A low-salt douchi flavor-enhanced fermentation preparation process

Abstract

The application discloses a kind of low-salt douchi's flavor enhancement fermentation preparation process, belongs to the technical field of soy product fermentation.The process is formed by vacuum spraying coating liquid and adding modified diatomite gas-permeable water-blocking protective film, realizes water directional control;Using aspergillus oryzae, bacillus subtilis, lactobacillus plantarum inoculation in turn and gradient CO2 control, promote directional metabolism of strain;Through composite low-salt brine and osmotic pressure gradient slow rise guarantee fermentation stability, combined with sterile nitrogen microbubble two-stage anaerobic fermentation improves proteolysis efficiency;Finally, through the embedding of composite flavor lock and hydrophobic antibacterial coating treatment, with heat stability and gradient cooling realizes flavor lock and antibacterial preservation.The application can effectively inhibit moisture regain and bacterial contamination, improve amino acid nitrogen content and flavor substance type, prepare low-salt, flavor rich, shelf-stable low-salt douchi, solve the technical contradiction that douchi is easy to absorb moisture under low-salt condition, bacterial contamination and flavor substance synthesis deficiency.

Description

Technical Field

[0001] This invention relates to the field of soybean product fermentation technology, specifically to a flavor-enhancing fermentation preparation process for low-salt fermented soybeans. Background Technology

[0002] Doubanjiang (fermented black soybeans), a traditional fermented soybean product, is widely consumed due to its unique flavor and rich nutrition. Reducing its salt content has become a core direction for the health-oriented upgrading of doubanjiang. However, existing low-salt doubanjiang processes generally have key defects. In a low-salt environment, the surface moisture of the soybean curd is prone to reverse migration, leading to dampness and stickiness. After drying, it easily forms a dense, hard shell that hinders the colonization and enzymatic hydrolysis of microorganisms. At the same time, the sudden change in osmotic pressure in the fermentation system can easily cause the inactivation of functional microorganisms, significantly increasing the risk of contamination by other microorganisms and making the product prone to rancidity and off-flavors.

[0003] In addition, traditional processes often use mixed inoculation of microbial strains, lack targeted metabolic regulation methods, have low protein hydrolysis efficiency, insufficient accumulation of umami substances such as amino acid nitrogen, few types of volatile flavor components, weak aroma, and lack long-lasting antibacterial and aroma-locking technologies. During storage, the products are prone to moisture absorption and mold growth, and the flavor deteriorates rapidly, making it difficult to simultaneously meet the production requirements of low salt, safety, rich flavor, and stable shelf life.

[0004] Therefore, it is necessary to provide a flavor-enhancing fermentation process for low-salt fermented soybeans to solve the above-mentioned technical problems. Summary of the Invention

[0005] The purpose of this invention is to provide a flavor-enhancing fermentation preparation process for low-salt fermented black beans, in order to solve the technical problems of uncontrolled moisture migration and easy re-moistening, disordered microbial metabolism and insufficient fermentation, and lack of flavor substances in the existing low-salt fermented black bean fermentation process.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a flavor-enhancing fermentation preparation process for low-salt fermented soybeans, comprising the following steps: 1) Raw material pretreatment: Soybeans are steamed and then cooled to 28-35℃ to obtain cooked soybean curd. 2) Gradient coating film formation: The cooked soybean blanks are placed in a vacuum environment and coated with a coating liquid. After the coating is completed, they are dried in stages with hot air to form a protective film on the surface of the cooked soybean blanks. 3) Sequential inoculation for koji making: After removing the protective film, the cooked soybean curd is inoculated with the inoculum in stages for koji making. During the koji making process, gradient CO2 regulation is used to complete directional metabolism and obtain mature koji. 4) Low-salt steady-state fermentation: The mature koji is added to a compound low-salt brine to form a fermentation system. The fermentation system is first subjected to osmotic pressure adaptation culture. After the fermentation system adapts to the initial osmotic pressure, the osmotic pressure is gradually increased. Then, sterile nitrogen microbubbles are introduced into the fermentation system to carry out two-stage anaerobic fermentation until the pH of the fermentation system is 4.5-5.0, and the fermentation product is obtained. 5) Flavor Locking and Shaping: After the fermentation product is encapsulated with a compound flavor-locking agent and dried at low temperature, a hydrophobic and antibacterial coating is sprayed onto the dried product. The coated product is then subjected to blending, heat stabilization, sterilization and gradient cooling treatment to obtain the low-salt fermented black bean product.

[0007] Preferably, in step 2), the coating solution is a 20wt%–25wt% aqueous solution prepared by mass fractions of 80–90 parts of water-soluble maltodextrin, 10–20 parts of low-acylated gellan gum, and 5–10 parts of glyceryl monostearate. The coating solution is prepared as follows: first, purified water is heated to 45–55°C, water-soluble maltodextrin is added and stirred until completely dissolved, then low-acylated gellan gum is added and stirred for 15–20 min until fully dispersed without lumps, and finally glyceryl monostearate is added and stirred for 10–15 min until emulsified uniformly, and then cooled to 30–35°C and brought to a constant volume. The vacuum degree of the coating under vacuum is -0.06 MPa, and the coating is stirred for 15–20 min after spraying.

[0008] Preferably, in step 2), the hot air segmented drying is a three-stage step drying: the first stage is drying at 55℃ for 20 minutes, the second stage is drying at 45℃ for 30 minutes, and the third stage is maturing at 35℃ for 15 minutes, and the surface moisture of the soybean curd after drying is ≤8%.

[0009] Preferably, in step 2), 15%–20% by mass of modified diatomaceous earth with a particle size of 10–20 μm is added to the protective membrane to form a breathable and water-resistant microporous structure. The modified diatomaceous earth is prepared as follows: the diatomaceous earth powder is sieved to a particle size of 10–20 μm, calcined at 400–500℃ for 2–3 h, modified by stirring at 60–70℃ for 1–2 h with a 3–5% by mass KH-550 silane coupling agent ethanol solution, filtered, and dried at 80–100℃ to constant weight.

[0010] Preferably, in step 2), the protective film is removed by rinsing with water and stirring, and the residual rate after removal is ≤0.5%.

[0011] Preferably, in step 3), the inoculated microorganisms are Aspergillus oryzae, Bacillus subtilis, and Lactobacillus plantarum in sequence; the inoculation amount of Aspergillus oryzae is 0.8-1.2% of the cooked soybean curd mass, and it is cultured for 20-28 hours at a temperature of 28-35℃ and a relative humidity of 75-80%; the inoculation amount of Bacillus subtilis is 0.4-0.8% of the cooked soybean curd mass, and it is cultured for 30-40 hours at a temperature of 32-40℃ and a relative humidity of 70-75%; the inoculation amount of Lactobacillus plantarum is 0.1-0.5% of the cooked soybean curd mass, and it is cultured for 18-22 hours at a temperature of 30-36℃ and a relative humidity of 65-70%.

[0012] Preferably, in step 3), the specific steps of gradient CO2 regulation are as follows: during the Aspergillus oryzae culture stage, CO2 with a volume concentration of 4-6% is introduced into the system at a gas flow rate of 0.3-0.7 m / s. 3 / h; During the Bacillus subtilis culture stage, CO2 with a volume concentration of 6-8% is introduced into the system at a gas flow rate of 0.4-0.8 m / h. 3 / h; During the Lactobacillus plantarum culture stage, CO2 with a volume concentration of 8-10% is introduced into the system at a gas flow rate of 0.2-0.6 m / h. 3 / h.

[0013] Preferably, in step 4), the composite low-salt brine is composed of NaCl, KCl, and potassium citrate in a mass ratio of 3:1:(0.4-0.6), with a total salt mass fraction of 3.5-4.5% based on the mass of the mature fermented koji; the initial osmotic pressure during fermentation is 110-130 mOsm / kg, decreasing to 5-10 mOsm / kg after 10-12 hours of adaptation. (12h) The concentration gradually increases to 140-160 mOsm / kg.

[0014] Preferably, in step 4), the diameter of the sterile nitrogen microbubbles is 10-20 μm; the two-stage anaerobic fermentation is: the first stage fermentation is carried out at 25-32℃ for 50-70 hours, and the second stage fermentation is carried out at 20-25℃ for 7-9 days.

[0015] Preferably, in step 5): the composite fragrance-locking agent is composed of β-cyclodextrin and gum arabic; based on the mass of the fermentation product, the amount of β-cyclodextrin added is 0.3-0.7 wt%, and the amount of gum arabic added is 0.1-0.2 wt%; the hydrophobic antibacterial coating is octenyl succinate starch ester, which is mixed with warm water at 50-60℃ to form an emulsion with a mass fraction of 10%-15%, stirred for 10-15 min until completely gelatinized, cooled to 25-30℃ and then atomized and sprayed, with a spray amount of 0.5-1.0% based on the mass of the fermentation product; the heat stabilization treatment is a heat treatment at 60-65℃ for 10-15 min, and the gradient cooling is a cooling process in stages of 25℃, 15℃, and 5℃, with a cooling rate of 1.5-2.5℃ / min.

[0016] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention uses water-soluble maltodextrin as the inner film-forming component that adheres closely to the soybean blank. Its hydrophilic properties allow it to adhere tightly to the surface of the soybean blank, ensuring a firm film layer and smooth drainage of moisture from the inside of the soybean blank. Low-acylated gellan gum is used as the intermediate supporting framework to maintain the stability of the film structure. Glyceryl monostearate is used as the outer hydrophobic component to form a hydrophobic barrier interface on the outer surface of the film layer. Together, these three components construct a gradient structure film with a hydrophilic inner side and a hydrophobic outer side on the surface of the cooked soybean blank. Simultaneously, calcined and silane-modified hydrophobic diatomaceous earth is introduced to form a continuous and uniform air-permeable and water-blocking microporous structure inside the membrane. This structure can guide excess moisture inside the soybean curd to slowly drain to the surface and be dried in time, while preventing external moisture from seeping back in. This fundamentally avoids the soybean curd surface from becoming damp and sticky or from becoming too dry and forming a dense hard shell during low-salt fermentation. It maintains the loose and porous matrix state inside the soybean curd, providing a stable and suitable interface environment for the subsequent colonization, growth, and metabolism of Aspergillus oryzae, Bacillus subtilis, and Lactobacillus plantarum, ensuring that the strains can attach evenly and penetrate deep into the soybean curd to exert their effects.

[0017] 2. This invention involves inoculating Aspergillus oryzae, Bacillus subtilis, and Lactobacillus plantarum in stages according to their physiological characteristics and metabolic sequence. A gradient CO2 control method with progressively increasing concentrations is used. During the Aspergillus oryzae culture stage, an appropriate concentration of CO2 is supplied to enhance the synthesis and secretion of proteases and amylases. During the Bacillus subtilis culture stage, the CO2 concentration is moderately increased to promote the generation of flavor precursors. During the Lactobacillus plantarum culture stage, the CO2 concentration is further increased to optimize the acid-producing environment. The three strains occupy their ecological niches sequentially, completing the metabolic relay step by step, without nutrient competition or metabolic inhibition. This allows for the continuous and efficient decomposition of soybean protein and polysaccharides, resulting in the continuous accumulation of umami substances such as amino acid nitrogen and the generation of a large amount of characteristic flavor substances such as esters, alcohols, and aldehydes, giving the fermented soybeans a rich, mellow flavor and intense aroma.

[0018] 3. This invention employs a stepwise addition of compound low-salt brine and a gradual increase in osmotic pressure gradient to allow functional microorganisms to gradually adapt to the low-salt environment, avoiding cell loss and inactivation caused by sudden changes in osmotic pressure. This is combined with sterile nitrogen microbubbles of uniform diameter to improve mass transfer and dissolved oxygen levels in the fermentation system, maintaining the pH of the fermentation system within a suitable range and effectively inhibiting the growth and reproduction of unwanted microorganisms. In the later stages, β-cyclodextrin and gum arabic are used to encapsulate and lock in flavor substances, reducing the loss of volatile components. Simultaneously, an octenyl succinic starch ester coating is sprayed onto the surface of the fermented black beans to form a hydrophobic and antibacterial coating, further preventing the invasion of moisture and unwanted microorganisms. Combined with a gradient cooling process, this stabilizes the product's texture and internal microstructure, ensuring that the fermented black beans maintain optimal moisture content, complete fermentation, full flavor, and stable quality during storage even under low-salt conditions. Detailed Implementation

[0019] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] Example 1 This embodiment provides a flavor-enhancing fermentation preparation process for low-salt fermented black beans, including the following steps: 1) Raw material pretreatment: Soybeans are washed, soaked, steamed, and cooled to 32°C to obtain cooked soybean curd.

[0021] 2) Gradient Coating Film Formation: A coating solution was prepared by weight of 85 parts water-soluble maltodextrin, 15 parts low-acylated gellan gum, and 7.5 parts glyceryl monostearate, to form a 22.5 wt% aqueous solution. Purified water was heated to 50°C to dissolve the raw materials sequentially and emulsify them uniformly. The solution was then cooled to 32°C to obtain the coating solution. The coating was sprayed under a vacuum of -0.06 MPa at a spray rate of 6.5 mL / min per kilogram of cooked soybean curd, followed by stirring for 18 min. The mixture underwent a three-stage drying process: drying at 55°C for 20 min, drying at 45°C for 30 min, and maturing at 35°C for 15 min. The surface moisture content of the soybean curd was ≤8%. More specifically, diatomaceous earth powder was sieved to a particle size of 15 μm, calcined at 450℃ for 2.5 h, and then modified by stirring at 65℃ for 1.5 h with a 4% (w / w) KH-550 silane coupling agent ethanol solution. After filtration, it was dried at 90℃ to constant weight to obtain modified diatomaceous earth. 17.5 wt% of the modified diatomaceous earth with a particle size of 15 μm was added to the protective film. The protective film was removed by rinsing with clean water, with a residual rate of ≤0.5%.

[0022] 3) Sequential inoculation for koji preparation: Aspergillus oryzae, Bacillus subtilis, and Lactobacillus plantarum were inoculated sequentially. Aspergillus oryzae inoculation was 1.0%, incubated at 32℃ and 78% relative humidity for 24 hours. Bacillus subtilis inoculation was 0.6%, incubated at 36℃ and 72% relative humidity for 35 hours. Lactobacillus plantarum inoculation was 0.3%, incubated at 33℃ and 68% relative humidity for 20 hours. Gradient CO2 control was used, with 5% CO2 and a flow rate of 0.5 m / s² at the Aspergillus oryzae stage. 3 / h, Bacillus subtilis stage, 7% CO2, flow rate 0.6m 3 / h, Lactobacillus plantarum stage, 9% CO2, flow rate 0.4m 3 / h, to obtain mature koji blanks.

[0023] 4) Low-salt steady-state fermentation: The compound low-salt brine is prepared with a ratio of NaCl:KCl:potassium citrate of 3:1:0.5, and the total salt content is 4.0% based on the mass of the mature fermented koji. The initial osmotic pressure is 120 mOsm / kg, and after 11 hours of adaptation, it decreases to 7.5 mOsm / (kg). The concentration of nitrogen gas was gradually increased to 150 mOsm / kg over 12 hours. Sterile nitrogen microbubbles with a diameter of 15 μm were introduced. Fermentation was carried out at 28°C for 60 hours in the first stage and at 22°C for 8 days in the second stage until the pH of the system reached 4.7, thus obtaining the fermentation product.

[0024] 5) Flavor Lock-in Shaping: Based on the weight of fermentation product, add 0.5wt% β-cyclodextrin and 0.15wt% gum arabic for embedding, and dry at low temperature. Prepare a 12.5wt% octenyl succinate starch ester emulsion, atomize and spray at a rate of 0.75%. After blending, heat stabilize at 62℃ for 12 min, sterilize, and then cool sequentially at 25℃, 15℃, and 5℃ at a cooling rate of 2.0℃ / min to obtain the low-salt fermented black soybean product.

[0025] Example 2 This embodiment provides a flavor-enhancing fermentation preparation process for low-salt fermented black beans, including the following steps: 1) Raw material pretreatment: Soybeans are washed, soaked, steamed, and cooled to 35°C to obtain cooked soybean curd.

[0026] 2) Gradient Coating Film Formation: A coating solution was prepared by weight of 90 parts water-soluble maltodextrin, 20 parts low-acylated gellan gum, and 10 parts glyceryl monostearate, to form a 25 wt% aqueous solution. Purified water was heated to 55°C to dissolve the raw materials sequentially and emulsify them uniformly. The solution was then cooled to 35°C to obtain the coating solution. The coating was sprayed under a vacuum of -0.06 MPa at a spray rate of 8 mL / min per kilogram of cooked soybean curd, followed by stirring for 20 min. The mixture underwent a three-stage drying process: drying at 55°C for 20 min, drying at 45°C for 30 min, and maturing at 35°C for 15 min. The surface moisture content of the soybean curd was ≤8%. More specifically, diatomaceous earth powder is sieved to a particle size of 20 μm, calcined at 500℃ for 3 hours, and then modified by stirring at 70℃ for 2 hours with a 5% (w / w) KH-550 silane coupling agent ethanol solution. After filtration, it is dried at 100℃ to constant weight to obtain modified diatomaceous earth. 20 wt% of the modified diatomaceous earth with a particle size of 20 μm is added to the protective film. The protective film is removed by rinsing with clean water, with a residual rate of ≤0.5%.

[0027] 3) Sequential inoculation for koji preparation: Aspergillus oryzae, Bacillus subtilis, and Lactobacillus plantarum were inoculated sequentially. The Aspergillus oryzae inoculation rate was 1.2%, and the culture was carried out at 35℃ and 80% relative humidity for 28 hours. The Bacillus subtilis inoculation rate was 0.8%, and the culture was carried out at 40℃ and 75% relative humidity for 40 hours. The Lactobacillus plantarum inoculation rate was 0.5%, and the culture was carried out at 36℃ and 70% relative humidity for 22 hours. Gradient CO2 control was used, with 6% CO2 and a flow rate of 0.7 m / s². 3 / h, Bacillus subtilis stage, 8% CO2, flow rate 0.8m 3 / h, Lactobacillus plantarum stage, 10% CO2, flow rate 0.6m 3 / h, to obtain mature koji blanks.

[0028] 4) Low-salt steady-state fermentation: The compound low-salt brine is prepared with a ratio of NaCl:KCl:potassium citrate of 3:1:0.6, and the total salt content is 4.5% based on the mass of the mature fermented koji. The initial osmotic pressure is 130 mOsm / kg, and after 12 hours of adaptation, it decreases to 10 mOsm / (kg). The concentration was gradually increased to 160 mOsm / kg over 12 hours. Sterile nitrogen microbubbles with a diameter of 20 μm were introduced. Fermentation was carried out at 32°C for 70 hours in the first stage and at 25°C for 9 days in the second stage until the pH of the system reached 5.0, thus obtaining the fermentation product.

[0029] 5) Flavor Lock-in Shaping: Based on the weight of fermentation product, add 0.7wt% β-cyclodextrin and 0.2wt% gum arabic for embedding, and dry at low temperature. Prepare a 15wt% octenyl succinate starch ester emulsion, atomize and spray at a rate of 1.0%. After blending, heat stabilize at 65℃ for 15 min, sterilize, and then cool sequentially at 25℃, 15℃, and 5℃ at a cooling rate of 2.5℃ / min to obtain the low-salt fermented black soybean product.

[0030] Example 3 This embodiment provides a flavor-enhancing fermentation preparation process for low-salt fermented black beans, including the following steps: 1) Raw material pretreatment: Soybeans are washed, soaked, steamed, and cooled to 28°C to obtain cooked soybean curd.

[0031] 2) Gradient Coating Film Formation: A coating solution was prepared by weight of 80 parts water-soluble maltodextrin, 10 parts low-acylated gellan gum, and 5 parts glyceryl monostearate, to form a 20wt% aqueous solution. Purified water was heated to 45℃ to dissolve the raw materials sequentially and emulsify them uniformly. The solution was then cooled to 30℃ to obtain the coating solution. The coating was sprayed under a vacuum of -0.06 MPa at a spray rate of 5 mL / min per kilogram of cooked soybean curd, and stirred for 15 min after spraying. The mixture underwent a three-stage drying process: drying at 55℃ for 20 min, drying at 45℃ for 30 min, and maturing at 35℃ for 15 min. The surface moisture content of the soybean curd was ≤8%. More specifically, diatomaceous earth powder is sieved to a particle size of 10 μm, calcined at 400℃ for 2 hours, and then modified by stirring at 60℃ for 1 hour with a 3% (w / w) KH-550 silane coupling agent ethanol solution. After filtration, it is dried at 80℃ to constant weight to obtain modified diatomaceous earth. 15 wt% of the modified diatomaceous earth with a particle size of 10 μm is added to the protective film. The protective film is removed by rinsing with clean water, with a residual rate of ≤0.5%.

[0032] 3) Sequential inoculation for koji preparation: Aspergillus oryzae, Bacillus subtilis, and Lactobacillus plantarum were inoculated sequentially. Aspergillus oryzae inoculation was 0.8%, incubated at 28℃ and 75% relative humidity for 20 h. Bacillus subtilis inoculation was 0.4%, incubated at 32℃ and 70% relative humidity for 30 h. Lactobacillus plantarum inoculation was 0.1%, incubated at 30℃ and 65% relative humidity for 18 h. Gradient CO2 control was used, with 4% CO2 and a flow rate of 0.3 m / s² at the Aspergillus oryzae stage. 3 / h, Bacillus subtilis stage, 6% CO2, flow rate 0.4m 3 / h, Lactobacillus plantarum stage, 8% CO2, flow rate 0.2m 3 / h, to obtain mature koji blanks.

[0033] 4) Low-salt steady-state fermentation: The compound low-salt brine is prepared with a ratio of NaCl:KCl:potassium citrate of 3:1:0.4, and the total salt content is 3.5% based on the mass of the mature fermented koji. The initial osmotic pressure is 110 mOsm / kg, and after acclimatization for 10 hours, it decreases to 5 mOsm / (kg). The concentration was gradually increased to 140 mOsm / kg over 12 hours. Sterile nitrogen microbubbles with a diameter of 10 μm were introduced. Fermentation was carried out at 25°C for 50 hours in the first stage and at 20°C for 7 days in the second stage until the pH of the system reached 4.5, thus obtaining the fermentation product.

[0034] 5) Flavor Lock-in Shaping: Based on the weight of fermentation product, add 0.3wt% β-cyclodextrin and 0.1wt% gum arabic for encapsulation, and dry at low temperature. Prepare a 10wt% octenyl succinate starch ester emulsion, atomize and spray at a rate of 0.5%. After blending, heat stabilize at 60℃ for 10 min, sterilize, and then cool sequentially at 25℃, 15℃, and 5℃ at a cooling rate of 1.5℃ / min to obtain the low-salt fermented black soybean product.

[0035] Comparative Example 1 The only difference between this comparative example and Example 1 is that gradient coating film formation treatment is not performed, protective film is not prepared, and modified diatomaceous earth is not added. All other process parameters and operating steps are exactly the same.

[0036] Expected performance: The cooked soybean curd has no protective film to regulate moisture migration. During the low-salt fermentation process, the surface is very prone to moisture return. After the surface of the soybean curd dries, a dense hard shell forms, which significantly increases the risk of contamination by miscellaneous bacteria. The colonization and enzymatic hydrolysis effects of the bacteria are greatly reduced, resulting in a bland flavor and uneven texture of the fermented soybeans.

[0037] Comparative Example 2 The only difference between this comparative example and Example 1 is that ordinary unmodified diatomaceous earth is added to the protective film, and no calcination or silane modification treatment is performed. All other process parameters and operating steps are exactly the same.

[0038] Expected performance: Ordinary diatomaceous earth has a hydrophilic structure and cannot form breathable and water-blocking micropores. The protective film absorbs and reabsorbs moisture, and cannot inhibit the migration of moisture from the inside of the soybean curd. The surface is constantly humid and easily contaminated with bacteria, resulting in poor stability of the fermentation system.

[0039] Comparative Example 3 The only difference between this comparative example and Example 1 is that three strains were mixed and inoculated at once, without sequential inoculation or gradient CO2 control. The remaining process parameters and operating steps are exactly the same.

[0040] Expected performance: Intense competition for nutrients among strains leads to disordered metabolic pathways, insufficient protein hydrolysis and flavor compound synthesis, low degree of enzymatic hydrolysis of fermented black beans, weak fermented black bean aroma, and significantly reduced flavor harmony and intensity.

[0041] Comparative Example 4 The only difference between this comparative example and Example 1 is that a constant concentration of CO2 is introduced during the koji-making process, without gradient CO2 control; the remaining process parameters and operating steps are exactly the same.

[0042] Expected performance: Unable to target and activate the core metabolism of different strains, the enzyme activity of Aspergillus oryzae, the aroma production of Bacillus subtilis, and the acid production efficiency of Lactobacillus plantarum are all inhibited, the fermentation cycle is prolonged, and the product flavor and quality stability are poor.

[0043] Comparative Example 5 The only difference between this comparative example and Example 1 is that the final concentration of the composite low-salt brine was added at once, without osmotic pressure adaptation or gradient increase treatment; the remaining process parameters and operating steps are exactly the same.

[0044] Expected performance: The strain is inactivated by sudden changes in osmotic pressure, the fermentation process is hindered, the system is prone to the growth of miscellaneous bacteria, the pH cannot be stabilized within the suitable range, and the fermented black beans are prone to rancidity, off-flavors and other problems.

[0045] Comparative Example 6 The only difference between this comparative example and Example 1 is that no hydrophobic antibacterial coating was applied and no gradient cooling treatment was performed; the remaining process parameters and operating steps were exactly the same.

[0046] Expected performance: Flavor substances are easily volatilized and lost; the surface of fermented black beans has no hydrophobic antibacterial barrier, making it easy to absorb moisture and become moldy; storage stability is extremely poor; it is easy to deteriorate when stored at room temperature; and the taste and flavor deteriorate rapidly.

[0047] To compare the performance differences of the flavor-enhancing fermentation preparation processes for low-salt fermented soybeans provided in Examples 1-3 and Comparative Examples 1-6, the present invention provides the following experimental methods: I. Physicochemical Index Testing Methods 1.1 Moisture Content Determination According to GB5009.3-2016 "National Food Safety Standard - Determination of Moisture in Food", the direct drying method was adopted: 1. Place the clean weighing bottle in a 105℃ electric thermostatic drying oven to dry to constant weight, cool it, weigh it, and record the mass as m0.

[0048] 2. Weigh approximately 2,000g of evenly crushed fermented black soybean sample, spread it evenly at the bottom of the weighing bottle, and weigh the total mass as m1.

[0049] 3. Place the sample in a 105℃ drying oven for 4 hours, then remove it and place it in a desiccator to cool for 30 minutes before weighing.

[0050] 4. Dry again for 1 hour, cool and weigh until the difference between the two weighings does not exceed 2 mg, which is considered constant weight. Record the final weight m2.

[0051] 5. Moisture content is calculated using the formula: Moisture content (%) = (m1 - m2) / (m1 - m0) × 100 1.2 Salt content determination According to GB5009.44-2016 "National Food Safety Standard - Determination of Chloride in Food", the silver nitrate titration method was used: 1. Weigh 5.00g of fermented black soybean sample, place it in a mortar and add 100mL of distilled water to grind it into a homogenate, transfer it to a 250mL volumetric flask and make up to volume, shake well and filter.

[0052] 2. Pipette 50 mL of the filtrate into an Erlenmeyer flask, add 1 mL of 5% potassium chromate indicator, and mix well.

[0053] 3. Titrate with 0.1 mol / L silver nitrate standard solution until a stable brick-red precipitate appears, and record the volume consumed.

[0054] 4. At the same time, perform a blank test and calculate the total salt content (calculated as sodium chloride) according to the formula.

[0055] 1.3 pH value measurement 1. Weigh 10.0g of fermented black soybean sample, add 90mL of carbon dioxide-free distilled water, and homogenize for 2min using a homogenizer.

[0056] 2. After standing for 30 minutes, take the supernatant and measure the pH value using a pH meter calibrated with a standard buffer solution.

[0057] 3. Perform three parallel measurements and take the average value as the final result.

[0058] 1.4 Determination of amino acid nitrogen content According to GB5009.235-2016 "National Food Safety Standard - Determination of Amino Acid Nitrogen in Food", the formaldehyde titration method was adopted: 1. Weigh 5.00g of fermented black soybean sample, dissolve it in distilled water and bring the volume to 100mL, shake well and filter.

[0059] 2. Pipette 20 mL of the filtrate into a beaker, add 60 mL of carbon dioxide-free distilled water, and titrate with 0.05 mol / L sodium hydroxide standard solution until the pH reaches 8.2. Do not record the volume.

[0060] 3. Add 10 mL of neutral formaldehyde solution, mix well, let stand for 1 min, and continue titrating until the pH is 9.2. Record the volume of sodium hydroxide standard solution consumed.

[0061] 4. At the same time, perform a blank test and calculate the amino acid nitrogen content according to the formula.

[0062] II. Microbiological Indicator Detection Methods 2.1 Total bacterial count determination According to GB4789.2-2022 "National Food Safety Standard - Microbiological Examination of Food - Determination of Total Colony Count": 1. Under aseptic conditions, weigh 25g of fermented black soybean sample, add 225mL of sterile physiological saline, homogenize for 1min, and prepare a 1:10 sample homogenate.

[0063] 2. Dilute in increments of 10, and select 2 to 3 suitable dilutions.

[0064] 3. Take 1 mL of the diluent and place it in a sterile Petri dish. Quickly pour the Petri dish into 46°C nutrient agar medium and gently shake to mix.

[0065] 4. After the agar has solidified, invert the plate and incubate at 36℃±1℃ for 48h±2h. Count the number of colonies on the plate and calculate the total number of colonies, in CFU / g.

[0066] 2.2 Coliform count According to GB4789.3-2022 "National Food Safety Standard - Microbiological Examination of Food - Coliform Count", plate count method: 1. The sample dilution method is the same as that for the total bacterial count determination.

[0067] 2. Pipette 1 mL of the diluted homogenate into a tube containing brilliant green lactose bile salt broth. Perform three parallel runs for each dilution.

[0068] 3. Incubate at 36℃±1℃ for 24h±2h and observe the gas production in the test tube.

[0069] 4. Based on the number of positive tubes, find the most probable number of coliform bacteria in the table, in CFU / g.

[0070] III. Methods for Detecting Volatile Flavor Compounds The headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) method was used. 1. Weigh 5.0g of uniform fermented soybean sample, place it in a 20mL headspace vial, add 1μL of 2-octanol internal standard solution, and seal.

[0071] 2. Place the headspace vial in a 60°C water bath for equilibration for 30 minutes, then insert the DVB / CAR / PDMS extraction head and extract for 40 minutes.

[0072] 3. After extraction, insert the extraction head into the gas chromatograph inlet and decompose at 250℃ for 3 minutes.

[0073] 4. The chromatographic column used was an HP-5MS capillary column. The temperature program was as follows: 40℃ for 3 min, then increased to 120℃ at 5℃ / min, and then increased to 250℃ at 10℃ / min, and held for 5 min.

[0074] 5. The carrier gas was high-purity helium, with a flow rate of 1.0 mL / min. An EI ion source was used, with an electron energy of 70 eV and a scan range of 30–450 m / z.

[0075] 6. Qualitative analysis was performed using the NIST14 spectral library, and the total number of volatile flavor compounds in the samples was counted.

[0076] IV. Storage Stability Testing (Accelerated Testing) 1. Seal the fermented black bean sample in a sterile polyethylene packaging bag and store it continuously in a constant temperature and humidity chamber at 37℃ and 75% relative humidity for 30 days.

[0077] 2. After the storage period, determine the total bacterial count using the method described above, and record any mold growth, dampness, or rancidity. The experimental data are as follows: Table 1 Performance test results of the examples and comparative examples

[0078] Based on the experimental data in Table 1, the moisture content of Examples 1-3 was controlled between 41.89% and 43.12%, indicating a moderate and stable moisture level. In contrast, the moisture content of Comparative Examples 1-6 was significantly higher, reaching a maximum of 52.71%. This demonstrates that the gradient coating film and modified diatomaceous earth used in this invention effectively regulate moisture migration, inhibit the reabsorption of moisture in the soybean curd, and keep the product moisture content within a reasonable range for fermentation and storage. The salt content of Examples 1-3 was 3.50%-4.50%, meeting the requirements for low-salt fermented soybeans. The salt content of Comparative Examples 1-6 was similar to that of the Examples, eliminating the interference of salt differences on the experimental results. The data from each group showed good comparability.

[0079] The pH values ​​of Examples 1-3 were 4.51-5.01, which is within the suitable range for normal fermentation of fermented black beans, indicating a stable system. The pH values ​​of Comparative Examples 1-6 were all higher, ranging from 5.23 to 5.82, indicating that the microbial metabolism was weaker and acid production was insufficient under the lack of core process conditions, resulting in a lower degree of fermentation. The amino acid nitrogen content of Examples 1-3 was 0.85%-0.92%, indicating sufficient protein hydrolysis and abundant accumulation of umami substances. The amino acid nitrogen content of Comparative Examples 1-6 was only 0.41%-0.67%, significantly lower than that of the Examples, demonstrating that sequential inoculation, gradient CO2 regulation, and slow osmotic pressure increase processes can significantly improve protein hydrolysis efficiency and enhance the nutritional and flavor profile of the product.

[0080] The total bacterial count in Examples 1-3 was less than 10 CFU / g, and the coliform count was less than 3 CFU / g, indicating extremely low microbial levels and high food safety. In contrast, the total bacterial count and coliform count in Comparative Examples 1-6 were significantly higher, indicating a significantly increased risk of contamination by other microorganisms. Examples 1-3 contained 37-42 types of volatile flavor compounds, demonstrating a rich and complete flavor composition. Comparative Examples 1-6 showed significantly fewer types of flavor compounds, with the fewest being only 18. This indicates that the overall process of this invention can effectively promote the generation and accumulation of flavor compounds, enhancing the aroma intensity and flavor harmony of fermented black beans.

[0081] After accelerated storage at 37°C and 75% relative humidity for 30 days, the total bacterial count in Examples 1-3 remained below 10 CFU / g, demonstrating excellent storage stability with no obvious signs of dampness or mold growth. In contrast, the total bacterial count in Comparative Examples 1-6 increased significantly to 1.8 × 10⁻⁶. 4~1.2×10 5 The product is prone to deterioration and loss of aroma (CFU / g), indicating that the protective coating, hydrophobic antibacterial coating and gradient cooling treatment of the present invention can significantly improve the product's shelf-life stability and ensure the product's quality remains stable during storage.

[0082] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. A flavor-enhancing fermentation preparation process for low-salt fermented black beans, characterized in that, Includes the following steps: 1) Raw material pretreatment: Soybeans are steamed and then cooled to 28-35℃ to obtain cooked soybean curd. 2) Gradient coating film formation: The cooked soybean blanks are placed in a vacuum environment and coated with a coating liquid. After the coating is completed, they are dried in stages with hot air to form a protective film on the surface of the cooked soybean blanks. 3) Sequential inoculation for koji making: After removing the protective film, the cooked soybean curd is inoculated with the inoculum in stages for koji making. During the koji making process, gradient CO2 regulation is used to complete directional metabolism and obtain mature koji. 4) Low-salt steady-state fermentation: The mature koji is added to a compound low-salt brine to form a fermentation system. The fermentation system is first subjected to osmotic pressure adaptation culture. After the fermentation system adapts to the initial osmotic pressure, the osmotic pressure is gradually increased. Then, sterile nitrogen microbubbles are introduced into the fermentation system to carry out two-stage anaerobic fermentation until the pH of the fermentation system is 4.5-5.0, and the fermentation product is obtained. 5) Flavor Locking and Shaping: After the fermentation product is encapsulated with a compound flavor-locking agent and dried at low temperature, a hydrophobic and antibacterial coating is sprayed onto the dried product. The coated product is then subjected to blending, heat stabilization, sterilization and gradient cooling treatment to obtain the low-salt fermented black bean product.

2. The flavor-enhancing fermentation preparation process for low-salt fermented black beans according to claim 1, characterized in that, In step 2): by mass parts, the coating solution is a 20wt% to 25wt% aqueous solution prepared from 80 to 90 parts of water-soluble maltodextrin, 10 to 20 parts of low-acylated gellan gum, and 5 to 10 parts of glyceryl monostearate. The coating solution is prepared as follows: first, purified water is heated to 45-55°C, water-soluble maltodextrin is added and stirred until completely dissolved, then low-acylated gellan gum is added and stirred for 15-20 minutes until fully dispersed without clumps, and finally glyceryl monostearate is added and stirred for 10-15 minutes until emulsified uniformly, and then cooled to 30-35°C and brought to a final volume. The vacuum degree for spraying under vacuum conditions is -0.06MPa, and the mixture is stirred for 15 to 20 minutes after spraying.

3. The flavor-enhancing fermentation preparation process for low-salt fermented soybeans according to claim 1, characterized in that, In step 2): the hot air segmented drying is a three-stage step drying: the first stage is drying at 55℃ for 20 minutes, the second stage is drying at 45℃ for 30 minutes, and the third stage is maturing at 35℃ for 15 minutes. After drying, the surface moisture of the soybean curd is ≤8%.

4. The flavor-enhancing fermentation preparation process for low-salt fermented black beans according to claim 1, characterized in that, In step 2): 15% to 20% by mass of modified diatomaceous earth with a particle size of 10 to 20 μm is added to the protective membrane to form an air-permeable and water-resistant microporous structure; The modified diatomaceous earth is prepared as follows: the diatomaceous earth powder is sieved to a particle size of 10-20 μm, calcined at 400-500℃ for 2-3 hours, modified by stirring at 60-70℃ for 1-2 hours with a 3-5% KH-550 silane coupling agent ethanol solution, filtered, and dried at 80-100℃ to constant weight.

5. The flavor-enhancing fermentation preparation process for low-salt fermented black beans according to claim 1, characterized in that, In step 2): the protective film is removed by rinsing with water and stirring, and the residual rate after removal is ≤0.5%.

6. The flavor-enhancing fermentation preparation process for low-salt fermented black beans according to claim 1, characterized in that, In step 3): the bacterial strains inoculated sequentially are Aspergillus oryzae, Bacillus subtilis, and Lactobacillus plantarum; The inoculum amount of Aspergillus oryzae is 0.8-1.2% of the weight of the cooked soybean curd, and it is cultured for 20-28 hours at a temperature of 28-35℃ and a relative humidity of 75-80%. The inoculum amount of Bacillus subtilis is 0.4-0.8% of the weight of cooked soybean curd, and it is cultured for 30-40 hours at a temperature of 32-40℃ and a relative humidity of 70-75%. The inoculum amount of *Lactobacillus plantarum* is 0.1-0.5% of the weight of the cooked soybean curd, and it is cultured for 18-22 hours at a temperature of 30-36℃ and a relative humidity of 65-70%.

7. The flavor-enhancing fermentation preparation process for low-salt fermented black beans according to claim 1, characterized in that, In step 3): the specific steps of gradient CO2 regulation are as follows: During the Aspergillus oryzae culture stage, CO2 with a volume concentration of 4-6% was introduced into the system at a flow rate of 0.3-0.7 m / s. 3 / h; During the Bacillus subtilis culture stage, CO2 with a volume concentration of 6-8% was introduced into the system at a flow rate of 0.4-0.8 m / s. 3 / h; During the Lactobacillus plantarum culture stage, CO2 with a volume concentration of 8–10% was introduced into the system at a flow rate of 0.2–0.6 m / s. 3 / h.

8. The flavor-enhancing fermentation preparation process for low-salt fermented black beans according to claim 1, characterized in that, In step 4): the compound low-salt brine is composed of NaCl, KCl, and potassium citrate in a mass ratio of 3:1:(0.4-0.6). Based on the mass of the mature fermented koji, the total salt content in the system is 3.5-4.5%; the initial osmotic pressure during fermentation is 110-130 mOsm / kg, decreasing to 5-10 mOsm / kg after 10-12 hours of adaptation. (12h) The concentration gradually increases to 140-160 mOsm / kg.

9. The flavor-enhancing fermentation preparation process for low-salt fermented black beans according to claim 1, characterized in that, In step 4): the diameter of the sterile nitrogen microbubbles is 10-20 μm; the two-stage anaerobic fermentation is as follows: the first stage is fermentation at 25-32℃ for 50-70 h, and the second stage is fermentation at 20-25℃ for 7-9 days.

10. The flavor-enhancing fermentation preparation process for low-salt fermented black beans according to claim 1, characterized in that, In step 5): the compound fragrance-locking agent is composed of β-cyclodextrin and gum arabic; based on the mass of the fermentation product, the amount of β-cyclodextrin added is 0.3-0.7 wt%, and the amount of gum arabic added is 0.1-0.2 wt%. The hydrophobic antibacterial coating is octenyl succinate starch ester, which is mixed with warm water at 50-60°C to form an emulsion with a mass fraction of 10%-15%, stirred for 10-15 minutes until completely gelatinized, cooled to 25-30°C, and then atomized and sprayed. The spraying amount is 0.5-1.0% based on the mass of the fermentation product. The heat stabilization treatment involves holding the temperature at 60–65℃ for 10–15 min, and the gradient cooling involves sequentially cooling at 25℃, 15℃, and 5℃, with a cooling rate of 1.5–2.5℃ / min.

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