Low-salt fermented soybean paste production process based on co-fermentation of compound microorganisms

Abstract

This invention relates to the field of broad bean paste technology and discloses a low-salt broad bean paste production process based on the synergistic fermentation of compound microorganisms. The process includes: uniformly mixing broad bean koji with drained chili embryos to obtain a solid mixture; spraying an A-phase initiation conditioning solution onto the solid mixture and inoculating it with *Lactobacillus plantarum*, mixing until moistened to obtain a primary fermentation mash; controlling the primary fermentation mash to undergo constant-temperature fermentation until the pH value drops to a preset acidic range to obtain an acidified mature mash; injecting a B-phase hypertonic regulating solution into the acidified mature mash and inoculating it with *Tetracoccus halophilus* and *Zygosacchariformis*, mixing uniformly to obtain a phase-inversion fermentation mash; cooling the phase-inversion fermentation mash and conducting anaerobic fermentation under air-isolated conditions to obtain the low-salt broad bean paste product. This invention solves the problems of insufficient umami and softening in low-salt fermentation by segmented salt control driving synergistic succession of microorganisms, combined with chemical regulation to protect enzyme activity and enhance texture, significantly improving the flavor and taste of the product.

Description

Technical Field

[0001] This invention relates to the field of fermented soybean paste technology, specifically to a low-salt fermented soybean paste production process based on the synergistic fermentation of compound microbial strains. Background Technology

[0002] Doubanjiang (fermented broad bean paste) is a traditional condiment made primarily from broad beans, chili peppers, and salt through microbial fermentation. It possesses a unique aroma of soy sauce and esters and is widely used in food preparation. Its production process mainly includes broad bean koji making, chili pepper pickling, and the subsequent fermentation of the mixture. The enzymatic hydrolysis of proteins and the synthesis of flavor compounds are key steps in determining product quality.

[0003] With the popularization of healthy eating concepts, reducing the salt content in food has become an industry trend. Existing low-salt fermented soybean paste production processes often employ compound microbial co-fermentation technology, which involves artificially inoculating multiple functional microorganisms such as Aspergillus oryzae and Lactobacillus plantarum to reduce salt content while utilizing the metabolic characteristics of different microbial strains to shorten the fermentation cycle and enhance the flavor complexity of the product.

[0004] However, existing low-salt fermentation processes still have shortcomings. First, in a low-salt environment, the growth and metabolism of lactic acid bacteria are uninhibited, leading to an excessively rapid acid production rate. This causes the pH value of the system to drop rapidly below the stable range of neutral proteases in the early stages of fermentation, severely inhibiting enzyme activity, resulting in incomplete protein hydrolysis, insufficient accumulation of amino acid nitrogen, and a bland umami flavor in the finished product. Second, the low osmotic pressure environment easily causes the fermented soybean granules to absorb excessive water and swell. Coupled with the degradation of cell wall pectin during fermentation, this leads to softening of the granule tissue, making it difficult to maintain the crisp and chewy texture that traditional fermented soybean paste should have. In addition, a sustained low-salt environment also faces the risks of high contamination by other microorganisms and easy oxidation and discoloration of the product, making it difficult to simultaneously ensure fermentation safety, physicochemical indicators, and sensory quality.

[0005] Therefore, this invention proposes a low-salt fermented soybean paste production process based on the synergistic fermentation of compound microbial strains to address the shortcomings of existing technologies. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a low-salt fermented soybean paste production process based on the synergistic fermentation of compound microorganisms. This process solves the problems in existing low-salt fermented soybean paste processes, such as the increased risk of contamination by miscellaneous bacteria due to the low-salt environment, and the tendency of low osmotic pressure to cause the soybean particles to absorb water and swell, resulting in softening of the tissue and poor chewiness of the product. At the same time, the rapid acid production by lactic acid bacteria in the early stage of low-salt fermentation can cause the pH value to drop rapidly below the stable range of neutral proteases, inhibiting enzyme activity, resulting in incomplete protein hydrolysis and ultimately insufficient umami flavor in the product.

[0007] To address the above problems, the present invention provides the following technical solution:

[0008] This invention provides a low-salt fermented soybean paste production process based on the synergistic fermentation of compound microbial strains, comprising the following steps:

[0009] The broad bean curd is mixed evenly with the drained chili embryos to obtain a solid mixture.

[0010] A phase A starting conditioning solution is sprayed onto the solid mixture, and Lactobacillus plantarum is inoculated. The mixture is then mixed until it is moistened to obtain primary fermentation mash. The primary fermentation mash is controlled to undergo constant temperature fermentation until the pH value drops to a preset acidic range to obtain acidified mature mash. The A phase starting conditioning solution is an aqueous solution containing sodium citrate dihydrate, magnesium sulfate heptahydrate, and sodium chloride.

[0011] A B-phase hypertonic control solution is injected into the acidified mature mash, and halophilic tetracocci and Rhodotorula roux are inoculated and mixed evenly to obtain phase-inverted fermentation mash; wherein, the B-phase hypertonic control solution is an aqueous solution containing sodium chloride, anhydrous calcium chloride and L-ascorbic acid sodium;

[0012] The phase-inversion fermentation mash is cooled and subjected to anaerobic fermentation under air-isolated conditions to obtain a low-salt fermented soybean paste product.

[0013] This invention significantly improves the quality and safety of low-salt fermented soybean paste through a strategy combining segmented salt control and chemical regulation. Its main mechanism of action and beneficial effects are as follows:

[0014] Firstly, this invention protects the activity of neutral protease by constructing a pH buffer system, thus solving the problem of insufficient umami flavor in low-salt fermentation. In the initial A-phase stage of fermentation, sodium citrate dihydrate forms a buffer pair with the acidic substances produced during fermentation, effectively mitigating the sudden drop in pH caused by the rapid metabolism of *Lactobacillus plantarum*, maintaining the system's acidity within the optimal range for neutral protease activity. Combined with magnesium sulfate heptahydrate providing magnesium ions to stabilize the enzyme's molecular conformation, this ensures that the protease derived from *Aspergillus oryzae* maintains high catalytic efficiency even in low-salt environments, promoting the deep hydrolysis of large protein molecules into amino acid nitrogen, thereby significantly enhancing the product's umami flavor.

[0015] Secondly, this invention utilizes salinity gradient changes to achieve directed succession of the microbial community. The process employs a low-salt initiation fermentation combined with high-salt phase inversion regulation. The low-salt environment of phase A promotes the rapid proliferation and acid production of *Lactobacillus plantarum* to inhibit contaminating bacteria. Once the pH reaches a preset range, phase B, a hypertonic regulating solution, is injected to cause a sudden change in osmotic pressure within the system. This change in physical environment inhibits the excessive metabolism of *Lactobacillus plantarum*, which is intolerant to high salt, preventing excessive acidity. Simultaneously, it activates the more salt-tolerant *Tetracoccus halophilus* and *Zygosacchariformis*, making them the dominant microbial community in the later stages of fermentation and promoting the synthesis of flavor compounds such as alcohols and esters.

[0016] Furthermore, this invention solves the textural softening problem of low-salt products through in-situ chemical cross-linking technology. In the acidic environment established during phase A fermentation, the protopectin in the cell walls of soybeans partially hydrolyzes into low-methoxyl pectin. Calcium ions introduced from the phase B regulating solution penetrate the tissue and undergo a chemical cross-linking reaction with the low-methoxyl pectin molecular chains, forming a dense calcium pectate gel network. This microstructure enhances the mechanical strength of the cell walls, effectively counteracting the hydration swelling effect caused by low osmotic pressure, and giving the finished product a pleasantly crisp texture.

[0017] Finally, the addition of sodium L-ascorbate to the B-phase control solution adjusted the redox potential of the system, blocked the oxidative browning pathway of polyphenols and amino acids, and enabled the product to maintain a bright reddish-brown color during the long-term fermentation process.

[0018] Preferably, the method for preparing the broad bean koji is as follows:

[0019] Peeled dried broad beans were soaked, steamed until fully cooked, and then cooled to below 38℃. Flour was added at 18% of the total weight of the dried beans, and 0.04% of Aspergillus oryzae spore powder was inoculated. After mixing, the mixture was aerated and fermented to maintain a koji layer thickness of 30cm. It was then incubated at 30-35℃ for 40-44 hours, with frequent turning during this period. The koji was ready when the neutral protease activity reached above 2000 U / g. By limiting the koji-making process parameters, sufficient neutral protease was accumulated in the koji, providing an adequate enzyme source for the subsequent protein hydrolysis in phase A.

[0020] Preferably, the solid mixture comprises 400-450 parts by weight of broad bean koji and 100-150 parts by weight of chili embryos; the A-phase starter conditioning solution comprises 270-380 parts by weight of process water, 3.0-5.0 parts by weight of sodium citrate dihydrate, 0.2-0.4 parts by weight of magnesium sulfate heptahydrate, and 40-48 parts by weight of sodium chloride; the inoculum amount of *Lactobacillus plantarum* is 0.5-1.0 parts by weight. This formulation ensures a suitable carbon-to-nitrogen ratio in the fermentation substrate, and the salt content and buffer concentration in the A-phase solution are well-matched, maintaining a moderately low salt level to facilitate lactic acid bacteria growth while providing sufficient buffer capacity.

[0021] Preferably, the B-phase hypertonic conditioning solution comprises 110-145 parts by weight of process water, 35-45 parts by weight of sodium chloride, 1.0-2.0 parts by weight of anhydrous calcium chloride, and 0.5-0.8 parts by weight of L-ascorbic acid sodium; the inoculum amount of the halophilic tetracoccus is 1.0-2.0 parts by weight, and the inoculum amount of the Leucobacter rumenella is 1.0-2.0 parts by weight. This formulation ensures that the salinity of the injected mixture is sufficient to inhibit lactic acid bacteria and act as a preservative, while the calcium ion concentration is sufficient to complete pectin cross-linking without producing bitterness.

[0022] Preferably, the preparation method of the B-phase hyperosmotic conditioning solution is as follows: process water is heated to 45-50℃, sodium chloride and anhydrous calcium chloride are added, and stirred until completely dissolved to obtain a primary composite salt solution; the primary composite salt solution is cooled to 25-32℃, L-ascorbic acid sodium is added, and stirred until completely dissolved to obtain the final solution; the B-phase hyperosmotic conditioning solution is used within 2 hours of preparation. A stepwise dissolution method is adopted, first using a higher temperature to promote the dissolution of inorganic salts, and then cooling to add heat-sensitive L-ascorbic acid sodium, thus avoiding the high-temperature oxidation and inactivation of ascorbic acid.

[0023] Preferably, the temperature for constant-temperature fermentation is controlled at 35-38℃; the fermentation mash is turned every 12 hours for the first 48 hours, and then allowed to stand for fermentation after 48 hours; the preset acidity range is pH 5.25-5.45. This temperature range takes into account both the growth of *Lactobacillus plantarum* and the catalytic requirements of neutral protease; the initial turning of the mash promotes the distribution of bacteria and aerobic proliferation, while the later standing creates a micro-anaerobic environment to promote acid production; the pH endpoint is controlled at 5.25-5.45, which ensures the action time of protease and provides a suitable initial acidity for subsequent yeast fermentation.

[0024] Preferably, the steps of injecting phase B hypertonic regulating solution into the acidified mature mash and inoculating with halophilic tetracocci and juvenile yeast are as follows: start stirring and control the speed at 20-30 rpm; uniformly inject the phase B hypertonic regulating solution into the acidified mature mash over 10-15 minutes and continue stirring for 30 minutes; inoculate with the halophilic tetracocci and juvenile yeast 5 minutes before the end of stirring. By controlling the stirring speed and injection time, a gentle mixing of high-salt liquid and viscous mash is achieved, avoiding severe shear force that could damage the integrity of the soybean particles; inoculating with yeast at the end of stirring reduces mechanical damage to the microorganisms.

[0025] Preferably, the step of cooling the phase-inversion fermentation mash and carrying out anaerobic fermentation under air-isolated conditions is as follows: the temperature of the phase-inversion fermentation mash is lowered to 25-28°C, the surface is covered with a thin film to isolate it from air, and it is kept at a constant temperature for 90 days for static fermentation. The cooling temperature is consistent with the optimal metabolic temperature for aroma production by *Zygosacchariformis*, and the anaerobic environment is conducive to alcohol fermentation and prevents surface oxidation and anthracnose.

[0026] Preferably, the viable count of the *Lactobacillus plantarum* is ≥1.0 × 10⁻⁶. 11 CFU / g; the viable count of the halophilic tetracoccus is ≥1.0×10⁻⁶. 9 CFU / mL; the viable count of the *Zygosacchariformis* ≥ 1.0 × 10⁻⁶ 8 CFU / mL.

[0027] Preferably, the chili embryo is a semi-finished product of chili peppers that have been washed, crushed and salted, with a moisture content of 65%-70%.

[0028] This invention provides a low-salt fermented soybean paste production process based on the synergistic fermentation of compound microbial strains. It has the following beneficial effects:

[0029] 1. This invention solves the problem of rapid pH drop caused by lactic acid bacteria metabolism and acid production in low-salt environments by constructing a buffer system of sodium citrate and magnesium sulfate during the fermentation initiation stage. This buffer system maintains the acidity at the initial stage of fermentation within the suitable range for neutral proteases, and, combined with the stabilizing effect of magnesium ions on the enzyme molecule conformation, maintains the catalytic activity of the proteases. This promotes the deep conversion of substrate proteins into amino acid nitrogen, solving the problems of low amino acid production and insufficient umami caused by inhibited enzyme activity in traditional low-salt fermentation, and improving the flavor quality of the product.

[0030] 2. This invention employs a segmented salt control strategy, utilizing changes in osmotic pressure to drive the directed succession of the microbial community. In the early stages of the process, a low-salt environment promotes the proliferation and acid production of *Lactobacillus plantarum*, establishing a dominant bacterial community to suppress other microorganisms. During the middle stages of fermentation, a hypertonic regulating solution is injected to create a salinity jump, inhibiting excessive metabolism by lactic acid bacteria to prevent rancidity, while simultaneously activating halophilic tetracocci and *Zygosacchariformis* to become the dominant bacterial community. This regulatory mechanism ensures the safety of low-salt fermentation while promoting the synthesis of alcohol and ester flavor compounds, thus optimizing the flavor characteristics of the finished product.

[0031] 3. This invention improves the textural properties and sensory color of low-salt fermented soybean paste by introducing calcium ions and antioxidants into the phase inversion control liquid. Calcium ions react with low-methoxyl pectin generated under acidic conditions to form an in-situ chemical cross-linking structure, enhancing the hardness and strength of the soybean particles and counteracting the hydration softening effect caused by low osmotic pressure, thus giving the product a crisp texture. Simultaneously, sodium ascorbate regulates the redox potential of the system, blocking non-enzymatic browning of polyphenols and amino acids, allowing the finished product to maintain a bright red color during long-term fermentation. Attached Figure Description

[0032] Figure 1 The following figures illustrate the dynamic monitoring of physicochemical indicators during the fermentation process in Examples 1-3: (a) Figure shows the kinetic change of pH during fermentation, and (b) Figure shows the accumulation of amino acid nitrogen during fermentation.

[0033] Figure 2 The following are monitoring diagrams of directional succession of microbial communities in Examples 1-3, where (a) shows the succession dynamics of lactic acid bacteria community and (b) shows the succession dynamics of yeast community. Detailed Implementation

[0034] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0035] The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products.

[0036] Sodium citrate dihydrate (CAS No. 6132-04-3), magnesium sulfate heptahydrate (CAS No. 10034-99-8), anhydrous calcium chloride (CAS No. 10043-52-4), and sodium L-ascorbate (CAS No. 134-03-2) are all commercially available food-grade products with a purity of ≥99.0% (of which anhydrous calcium chloride is ≥95.0%); the sodium chloride is commercially available food-grade non-iodized refined salt (CAS No. 7647-14-5).

[0037] The broad beans are commercially available peeled and dried broad beans with a starch content of ≥45% and a protein content of ≥25%; the flour is commercially available ordinary wheat flour; the chili pepper embryos are semi-finished products made from fresh Erjingtiao or millet chilies that have been washed, crushed, and salted for more than 3 months, with a drained moisture content of 65%-70%; the process water meets the hygiene standards for drinking water.

[0038] Aspergillus oryzae was selected from Hu Niang 3.042, with an enzyme activity ≥2000U / g after koji preparation; Lactobacillus plantarum was a commercially available direct-inoculation freeze-dried powder with a viable count ≥1.0×10⁻⁶. 11 CFU / g; both *Hydroxytetracoccus* and *Zygosaccharidus rhus* were commercially available liquid seed cultures, with viable counts ≥1.0 × 10⁻⁶. 9 CFU / mL and ≥1.0×10 8 CFU / mL.

[0039] Preparation Example 1:

[0040] This preparation example provides a method for preparing broad bean koji, including the following steps:

[0041] Peeled and dried broad beans were selected, washed to remove impurities, and soaked in water until the beans absorbed 45% of their water content. The soaked beans were then drained. The soaked beans were then pressure-cooked in a steamer at 0.1 MPa for 15 minutes, until fully cooked and the beans were intact without any rotten core. The cooked broad beans were then cooled to 38°C. Flour was added at 18% of the dry bean weight and mixed thoroughly. 0.04% of Aspergillus oryzae spore powder (3.042) was added to the mixture. After thorough mixing, the mixture was transferred to a thick-layer, ventilated koji-making tank, maintaining a koji layer thickness of 30 cm. The mixture was incubated at 30-35°C for 42 hours, with mechanical turning performed at the 16th and 26th hours to adjust the temperature and ventilation. When the koji material clumps together, is covered with yellow-green spores, and has a strong aroma, it is ready. The koji was then crushed and the neutral protease activity was measured to be 2150 U / g (dry basis), yielding the finished broad bean koji.

[0042] Preparation Example 2:

[0043] This preparation example provides a method for preparing a phase B hypertonic conditioning solution, including the following steps:

[0044] Add 125 parts by weight of process water to the mixing tank and raise the water temperature to 48°C to obtain preheated process water. Add 40 parts by weight of sodium chloride and 1.5 parts by weight of anhydrous calcium chloride to the preheated process water, and turn on the agitator to stir at 60 rpm for 10 minutes until the solids are completely dissolved to obtain the primary composite salt solution. Cool the temperature of the primary composite salt solution to 30°C to obtain the cooling base solution. Add 0.6 parts by weight of L-ascorbic acid sodium to the cooling base solution and maintain stirring for 5 minutes until completely dissolved to obtain the B-phase hypertonic control solution. This B-phase hypertonic control solution must be used within 2 hours of preparation.

[0045] Preparation Example 3:

[0046] This preparation example provides a method for preparing a phase B hypertonic conditioning solution, including the following steps:

[0047] Add 110 parts by weight of process water to the mixing tank and raise the water temperature to 45°C to obtain preheated process water. Add 35 parts by weight of sodium chloride and 1.0 part by weight of anhydrous calcium chloride to the preheated process water, and stir with a stirrer until the solids are completely dissolved to obtain a primary composite salt solution. Lower the temperature of the primary composite salt solution to 25°C to obtain a cooling base solution. Add 0.5 parts by weight of L-ascorbic acid sodium to the cooling base solution and maintain stirring until completely dissolved to obtain the B-phase hypertonic control solution. This B-phase hypertonic control solution must be used within 2 hours of preparation.

[0048] Preparation Example 4:

[0049] This preparation example provides a method for preparing a phase B hypertonic conditioning solution, including the following steps:

[0050] Add 145 parts by weight of process water to the mixing tank and raise the water temperature to 50°C to obtain preheated process water. Add 45 parts by weight of sodium chloride and 2.0 parts by weight of anhydrous calcium chloride to the preheated process water, and stir with a stirrer until the solids are completely dissolved to obtain a primary composite salt solution. Lower the temperature of the primary composite salt solution to 32°C to obtain a cooling base solution. Add 0.8 parts by weight of L-ascorbic acid sodium to the cooling base solution and maintain stirring until completely dissolved to obtain a B-phase hypertonic conditioning solution. This B-phase hypertonic conditioning solution must be used within 2 hours of preparation.

[0051] Example 1:

[0052] This embodiment provides a low-salt fermented soybean paste production process based on the synergistic fermentation of compound microbial strains, including the following steps:

[0053] Step 1: Weigh 420 parts by weight of the broad bean koji prepared in Preparation Example 1 and 130 parts by weight of the drained chili embryos. Put the two raw materials into the fermentation tank, turn on the stirring equipment and mix evenly to obtain a solid mixture.

[0054] Step 2: Measure 320 parts by weight of process water, add 4.0 parts by weight of sodium citrate dihydrate, 0.3 parts by weight of magnesium sulfate heptahydrate, and 45 parts by weight of sodium chloride, and stir until completely dissolved to obtain phase A starting conditioning solution. Spray phase A starting conditioning solution evenly onto the solid mixture, and simultaneously add 0.8 parts by weight of *Lactobacillus plantarum* freeze-dried powder. Start mechanical stirring for 30 minutes until the material is moistened to obtain primary fermentation mash. Control the temperature of the primary fermentation mash at 36℃ for constant temperature fermentation. Perform a turning operation every 12 hours for the first 48 hours of constant temperature fermentation, and then allow it to ferment statically after 48 hours. On the 5th day of fermentation, take a sample and measure the pH value to obtain acidified mature mash of 5.35.

[0055] Step 3: Turn on the agitator in the fermentation tank and control the rotation speed to 25 rpm. Inject the B-phase hypertonic conditioning solution obtained in Preparation Example 2 evenly into the acidified mature mash within 12 minutes, and continue stirring for 30 minutes. Five minutes before the end of stirring, add 1.5 parts by weight of halophilic tetracoccus liquid seed and 1.5 parts by weight of Leuciscus rouxii liquid seed to the mash, mix thoroughly, and immediately stop stirring to obtain the phase-inversion fermentation mash.

[0056] Step 4: Quickly adjust the fermentation environment temperature, lowering the temperature of the phase-inversion fermentation mash to 25℃. Cover the surface of the fermentation tank with a polyethylene film and compact it to isolate it from air, thus obtaining an anaerobic fermentation system. Control this anaerobic fermentation system to ferment statically at a constant temperature of 25℃ for 90 days. After fermentation, samples are taken and tested to ensure that the physicochemical indicators are qualified, yielding the low-salt fermented soybean paste product.

[0057] Example 2:

[0058] This embodiment provides a low-salt fermented soybean paste production process based on the synergistic fermentation of compound microbial strains, including the following steps:

[0059] Step 1: Weigh 400 parts by weight of the broad bean koji prepared in Preparation Example 1 and 100 parts by weight of the drained chili embryos. Put the above raw materials into the fermentation tank, turn on the stirring equipment and mix evenly to obtain a solid mixture.

[0060] Step 2: Measure 380 parts by weight of process water, add 3.0 parts by weight of sodium citrate dihydrate, 0.2 parts by weight of magnesium sulfate heptahydrate, and 48 parts by weight of sodium chloride, and stir until completely dissolved to obtain phase A starting conditioning solution. Spray phase A starting conditioning solution evenly onto the solid mixture, and simultaneously add 0.5 parts by weight of *Lactobacillus plantarum* freeze-dried powder. Start mechanical stirring for 30 minutes until the material is moistened to obtain primary fermentation mash. Control the temperature of the primary fermentation mash at 35℃ for constant temperature fermentation. Perform a turning operation every 12 hours for the first 48 hours of constant temperature fermentation, and then allow it to stand for fermentation after 48 hours. On the 5th day of fermentation, take a sample and measure the pH value to obtain acidified mature mash of 5.25.

[0061] Step 3: Turn on the agitator in the fermentation tank and control the rotation speed to 20 rpm. Inject the B-phase hypertonic conditioning solution obtained in Preparation Example 3 evenly into the acidified mature mash within 10 minutes, and continue stirring for 30 minutes. Five minutes before the end of stirring, add 1.0 part by weight of *Tetracoccus halophilus* liquid seed and 1.0 part by weight of *Zygosacchariformis* liquid seed to the mash, mix thoroughly, and immediately stop stirring to obtain the phase-inversion fermentation mash.

[0062] Step 4: Quickly adjust the fermentation environment temperature, lowering the temperature of the phase-inversion fermentation mash to 25℃. Cover the surface of the fermentation tank with a polyethylene film and compact it to isolate it from air, thus obtaining an anaerobic fermentation system. Control this anaerobic fermentation system to ferment statically at a constant temperature of 25℃ for 90 days. After fermentation, samples are taken and tested to ensure that the physicochemical indicators are qualified, yielding the low-salt fermented soybean paste product.

[0063] Example 3:

[0064] This embodiment provides a low-salt fermented soybean paste production process based on the synergistic fermentation of compound microbial strains, including the following steps:

[0065] Step 1: Weigh 450 parts by weight of the broad bean koji prepared in Preparation Example 1 and 150 parts by weight of the drained chili embryos. Put the above raw materials into the fermentation tank, turn on the stirring equipment and mix evenly to obtain a solid mixture.

[0066] Step 2: Measure 270 parts by weight of process water, add 5.0 parts by weight of sodium citrate dihydrate, 0.4 parts by weight of magnesium sulfate heptahydrate, and 40 parts by weight of sodium chloride, and stir until completely dissolved to obtain phase A starting conditioning solution. Spray phase A starting conditioning solution evenly onto the solid mixture, and simultaneously add 1.0 part by weight of *Lactobacillus plantarum* freeze-dried powder. Start mechanical stirring for 30 minutes until the material is moistened to obtain primary fermentation mash. Control the temperature of the primary fermentation mash at 38℃ for constant temperature fermentation. Perform a turning operation every 12 hours for the first 48 hours of constant temperature fermentation, and then allow it to stand for fermentation after 48 hours. On the 5th day of fermentation, take a sample and measure the pH value to be 5.45, obtaining acidified mature mash.

[0067] Step 3: Turn on the agitator in the fermentation tank and control the rotation speed to 30 rpm. Inject the B-phase hypertonic conditioning solution obtained in Preparation Example 4 evenly into the acidified mature mash within 15 minutes, and continue stirring for 30 minutes. Five minutes before the end of stirring, add 2.0 parts by weight of halophilic tetracoccus liquid seed and 2.0 parts by weight of Leuciscus rouxii liquid seed to the mash, mix thoroughly, and immediately stop stirring to obtain the phase-inversion fermentation mash.

[0068] Step 4: Quickly adjust the fermentation environment temperature, lowering the temperature of the phase-inversion fermentation mash to 28℃. Cover the surface of the fermentation tank with a polyethylene film and compact it to isolate it from air, thus obtaining an anaerobic fermentation system. Control this anaerobic fermentation system to ferment statically at a constant temperature of 28℃ for 90 days. After fermentation, samples are taken and tested to ensure that the physicochemical indicators are qualified, yielding the low-salt fermented soybean paste product.

[0069] Comparative Example 1:

[0070] Compared with Example 1, the difference is that sodium citrate dihydrate and magnesium sulfate heptahydrate were not added in step two when preparing the A-phase start-up conditioning solution. The remaining operation steps and parameters are the same as in Example 1.

[0071] Comparative Example 2:

[0072] Compared to Example 1, the difference lies in the method of adding sodium chloride. Specifically, in step two, the total amount of sodium chloride required in steps two and three of Example 1 (i.e., 45 + 40 = 85 parts) is added to the A-phase start-up conditioning solution all at once; sodium chloride is no longer added to the B-phase hypertonic conditioning solution in step three. All other operating steps and parameters are the same as in Example 1.

[0073] Comparative Example 3:

[0074] Compared with Example 1, the difference is that L-ascorbic acid sodium was not added when preparing phase B hypertonic conditioning solution in step three, while the other operation steps and parameters were the same as in Example 1.

[0075] Comparative Example 4:

[0076] Compared with Example 1, the difference is that anhydrous calcium chloride was not added when preparing the B-phase hyperosmolarity conditioning solution in step three, while the other operation steps and parameters were the same as in Example 1.

[0077] Test Example 1:

[0078] 1. Experiment Description

[0079] This test aims to monitor the pH change trajectory and amino acid nitrogen accumulation in the system from the initial fermentation stage (A-phase start-up stage) to the phase transition stage (B-phase regulation stage) to verify the protective effect of the sodium citrate buffer system on the activity of neutral protease and the kinetic effect of low-salt environment on substrate degradation.

[0080] The fermentation materials used in the processes of Examples 1, 2, and 3 were used as test subjects in the experiment. The specific experimental steps and sampling points are as follows:

[0081] Fermentation start time (0h, i.e. the time when the primary fermentation mash is obtained in step two): Samples were taken from the center point and the surrounding edge points of the fermentation tanks corresponding to the three examples, and the samples were mixed to serve as the base samples at that time point. The initial pH value and amino acid nitrogen content were measured.

[0082] Phase A fermentation stage (0h-120h, corresponding to the isothermal fermentation process in step two): In the first 72 hours, the primary fermentation mash is sampled and monitored every 12 hours; from the 72nd hour to the 120th hour, samples are taken every 24 hours.

[0083] Phase transition point and B-phase fermentation stage (120h-360h, corresponding to the initial stages of steps three and four): After the phase transition operation (injection of B-phase hypertonic control liquid and completion of stirring) at the 120th hour, the material is transformed into phase-inverted fermentation mash. Sampling and monitoring of this phase-inverted fermentation mash (or anaerobic fermentation system) continues at the 126th, 144th, 240th, and 360th hours.

[0084] Measurement methods: pH values ​​were measured using a high-precision acidity meter (Mettler-Toledo FE28), calibrated with standard buffer solutions of pH 4.01 and pH 6.86 before measurement; amino acid nitrogen content was determined according to the formaldehyde titration method in GB 5009.235-2016 "National Food Safety Standard - Determination of Amino Acid Nitrogen in Food", and the results are expressed as g / 100g. All samples were measured in triplicate, and the arithmetic mean was recorded as the final data.

[0085] 2. Experimental data (see Table 1)

[0086] Table 1: Kinetic monitoring data of pH value and amino acid nitrogen content at the initial stage of fermentation in Examples 1-3

[0087]

[0088] Note: AAN represents the amino acid nitrogen content.

[0089] 3. Conclusion Analysis

[0090] According to Table 1 and Figure 1 The monitoring data was analyzed:

[0091] In Example 1, during the A-phase fermentation stage from 0 to 120 hours, the pH value exhibited a clear non-linear decreasing trend. Between 24 and 72 hours, the rate of pH decrease slowed, remaining within the range of 5.38 to 5.78. This indicates that the buffer system formed by sodium citrate dihydrate played a role in chemical homeostasis regulation, effectively delaying the sudden pH drop caused by the accumulation of lactic acid produced by the rapid metabolism of *Lactobacillus plantarum*. In contrast, Example 2 (3.0 parts by weight), with a lower sodium citrate addition, showed a slightly faster rate of pH decrease, reaching 5.25 at 120 hours, but still not falling below the critical value of 5.0; while Example 3, with a higher addition, maintained a higher pH level (5.45 at 120 hours).

[0092] Data shows that the pH value remained within a buffer plateau period (stable range) of 5.2-5.8, corresponding to the rapid accumulation period of amino acid nitrogen. In Example 1, the amino acid nitrogen content reached 1.15 g / 100 g before phase inversion on day 5 (120 h). This indicates that the neutral protease derived from Aspergillus oryzae maintained high catalytic activity under these pH conditions and was not inhibited or denatured by the strong acid environment.

[0093] After adding the B-phase hypertonic control solution at 120 hours, the system underwent a salinity jump (a sudden change in salinity). Data monitoring showed that the rate of amino acid nitrogen production slowed slightly from 126 to 144 hours, which is consistent with the inhibitory effect of high-salt environments on enzyme activity. However, it is noteworthy that the pH values ​​in all three examples remained stable after phase inversion, and the total amino acid nitrogen content reached over 1.35 g / 100 g at 360 hours (1.48 g / 100 g in Example 1). This indicates that the initial low-salt buffer hydrolysis strategy had completed the deep degradation of most proteins, and the subsequent high-salt environment was mainly used for flavor synthesis and preservation. This two-stage process solves the problem of low enzymatic hydrolysis efficiency in traditional high-salt fermentation, while also avoiding the risk of microbial contamination from continuous low-salt conditions. Example 1 showed the best overall performance, demonstrating that this formulation achieved an optimized balance between enzyme activity protection and fermentation safety.

[0094] Test Example 2:

[0095] 1. Experiment Description

[0096] This test aims to verify whether the osmotic pressure regulation mechanism set in the process effectively realizes the directional succession of the microbial community, that is, to inhibit the growth of the dominant bacteria Lactobacillus plantarum in the early stage through salinity jump, while activating the salt-tolerant yeast community to become the dominant bacteria in the later stage of fermentation.

[0097] The fermentation materials used in the processes of Examples 1, 2, and 3 were used as test subjects in the experiment. The specific experimental steps and sampling points are as follows:

[0098] Three key sampling time points were set: the first time point was the 96th hour of fermentation (day 4), when the material was in the late stage of low-salt fermentation of phase A and had not yet undergone phase inversion; the second time point was the 144th hour of fermentation (day 6), when the injection of high-osmotic conditioning solution for phase B and the inoculation of bacteria had been completed and a 24-hour adaptation period had been completed; the third time point was the 720th hour of fermentation (day 30), when the material was in the anaerobic ripening and stabilization period.

[0099] At the aforementioned time points, approximately 200g of samples were collected from different depths (upper, middle, and lower) of the fermentation tanks in the three embodiments. After being mixed evenly, 25g of the sample was aseptically weighed and placed in a homogenizing bag containing 225mL of sterile physiological saline (containing 0.1% peptone). The sample was homogenized by tapping for 2 minutes to prepare a 1:10 sample homogenate.

[0100] The sample homogenate was serially diluted 10-fold using a gradient dilution method. Plate samples of the appropriate dilutions were then used for counting.

[0101] Lactic acid bacteria were counted using MRS agar medium (with 0.1% actinomycete ketone added to inhibit fungi), and were anaerobically cultured at 37°C for 48 hours before counting. Yeast was counted using Bengal red medium (with 0.1% chloramphenicol added to inhibit bacteria), and was cultured at 28°C for 72 hours before counting.

[0102] Colony count results are expressed as colony forming units (CFU / g). Three parallel dishes were prepared for each sample, and the average value was taken.

[0103] 2. Experimental data (see Table 2)

[0104] Table 2: Monitoring data of microbial community succession during fermentation in Examples 1-3 (CFU / g)

[0105]

[0106] 3. Conclusion Analysis

[0107] According to Table 2 and Figure 2 The monitoring data was analyzed:

[0108] At the 96th hour of fermentation (phase A, low-salt period), the total number of lactic acid bacteria in all three examples reached 10. 8 The CFU / g level indicates that these bacteria become the dominant microbial population. The high-density metabolism of these lactic acid bacteria at this stage is the main driving force for lactic acid production and pH reduction. In Example 2, the lactic acid bacteria proliferation rate fluctuated slightly due to differences in the salt concentration setting of phase A, while Examples 1 and 3 maintained a high biomass. At this point, the yeast count remained at 10... 3The background level of around CFU / g indicates that the environment under the process conditions in step two is not yet suitable for large-scale yeast proliferation.

[0109] At 144 hours, after the injection of phase B hypertonic conditioning solution and completion of phase inversion, the microbial community structure underwent significant changes. Data from Example 1 showed that the number of lactic acid bacteria increased from 4.21 × 10⁻⁶. 8 CFU / g dropped sharply to 3.54×10 5 The CFU / g concentration decreased by three orders of magnitude. This confirms that the high osmotic pressure environment introduced by phase B solution significantly inhibited *Lactobacillus plantarum*, causing it to no longer occupy the dominant ecological niche. Simultaneously, artificially introduced salt-tolerant yeasts (*Halophilic Tetracoccus* and *Zygosacchariformis*) rapidly adapted and multiplied to 10⁻⁶ CFU / g with the assistance of magnesium ion preloading in phase A and the reducing environment in phase B. 5 CFU / g level.

[0110] By the 720th hour (post-ripening stationary phase), the bacterial succession was complete. The number of lactic acid bacteria in Example 1 further decreased to 10. 3 The level of cells is reduced to a low level of survival, no longer dominating metabolism; while the number of yeast cells has increased to 3.78 × 10⁻⁶. 7 CFU / g became the absolutely dominant bacterial group. In contrast, in Example 3, due to differences in salt supplementation or dilution in step three, the lactic acid bacteria still retained 10 CFU / g after 144 hours. 6 The residual level is high, posing a risk of excessive acidity in the later stages.

[0111] In summary, the parameter combination in Example 1 effectively played a role in osmotic pressure regulation, utilizing lactic acid bacteria to produce acid in the early stage and driving the replacement of strains through physicochemical means at the phase transition node, ensuring the efficient production of alcohol and aroma by yeast under low competitive pressure in the later stage, and verifying the feasibility of the niche control strategy under non-sterilization conditions.

[0112] Test Example 3:

[0113] 1. Experiment Description

[0114] This test aims to quantitatively analyze the key physicochemical indicators of the final products (fermented for 90 days) obtained from each embodiment and comparative example using national standard testing methods, in order to evaluate the comprehensive impact of different process parameters and material components on fermentation efficiency, substrate utilization, and the chemical composition of the final product.

[0115] The experiments used the low-salt fermented soybean paste products prepared in Examples 1 to 3, and Comparative Examples 1 to 4 as test subjects. The specific experimental steps are as follows:

[0116] After fermentation, samples were randomly taken from multiple points in each fermentation tank, mixed thoroughly, and then 500g of sample was collected using the quartering method. The samples were then homogenized in a tissue homogenizer to prepare a homogenate for testing.

[0117] Determination of amino acid nitrogen content: The determination was performed according to Method II (pH meter method) of GB 5009.235-2016 "National Food Safety Standard - Determination of Amino Acid Nitrogen in Food". 5.0 g of homogenized sample was weighed, diluted with distilled water, filtered, and the amino groups were fixed with formaldehyde solution. The solution was then titrated with 0.05 mol / L sodium hydroxide standard solution to the endpoint pH 9.2, and the content was calculated.

[0118] Total acid content determination: Performed according to GB 12456-2021 "National Food Safety Standard - Determination of Total Acid in Food". After weighing and soaking the sample for extraction, titrate with 0.1 mol / L sodium hydroxide standard solution until a faint red color appears, using phenolphthalein as an indicator. The result is expressed as lactic acid (conversion factor 0.090).

[0119] Determination of reducing sugar content: The determination was performed using the direct titration method according to GB 5009.7-2016 "National Food Safety Standard - Determination of Reducing Sugars in Food". The glucose equivalent was determined using Fehling's reagent redox reaction and methylene blue as an indicator.

[0120] Salt (sodium chloride) content determination: determined according to the silver nitrate titration method of GB 5009.44-2016 "National Food Safety Standard for Chlorides in Food".

[0121] All physicochemical indicators were measured in triplicate, and the results were retained to two decimal places.

[0122] 2. Experimental data (see Table 3)

[0123] Table 3: Test results of physicochemical properties of finished products from each example and comparative example

[0124]

[0125] 3. Conclusion Analysis

[0126] The analysis based on the test data in Table 3 is as follows:

[0127] Regarding amino acid nitrogen, Example 1 showed a content of 1.58 g / 100 g, which was superior to Examples 2 and 3, and significantly higher than Comparative Example 1 (0.86 g / 100 g). Comparative Example 1, lacking a sodium citrate buffer system and magnesium ions, experienced a pH drop below the optimal range for acidic proteases during the initial fermentation stage, resulting in limited enzymatic hydrolysis and ineffective protein conversion. The data from Example 1 demonstrate that maintaining a suitable pH environment is crucial for improving amino acid yield in low-salt fermentation.

[0128] Regarding the total acid content, Example 1 showed a value of 1.85 g / 100 g, indicating sufficient lactic acid fermentation. In contrast, Comparative Example 2 had a total acid content of only 0.68 g / 100 g, a significantly lower value. This is because Comparative Example 2 employed a one-time salt addition process, where the initial high-salt environment (>10%) inhibited the growth and metabolism of the salt-tolerant *Lactobacillus plantarum*, thus hindering acid production. This data verifies the effectiveness of the segmented salt control process of this invention in ensuring the acid production performance of lactic acid bacteria.

[0129] Regarding the reducing sugar content, Example 1 (10.42 g / 100 g) was significantly higher than Comparative Example 3 (7.55 g / 100 g). Comparative Example 3 did not contain sodium L-ascorbate, resulting in a higher redox potential. The dissolved oxygen introduced by stirring and tank turning induced the *Zygosacchariformis* to undergo the Pasteur effect, switching to aerobic respiration and consuming substrate sugars to produce carbon dioxide and water. Example 1, by adding ascorbic acid to regulate the system's ORP, effectively reduced aerobic sugar consumption and improved the reducing sugar retention rate in the final product.

[0130] Furthermore, the physicochemical data of Comparative Example 4 (amino acid nitrogen 1.56 g / 100 g, total acid 1.82 g / 100 g) showed no significant difference from those of Example 1. This indicates that the absence of calcium ions does not affect the biochemical reaction processes such as enzymatic hydrolysis, acid production, and sugar metabolism. Therefore, the difference in final quality between Comparative Example 4 and Example 1 mainly exists at the level of physical texture, rather than changes in chemical composition, which corresponds to the results of subsequent texture analysis.

[0131] In summary, the process parameters of Example 1 achieved high levels of accumulation of amino acid nitrogen, total acid and reducing sugar while ensuring low salt content (approximately 10.8%), and the synergistic mechanism of each component was supported by data.

[0132] Test Example 4:

[0133] 1. Experiment Description

[0134] This test aims to quantitatively characterize the physical and mechanical properties of fermented products using a texture analyzer, specifically comparing the effects of adding anhydrous calcium chloride versus not adding this component on the texture and chewing texture of fermented soybean granules under low-salt conditions.

[0135] The experiments used the fermented soybean paste products prepared in Example 1 and Comparative Example 4 as test subjects. The specific experimental steps are as follows:

[0136] Approximately 100g of sample was randomly selected from the fermented soybean paste, and whole, undamaged soybean particles were manually selected. The surface of the particles was quickly rinsed with deionized water to remove any remaining sauce, then the surface moisture was blotted dry with filter paper and placed in petri dishes for later use. Twenty soybean particles were randomly selected from each test subject for parallel testing; the maximum and minimum values ​​were discarded, and the arithmetic mean was taken.

[0137] The TA.XTPlus texture analyzer was used for full texture analysis. A P / 36R cylindrical probe (36mm in diameter) was selected, and the test parameters were set as follows: pre-test speed 2.0mm / s, test speed 1.0mm / s, post-test speed 1.0mm / s; compression ratio set to 50%, interval between two compressions 5.0 seconds; trigger force set to 5.0g.

[0138] A single soybean granule is placed horizontally at the center of the stage, and the probe is pressed vertically downwards to perform two compression cycles. The instrument automatically records the force-time curve and calculates the texture parameters.

[0139] Parameter calculation instructions:

[0140] Hardness: Take the maximum peak force in the first compression cycle.

[0141] Elasticity: The ratio of the second compression height to the first compression height (length ratio).

[0142] Chewing properties: The calculation formula is hardness × elasticity × cohesiveness.

[0143] Note: Cohesion refers to the ability of a sample to resist damage and maintain its integrity. It is obtained by the ratio of the second compression area to the first compression area in the TPA curve. In this test, it is used as an intermediate parameter for calculating chewiness and will not be listed separately.

[0144] 2. Experimental data (see Table 4)

[0145] Table 4: Test results of the textural properties (TPA) of the finished soybean paste particles in Example 1 and Comparative Example 4

[0146]

[0147] 3. Conclusion Analysis

[0148] The analysis based on the texture test data in Table 4 is as follows:

[0149] Regarding the hardness index, the measured value for Example 1 was 984.52 g, while that for Comparative Example 4 was 387.65 g, showing a significant difference. In a low-salt fermentation system (salinity <11%), due to the relatively low osmotic pressure, the soybean granule cells absorb water and swell. Simultaneously, sodium ions displace calcium ions in the cell walls, leading to pectin dissolution and a loosening of the tissue structure. Comparative Example 4, without the addition of exogenous calcium, reflects the matrix softening phenomenon under low-salt conditions. Example 1 introduced a calcium source into the process, utilizing the acidic environment of the system to promote the release of calcium ions, which then cross-link with the low-methoxyl pectin in the cell walls, enhancing the mechanical strength of the cell walls and thus counteracting the hydration softening effect caused by the low-salt environment.

[0150] In terms of chewiness and elasticity, Example 1 (chewiness 412.33g, elasticity 0.68) was higher than Comparative Example 4 (chewiness 105.89g, elasticity 0.42). Chewiness is a comprehensive parameter determined by hardness, elasticity, and cohesion, representing the energy required to chew solid food to a swallowable state. The higher values ​​of Example 1 indicate that its particles maintain a relatively dense gel network structure, exhibiting a certain degree of resilience under stress. The lower values ​​of Comparative Example 4 indicate insufficient internal structural support, resulting in primarily plastic deformation under stress.

[0151] Based on the physicochemical data of Test Example 3 (where there was no significant difference in chemical composition between the two), this test confirmed that the difference in finished product quality stemmed from changes in physical microstructure. Example 1, through in-situ calcium bridge cross-linking technology, improved the texture characteristics of fermented soybean granules under low-salt, high-water-activity conditions, solving the common problem of tissue softening in low-salt fermented products.

[0152] Test Example 5:

[0153] 1. Experiment Description

[0154] This test aims to quantitatively score the finished soybean paste products prepared under different process conditions through a sensory evaluation panel, and to evaluate the impact of process parameters on the flavor quality of the final product from four dimensions: color, aroma, taste, and appearance.

[0155] The experiments used the fermented soybean paste products prepared in Examples 1 to 3, and Comparative Examples 1 to 3 as test subjects. The specific experimental steps are as follows:

[0156] A sensory evaluation team was formed, consisting of 10 people who had received sensory evaluation training and were required to be non-colorblind and have normal sense of smell and taste.

[0157] A 100-point evaluation system was established, with four evaluation indicators: color (20 points, evaluating reddish-brown color and gloss), aroma (30 points, evaluating soy sauce aroma, ester aroma and absence of off-odor), taste (30 points, evaluating umami, saltiness and palatability and aftertaste), and texture (20 points, evaluating particle integrity, crispness and viscosity).

[0158] Take 30g of each group of fermented and matured samples and place them in a white ceramic dish for random coding.

[0159] The evaluation was conducted in a separate sensory evaluation room, with the room temperature controlled at 25±2℃. The evaluators observed the color and shape, smelled the aroma, and finally tasted the flavor.

[0160] After tasting each sample, the evaluator rinsed their mouth with purified water and waited 2 minutes before evaluating the next sample.

[0161] When compiling statistics, the highest and lowest scores are removed, and the arithmetic mean of the remaining scores is taken as the final result, rounded to one decimal place.

[0162] 2. Experimental Data

[0163] Table 5: Sensory Evaluation Scores of Finished Products in Each Group

[0164]

[0165] 3. Conclusion Analysis

[0166] The sensory evaluation data in Table 5 are analyzed as follows:

[0167] Example 1 achieved a total score of 93.7, ranking at the best level across all four evaluation dimensions. Its flavor score of 28.2 points was positively correlated with the high amino acid nitrogen content measured in Example 3, indicating that the sodium citrate buffer system effectively maintained neutral protease activity, promoted deep hydrolysis of substrate proteins, and thus significantly enhanced the umami intensity of the product. Its texture score of 18.5 points validated the high hardness and chewiness data measured by the texture analyzer in Example 4, demonstrating that the addition of calcium ions maintained the crisp texture of the fermented soybean particles through chemical cross-linking.

[0168] Comparative Example 1 scored 20.8 points, the lowest among all groups. Due to the lack of pH buffering and magnesium ion assistance, the sudden drop in acidity in the early stages of fermentation inhibited enzyme activity and hindered protein degradation, resulting in insufficient accumulation of flavor compounds in the final product. This indicates that the degree of biochemical reactions during fermentation directly determines the taste indicators of the final product.

[0169] Comparative Example 2 scored 21.5 points in aroma, significantly lower than Example 1 (28.4 points). Comparative Example 2 employed a one-step high-salt method, where the continuously high osmotic pressure environment inhibited the growth and metabolism of salt-tolerant yeast, hindering the synthesis of volatile flavor compounds such as alcohols and esters. In contrast, the segmented salt control process in Example 1 provided a suitable metabolic window for yeast during the A phase and phase transition adaptation period, promoting the transformation and accumulation of characteristic aroma compounds.

[0170] Comparative Example 3 scored only 13.4 points for color. Because L-ascorbic acid sodium was not added, the redox potential of the fermentation system was out of control, leading to excessive non-enzymatic browning and oxidation of polyphenols and amino acids, resulting in a dull color in the finished product. Example 1, by creating a reducing environment, slowed down the oxidation process and effectively maintained the bright reddish-brown sensory characteristics of the product.

[0171] In summary, the sensory evaluation results corroborate the aforementioned physicochemical indicators, textural data, and microbial succession patterns. The combination of process parameters in Example 1, while ensuring fermentation safety, achieved overall optimization of the product's sensory quality through multi-dimensional metabolic regulation.

[0172] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A low-salt fermented soybean paste production process based on synergistic fermentation of compound microbial strains, characterized in that, Includes the following steps: The broad bean curd is mixed evenly with the drained chili embryos to obtain a solid mixture. A-phase starter conditioning liquid is sprayed onto the solid mixture, and Lactobacillus plantarum is inoculated. The mixture is then mixed until it is moistened to obtain primary fermentation mash. The primary fermentation mash is controlled to undergo constant temperature fermentation until the pH value drops to a preset acidic range to obtain acidified mature mash. Inject phase B hypertonic control solution into the acidified mature mash, and inoculate with halophilic tetracoccus and Rhodotorula glutinis, mix evenly to obtain phase inversion fermentation mash; The phase-inversion fermentation mash was cooled and subjected to anaerobic fermentation under air-isolated conditions to obtain a low-salt fermented soybean paste product. The solid mixture comprises 400-450 parts by weight of broad bean koji and 100-150 parts by weight of chili embryos; The A-phase start-up conditioning solution includes 270-380 parts by weight of process water, 3.0-5.0 parts by weight of sodium citrate dihydrate, 0.2-0.4 parts by weight of magnesium sulfate heptahydrate, and 40-48 parts by weight of sodium chloride. The inoculation amount of *Lactobacillus plantarum* is 0.5-1.0 parts by weight; The B-phase hypertonic conditioning solution comprises 110-145 parts by weight of process water, 35-45 parts by weight of sodium chloride, 1.0-2.0 parts by weight of anhydrous calcium chloride, and 0.5-0.8 parts by weight of L-ascorbic acid sodium. The inoculation amount of the halophilic tetracoccus is 1.0-2.0 parts by weight, and the inoculation amount of the zygosaccharidobacterium rouxii is 1.0-2.0 parts by weight.

2. The low-salt fermented soybean paste production process based on synergistic fermentation of compound microorganisms according to claim 1, characterized in that, The preparation method of the broad bean koji is as follows: Select peeled dried broad beans, soak and steam until fully cooked, then cool to below 38℃; Add flour at 18% of the weight of dried bean raw material and mix, and add 0.04% of Aspergillus oryzae spore powder of the total weight of dried bean raw material; After mixing, aeration is carried out to make koji, and the thickness of the koji layer is controlled at 30cm. It is then incubated at a constant temperature of 30-35℃ for 40-44 hours, during which the koji is turned over. When the neutral protease activity reaches 2000U / g or more, the koji is removed from the mold.

3. The low-salt fermented soybean paste production process based on synergistic fermentation of compound microorganisms according to claim 1, characterized in that, The preparation method of the B-phase hyperosmotic conditioning solution is as follows: Heat the process water to 45-50℃, add sodium chloride and anhydrous calcium chloride, and stir until completely dissolved to obtain a primary composite salt solution. The primary composite salt solution is cooled to 25-32℃, L-ascorbic acid sodium is added, and stirred until completely dissolved to obtain the solution; the B-phase hyperosmolarity regulating solution is used up within 2 hours after preparation.

4. The low-salt fermented soybean paste production process based on synergistic fermentation of compound microorganisms according to claim 1, characterized in that, The temperature for constant temperature fermentation is controlled at 35-38℃; the mash is turned over every 12 hours for the first 48 hours of constant temperature fermentation, and then left to ferment statically after 48 hours. The preset acidity range is pH 5.25-5.

45.

5. The low-salt fermented soybean paste production process based on synergistic fermentation of compound microorganisms according to claim 1, characterized in that, The steps of injecting phase B hypertonic control solution into the acidified mature mash and inoculating it with halophilic tetracocci and Lewy's zygosacchari are as follows: Turn on the stirrer and set the speed to 20-30 rpm. The B-phase hypertonic regulating solution is uniformly injected into the acidified mature mash within 10-15 minutes and stirred continuously for 30 minutes. Five minutes before the end of the stirring process, the halophilic tetracoccus and Ludwig's zygosacchari were inoculated.

6. The low-salt fermented soybean paste production process based on synergistic fermentation of compound microorganisms according to claim 1, characterized in that, The steps for cooling the phase-inversion fermentation mash and carrying out anaerobic fermentation under air-isolated conditions are as follows: The temperature of the phase-inversion fermentation mash was lowered to 25-28℃, the surface was covered with a thin film to isolate it from the air, and it was kept at a constant temperature for 90 days for static fermentation.

7. The low-salt fermented soybean paste production process based on synergistic fermentation of compound microorganisms according to claim 1, characterized in that, The viable count of *Lactobacillus plantarum* is ≥1.0 × 10⁻⁶. 11 CFU / g; the viable count of the halophilic tetracoccus is ≥1.0×10⁻⁶. 9 CFU / mL; the viable count of the *Zygosacchariformis* ≥ 1.0 × 10⁻⁶ 8 CFU / mL.

8. The low-salt fermented soybean paste production process based on synergistic fermentation of compound microorganisms according to claim 1, characterized in that, The chili embryo is a semi-finished product of chili peppers that have been washed, crushed, and salted, with a moisture content of 65%-70%.

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