 is: intermittent aeration mode, with a 12-hour aeration cycle followed by a 12-hour shutdown, a fermentation temperature of 28-30℃, and fermentation for 30-40 days until maturity.

7. A sodium-reduced, sugar-free soy sauce prepared by any one of claims 1-6, characterized in that, The total sugar content of the soy sauce is ≤0.5g / 100mL; the sodium content of the soy sauce is 4400-4800mg / 100ml.

8. A compound enzyme preparation, characterized in that, The compound enzyme preparation is composed of glucose isomerase and mannanase, and the mass ratio of glucose isomerase to mannanase is (5-1):

1. Alternatively, the compound enzyme preparation is composed of glucose oxidase and acidic protease, with a mass ratio of glucose oxidase to acidic protease of 1:(1-4).

9. A compound microbial agent, characterized in that, The compound bacterial agent is composed of *Hansenula anomala*, *Saccharomyces cerevisiae*, and *Pichia pastoris*, with the mass ratio of *Hansenula anomala*, *Saccharomyces cerevisiae*, and *Pichia pastoris* being (7-2):3:

3.

10. The application of the compound enzyme preparation as described in claim 8 or the compound microbial agent as described in claim 9 in the preparation of sodium-reduced and sugar-free soy sauce.

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