Low-salt fermented soybean paste production process based on temperature control by section

Abstract

The application relates to the field of food biotechnology, and discloses a low-salt bean paste production process based on segmented temperature control fermentation, which is made of fava bean koji, water, edible salt, sodium hexametaphosphate, lactococcus lactis nisin, edible alcohol, granulated coarse granular calcium carbonate and glucono-delta-lactone. The production process comprises the following steps: constructing a composite matrix, and first performing buffer-type medium-temperature enzymolysis; then adding a glucono-delta-lactone aqueous solution in a flow manner, utilizing the stoichiometric coupling reaction of the glucono-delta-lactone aqueous solution and the granulated coarse granular calcium carbonate to generate carbon dioxide in situ to construct a micropore structure and regulate the pH value of the system; and finally, connecting a Zygosaccharomyces rouxii to perform low-temperature aroma-producing after-ripening. Through the synergistic effect of the components, the application effectively solves technical problems in the low-salt fermentation process, such as low protein solubility, easy compaction of the matrix and breeding of miscellaneous bacteria, and improves the amino acid nitrogen content and protein hydrolysis rate of the finished product.

Description

Technical Field

[0001] This invention relates to the field of food biotechnology, specifically to a low-salt fermented soybean paste production process based on segmented temperature-controlled fermentation. Background Technology

[0002] Doubanjiang (fermented broad bean paste) is a traditional condiment made primarily from broad beans through microbial fermentation. Its unique flavor originates from the enzymatic hydrolysis of proteins and complex microbial metabolism. Traditional doubanjiang production typically employs a high-salt, low-temperature fermentation process, with a salt content generally exceeding 18%. This high salt concentration not only acts as a preservative, inhibiting the growth of unwanted bacteria, but also promotes the dissolution of broad bean globulins through the salt-soluble effect, facilitating the action of proteases. However, with the increasing popularity of healthy eating concepts, the health risks associated with high salt intake are receiving growing attention, making the development of low-salt doubanjiang an inevitable trend in the industry.

[0003] Reducing the salt content in the fermentation system disrupts the microecological and biochemical balance established by traditional processes, bringing a series of technical challenges to actual production. In a low-salt environment, the system's osmotic pressure decreases, weakening its natural ability to inhibit spoilage microorganisms and making it highly susceptible to the growth of contaminating bacteria and product rancidity. Simultaneously, the low ionic strength environment significantly reduces the solubility of broad bean protein, causing the protein to remain in a folded and aggregated state, making it difficult for proteases to reach substrate cleavage sites, resulting in low raw material hydrolysis rates and insufficient amino acid nitrogen production.

[0004] Furthermore, to reduce the discharge of high-salt wastewater, modern low-salt processes often employ solid-state or semi-solid-state fermentation. However, this leads to increased viscosity of the fermentation substrate, causing the mash to easily become physically compacted during fermentation. This severely hinders oxygen transfer, heat dissipation, and material exchange within the system, resulting in uneven fermentation, inhibited enzyme activity, and impaired flavor compound synthesis. Existing technologies mostly focus on single-level improvements, such as simply adding preservatives to control unwanted microorganisms or adding exogenous enzyme preparations to supplement enzyme activity. These methods fail to systematically address the synergistic challenges of dense substrate structure, difficulty in protein dissolution, and unstable enzymatic hydrolysis environment in low-salt fermentation.

[0005] Therefore, this invention proposes a low-salt fermented soybean paste production process based on segmented temperature-controlled fermentation 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 segmented temperature-controlled fermentation. This process solves the problems of low protein solubility and incomplete enzymatic hydrolysis due to low ionic strength, mass transfer obstruction due to the easy caking of high-viscosity matrix, and product spoilage due to the easy growth of miscellaneous bacteria in the low osmotic pressure environment during the solid-state fermentation of low-salt fermented soybean paste.

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

[0008] In a first aspect, the present invention provides a composite matrix composition for fermentation of low-salt fermented soybean paste, employing the following technical solution:

[0009] A composite matrix composition for low-salt fermented broad bean paste is made from raw materials comprising the following weight ratios: 550-650 parts broad bean koji; 250-400 parts water; 65-75 parts edible salt; 0.8-1.2 parts sodium hexametaphosphate; 0.1-0.25 parts nisin; 12-20 parts edible alcohol; 0.5-0.8 parts granulated coarse calcium carbonate; and 8-12 parts glucono-δ-lactone.

[0010] By adopting the above technical solution, this invention solves the problems of physical structure, biochemical environment and microbial control in low-salt fermentation by utilizing the stoichiometric coupling and synergistic effect between the components. The specific mechanism is as follows:

[0011] Firstly, addressing the problem of enzyme activity loss due to paste compaction and pH fluctuations during low-salt fermentation, this invention constructs a stoichiometric coupling system of granulated coarse-particle calcium carbonate and glucono-δ-lactone. Glucono-δ-lactone hydrolyzes in the aqueous phase to generate gluconic acid and release hydrogen ions. These hydrogen ions act on the surface of the granulated coarse-particle calcium carbonate, initiating controlled disintegration of the solid-phase calcium carbonate and generating calcium ions, water, and carbon dioxide gas. The in-situ generated carbon dioxide gas forms bubble cavities in the protein gel network, constructing a physical microporous structure, blocking the dense packing of protein micelles, reducing matrix hardness, and improving mass transfer efficiency. Simultaneously, the released calcium ions bind to the calcium-binding sites of neutral proteases, maintaining the conformational stability of the enzyme molecules; the gradual consumption of calcium carbonate forms a dynamic pH buffer system, preventing enzyme inactivation caused by excessively high local acidity.

[0012] Secondly, to address the problem of low solubility of broad bean globulin in low-salt environments, the polyelectrolyte effect and ion chelation properties of sodium hexametaphosphate are utilized to increase the ionic strength of the microenvironment in the low-salt system, inducing the protein to undergo salt dissolution, allowing the protein structure to expand and expose internal enzyme cleavage sites, thereby improving the hydrolysis efficiency of the protease.

[0013] Finally, to address the challenge of preservation in low-salt environments, the synergistic antibacterial mechanism of nisin and edible alcohol was utilized. Edible alcohol alters the permeability of bacterial cell membranes, reducing the tolerance of putrefactive bacteria to nisin; nisin binds to lipid II precursors on the cell membranes of Gram-positive bacteria, leading to the leakage of intracellular substances. The combination of these two substances inhibits common putrefactive bacteria such as thermostable Bacillus, without affecting the growth of alcohol-resistant Zygomyces rouxii.

[0014] Preferably, the weight ratio of the raw materials is as follows: 580 parts broad bean koji; 320 parts water; 70 parts edible salt; 1.0 part sodium hexametaphosphate; 0.15 to 0.2 parts nisin; 15 parts edible alcohol; 0.6 parts granulated coarse calcium carbonate; and 10 parts glucono-δ-lactone.

[0015] Preferably, the particle size of the granulated coarse calcium carbonate is 40 mesh to 60 mesh; the granulated coarse calcium carbonate is prepared by a method including the following steps: weighing light calcium carbonate powder, adding deionized water to mix and form a soft material; granulating the soft material through a 20-mesh sieve, drying it until the moisture content is less than 1 wt%; and sieving to retain particles between 40 mesh and 60 mesh.

[0016] Preferably, the nisin is added in the form of a pre-dissolved solution; the pre-dissolved solution is prepared by adding the nisin to sterile water, adding citric acid or dilute hydrochloric acid dropwise to adjust the pH of the system to 3.0-3.5, and stirring until the active ingredient is dissolved or a uniform suspension is formed.

[0017] Secondly, the present invention provides a low-salt fermented soybean paste production process based on segmented temperature-controlled fermentation, employing the following technical solution:

[0018] A low-salt fermented soybean paste production process based on segmented temperature-controlled fermentation utilizes the composite substrate composition for low-salt fermented soybean paste fermentation described in the first aspect for fermentation, comprising the following steps:

[0019] S1. Construction of composite fermentation substrate: Mix broad bean koji, water, edible salt, sodium hexametaphosphate, nisin, edible alcohol and granulated coarse calcium carbonate evenly, and adjust the initial pH of the system to 6.2-6.4.

[0020] S2, First stage buffered mesophilic enzymatic hydrolysis: Control the temperature of the fermented mash at 35℃~37℃ and ferment at a constant temperature for 36h~42h; utilize the solubilizing effect of the sodium hexametaphosphate and the solid-phase buffering characteristics of the granulated coarse calcium carbonate to maintain the enzymatic hydrolysis environment;

[0021] S3. Second stage of gluconate-δ-lactone addition and micropore construction: When fermentation is in progress for 36-42 hours, the aqueous solution of gluconate-δ-lactone is added to the system. The addition time is controlled to be completed within 20-30 hours. The internal micropores are constructed using carbon dioxide gas generated in situ, and the pH value of the system is reduced to 4.8-5.0.

[0022] S4. Third stage of low-temperature aroma production and post-ripening: The product temperature is lowered to 28℃~30℃, and Lutein's yeast is introduced. The fermentation is carried out at a constant temperature for 20d~30d.

[0023] S5. Post-processing: After fermentation, the product is obtained by sterilization and filling.

[0024] By adopting the above technical solution, this invention establishes a staged control strategy, resolving the contradiction between enzymatic hydrolysis efficiency and acidification rate during low-salt fermentation. The specific mechanism of action is as follows:

[0025] First, in stages S1 and S2, gluconate-δ-lactone is added with a delay, and sodium hexametaphosphate and poorly soluble granulated coarse-grained calcium carbonate are introduced at the beginning of fermentation to create a high pH (6.2–6.4) and calcium-stable enzymatic hydrolysis environment. During this stage: sodium hexametaphosphate regulates ionic strength and promotes the dissolution of broad bean protein; the granulated coarse-grained calcium carbonate remains in an unreacted solid phase, neither consuming acidity nor producing gas, serving as a potential buffer reserve; neutral proteases hydrolyze the dissolved substrate protein within a suitable pH range, accumulating amino acid nitrogen.

[0026] Secondly, in the S3 stage, the addition of glucono-δ-lactone initiates acidification and micropore construction. Glucono-δ-lactone hydrolyzes to release hydrogen ions, which neutralize with the coarse granulated calcium carbonate particles. The carbon dioxide gas produced in the reaction forms micropores in the fermentation mash, reducing mass transfer resistance in the later stages of fermentation. The released calcium ions compensate for the decreased enzyme stability caused by the drop in pH, prolonging the action time of the protease.

[0027] Finally, the S4 stage utilizes the microporous structure constructed in the S3 stage and a suitable weakly acidic environment (pH 4.8–5.0) to provide growth and metabolic conditions for *Zygosacchariformis rouxae*, promoting the synthesis of flavor compounds such as alcohols and esters.

[0028] Preferably, in step S1, the method for preparing the broad bean koji includes: peeling, soaking, draining, and steaming dried broad beans, then cooling to 38°C; mixing with flour, and inoculating with Aspergillus oryzae spore powder 3.042; and culturing at 32°C to 35°C for 40 to 44 hours to obtain the broad bean koji; wherein the protein content of the dried broad beans is 24.0 wt% to 25.0 wt%, and the protein content of the flour is 11.0 wt%.

[0029] Preferably, in step S2, the specific conditions for the constant temperature fermentation are: the temperature is controlled at 36℃~37℃, and the fermentation is allowed to stand for 40 hours; during this period, the mixture is stirred at low speed for 5 minutes every 12 hours, and the stirring speed is 10 rpm.

[0030] Preferably, in step S3, the concentration of the aqueous solution of gluconate-δ-lactone is 30 wt%; the feeding control strategy is to continuously feed at a constant rate so that the hydrolysis acid production rate of gluconate-δ-lactone matches the disintegration and neutralization rate of the granulated coarse calcium carbonate, so that the pH value of the system stabilizes between 4.8 and 5.0 at the end of the feeding.

[0031] Preferably, in step S4, the amount of *L. roux* added is 2v / v% to 4v / v of the total system volume; and the isothermal fermentation time is 25 days.

[0032] Preferably, in step S5, the sterilization conditions are heating to 85°C and holding for 20 minutes; the resulting low-salt broad bean paste has an amino acid nitrogen content greater than or equal to 0.95 g / 100 g and a protein hydrolysis rate greater than or equal to 57.0 wt%.

[0033] This invention provides a low-salt fermented soybean paste production process based on segmented temperature-controlled fermentation. It has the following beneficial effects:

[0034] 1. This invention utilizes the coupling effect of granulated coarse calcium carbonate and gluconate-δ-lactone, combined with a fed-batch strategy, to generate gas in situ during fermentation, constructing a microporous structure inside the fermented mash, reducing mass transfer resistance, and improving the problem of matrix compaction; at the same time, it utilizes the calcium ions released by the reaction and the solid-phase buffering properties of calcium carbonate to maintain the conformation and activity of proteases and increase the amino acid nitrogen content.

[0035] 2. This invention utilizes the polyelectrolyte effect of sodium hexametaphosphate to induce the dissolution and expansion of broad bean globulin through chelation and ionic strength regulation, thereby exposing internal enzyme cleavage sites, improving the problem of low protein solubility under low-salt conditions, and increasing the protein hydrolysis rate and the flavor of the finished product.

[0036] 3. This invention employs a synergistic preservative system of nisin and edible alcohol. Edible alcohol increases the permeability of bacterial cell membranes, promotes the function of nisin, inhibits the growth of other bacteria, and does not affect the normal metabolism of Zygosacchariformis, thus ensuring the smooth progress of the low-salt fermentation process. Attached Figure Description

[0037] Figure 1 The figures show the kinetic monitoring curves of different fermentation systems of the present invention during the acidification stage, wherein (a) shows the trend of pH value change over time; and (b) shows the trend of neutral protease activity change over time.

[0038] Figure 2 The figure shows the kinetic curves of substrate state changes during the initial fermentation stage of the present invention, wherein (a) the figure shows the change of nitrogen solubility index over time; and (b) the figure shows the amino acid nitrogen generation rate curve.

[0039] Figure 3 The graph shows the monitoring of microbial growth and chemical spoilage indicators during the mesophilic fermentation stage of this invention. (a) The graph shows the growth curve of Bacillus spp. over time; (b) The graph shows the trend of volatile basic nitrogen (TVB-N) content. The dashed line marks the thresholds of initial spoilage and severe deterioration.

[0040] Figure 4 This is a comprehensive comparison chart of the physicochemical properties of the main experimental components of this invention;

[0041] Figure 5 Bubble distribution diagrams showing the physical texture properties of each group of fermented soybean paste products according to the present invention;

[0042] Figure 6 This is a principal component analysis biplot of the present invention based on electronic tongue taste index and color difference parameter. Detailed Implementation

[0043] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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.

[0044] 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.

[0045] The broad beans, flour, edible salt, and production water are all commercially available food-grade products; the protein content of the broad beans is 24.5% (w / w), and the protein content of the flour is 11.0% (w / w).

[0046] Sodium hexametaphosphate is a commercially available food-grade product, CAS No.: 10124-56-8; light calcium carbonate is a commercially available food-grade product, CAS No.: 471-34-1; glucono-δ-lactone (GDL) is a commercially available food-grade product, CAS No.: 90-80-2; nisin is a commercially available food-grade product, CAS No.: 1414-45-5; edible alcohol (main component ethanol) is a commercially available food-grade product, CAS No.: 64-17-5.

[0047] Aspergillus oryzae and Zygosacchariformis are both commercially available brewing agents.

[0048] Calcium chloride (CAS No.: 10043-52-4) and L-lactic acid (CAS No.: 79-33-4) are both commercially available food-grade products.

[0049] Preparation Example 1: This preparation example provides a method for preparing granulated coarse-grained calcium carbonate used as a pH buffer and micropore building agent in the embodiments, including the following steps:

[0050] Weigh 1000g of commercially available food-grade light calcium carbonate powder, add an appropriate amount of deionized water (about 300-400mL) as a binder, and mix in a mixer until a uniform moist soft material is formed; granulate the soft material through a 20-mesh (850μm aperture) sieve, and dry the obtained wet granules in a 60℃ oven for 4-6 hours until the moisture content is less than 1%; place the dried granules on a standard test sieve vibrating sieve for granulation and sieving; collect the granules retained between a 40-mesh and 60-mesh (particle size about 250μm-425μm) sieve to obtain the target coarse calcium carbonate particles, and seal for later use.

[0051] Preparation Example 2: This preparation example provides a method for preparing micronized calcium carbonate for verifying the effect of particle size in a comparative example, comprising the following steps:

[0052] Take 500g of commercially available food-grade light calcium carbonate powder and sieve it on a 200-mesh standard sieve (approximately 75μm aperture). Collect the material passing through the 200-mesh sieve to obtain micro-powdered calcium carbonate with a particle size of less than 75μm. Seal and store for later use.

[0053] Preparation Example 3: This preparation example provides a pretreatment method for the composite solubilizing and preservative component used in the construction of the fermentation substrate in the examples, including the following steps:

[0054] Preparation of component A (solution): Weigh 300g of sodium hexametaphosphate (SHMP), add it to 2500mL of sterile water, heat to 40-50℃ and stir to aid dissolution. After complete dissolution, cool to room temperature and make up to 3000mL to obtain a 10% SHMP aqueous solution.

[0055] Preparation of component B (preservative solution): Weigh 60g of nisin and add it to 600mL of sterile water; add an appropriate amount of food-grade citric acid or dilute hydrochloric acid to adjust the pH to 3.0-3.5 to aid dissolution, and continue stirring on a magnetic stirrer until the active ingredient is completely dissolved or a uniform and stable suspension is formed to obtain the Nisin liquid phase component (because the solubility of high-concentration Nisin is limited, it needs to be prepared and used immediately while maintaining stirring).

[0056] Example 1: This example provides a low-salt fermented soybean paste production process based on segmented temperature-controlled fermentation, including the following steps:

[0057] Raw material pretreatment and koji making: Peel 100kg of dried broad beans, soak them until the water absorption rate is about 95%, drain them, and then steam them at 0.12MPa pressure for 12 minutes. Cool them to 38℃. Add 20kg of flour and mix well, then inoculate with 0.4kg of Aspergillus oryzae (Hu Niang 3.042) spore powder. Place the mixture in a koji tank and incubate at a controlled temperature of 32-35℃ for 42 hours, turning the koji twice during the incubation period, until the koji material clumps together and is covered with yellow-green mycelium, thus obtaining the koji.

[0058] Construction of the compound fermentation substrate: The fermented koji is transferred into a fermentation tank equipped with temperature control and stirring devices. Pre-prepared sterile brine is added, adjusting the total mass of the fermented mash to approximately 250 kg (controlling the total moisture content to approximately 48%). Then, the following are added sequentially:

[0059] (1) Edible salt, to make the final concentration of the system 7.0% (w / w);

[0060] (2) The amount of sodium hexametaphosphate (SHMP) added to the solution A prepared according to the method of Preparation Example 3 was equivalent to a final concentration of 0.10% (w / w) of sodium hexametaphosphate (SHMP).

[0061] (3) The amount of preservative solution B prepared according to the method of Preparation Example 3 was equivalent to a final concentration of nisin of 0.02% (w / w).

[0062] (4) Edible alcohol, the amount added is 1.5 mL / 100 g (v / w) of the total mass of the system.

[0063] (5) The amount of 40-60 mesh granulated coarse calcium carbonate prepared according to the method of Preparation Example 1 is 0.06% (w / w) of the total mass of the system.

[0064] After the above materials are added, they are mixed at a low speed until homogeneous. At this point, the initial pH of the system is approximately 6.4.

[0065] First stage (buffered mesophilic hydrolysis): Turn on the constant temperature circulating water and control the temperature of the mash at 36-37℃, and let it ferment for 40 hours. During this period, stir at low speed for 5 minutes (10 rpm) every 12 hours to keep the temperature uniform. This stage utilizes the solubilizing effect of SHMP and the phase isolation characteristics of CaCO3 to efficiently hydrolyze proteins in an environment of pH 6.2-6.4.

[0066] The second stage (GDL addition and microchannel construction): When fermentation reaches the 40th hour, a 30% (w / w) concentration of GDL aqueous solution is added. The addition rate is controlled and completed at a uniform rate within 24 hours. The total amount of GDL added (on a dry basis) is 1.0% (w / w) of the total mass of the fermented mash. As the acid produced by GDL hydrolysis undergoes a microscopic neutralization reaction with the coarse CaCO3 particles, CO2 bubbles are generated in situ to construct the internal microchannels. The addition is stopped at the 64th hour, at which point the pH of the system is stable at 4.9±0.1.

[0067] The third stage (low-temperature aroma production and post-ripening): the product temperature is lowered to 30℃, and 3% (v / w) of *Gnaphalium affine* culture medium is introduced; fermentation is maintained at a constant temperature of 30℃ for 25 days.

[0068] Post-processing: After fermentation, heat the fermented mash to 85℃ for 20 minutes to sterilize it. After cooling, it is bottled to obtain the finished low-salt broad bean paste.

[0069] Example 2: This example provides a low-salt fermented soybean paste production process based on segmented temperature-controlled fermentation, including the following steps:

[0070] Raw material pretreatment and koji making: The operation steps are the same as in Example 1.

[0071] Construction of the composite fermentation substrate: Except for adjustments to the following component addition amounts, the remaining operations are the same as in Example 1:

[0072] (1) The final concentration of sodium hexametaphosphate (SHMP) was adjusted to 0.08% (w / w);

[0073] (2) The amount of coarse granulated calcium carbonate prepared according to the method of Preparation Example 1 was adjusted to 0.05% (w / w);

[0074] (3) The final concentration of nisin was adjusted to 0.015% (w / w);

[0075] (4) The amount of edible alcohol added is adjusted to 1.2 mL / 100 g (v / w) of the total mass of the system.

[0076] (5) The salt concentration should be maintained at 7.0%.

[0077] First stage (buffered mesophilic enzymatic hydrolysis): The temperature is controlled at 35℃ and the fermentation time is 36 hours.

[0078] The second stage (GDL addition and micropore construction): GDL solution was added starting at hour 36, with the total GDL addition (on a dry basis) being 0.8% (w / w) of the total mass of the fermented mash. The addition was completed within 20 hours. The pH was measured to be approximately 5.0 after the addition was completed.

[0079] The third stage (low-temperature aroma production and post-fermentation): the temperature is lowered to 28℃, 2% (v / w) of Ludwig's conjugate yeast is introduced, and fermentation is carried out for 20 days.

[0080] Post-processing: The operation is the same as in Example 1.

[0081] Example 3: This example provides a low-salt fermented soybean paste production process based on segmented temperature-controlled fermentation, including the following steps:

[0082] Raw material pretreatment and koji making: The operation steps are the same as in Example 1.

[0083] Construction of the composite fermentation substrate: Except for adjustments to the following component addition amounts, the remaining operations are the same as in Example 1:

[0084] (1) The final concentration of sodium hexametaphosphate (SHMP) was adjusted to 0.12% (w / w);

[0085] (2) The amount of coarse calcium carbonate prepared according to the method of Preparation Example 1 was adjusted to 0.08% (w / w);

[0086] (3) The final concentration of nisin was adjusted to 0.025% (w / w);

[0087] (4) The amount of edible alcohol added is adjusted to 1.5 mL / 100 g (v / w) of the total mass of the system.

[0088] First stage (buffered mesophilic enzymatic hydrolysis): The temperature is controlled at 37℃ and the fermentation time is 42 hours.

[0089] The second stage (GDL addition and micropore construction): GDL solution was added starting at hour 42. To overcome the high protein buffer capacity and calcium carbonate neutralization, the total GDL addition (on a dry basis) was adjusted to 1.2% (w / w) of the total mass of the fermented mash. The addition time was controlled to be completed within 30 hours. The pH was measured to be approximately 4.8 after the addition was completed.

[0090] The third stage (low-temperature aroma production and post-fermentation): the temperature is lowered to 30℃, 4% (v / w) of Ludwig's conjugate yeast is introduced, and fermentation is carried out for 30 days.

[0091] Post-processing: The operation is the same as in Example 1.

[0092] Example 4: This example provides a low-salt fermented soybean paste production process based on segmented temperature-controlled fermentation, including the following steps:

[0093] This embodiment aims to verify the feasibility of reducing the amount of Nisin at high ethanol concentrations.

[0094] Raw material pretreatment and koji making: The operation steps are the same as in Example 1.

[0095] Construction of composite fermentation substrate: The component addition amounts are adjusted as follows:

[0096] (1) The amount of edible alcohol added is increased to 2.0 mL / 100 g (v / w) of the total mass of the system.

[0097] (2) The amount of nisin added was reduced to 0.01% (w / w);

[0098] (3) Sodium hexametaphosphate (SHMP) 0.10%, coarse granulated calcium carbonate 0.06%, edible salt 7.0%, GDL 1.0%, all consistent with Example 1.

[0099] Subsequent steps: The enzymatic hydrolysis temperature, GDL feeding strategy, and post-ripening process were all performed exactly as in Example 1. This parameter combination was intended to verify the compensatory effect of higher concentrations of ethanol on the inhibition of contaminating bacteria in the early stage of enzymatic hydrolysis, and its synergistic effect with low doses of Nisin.

[0100] Comparative Example 1: Compared with Example 1, the difference is as follows:

[0101] (1) When constructing the composite fermentation substrate, the amount of edible salt added is adjusted to 16.0% (w / w) of the total mass of the system. This can inhibit miscellaneous bacteria through high osmotic pressure and ensure the safety of long-term fermentation.

[0102] (2) No sodium hexametaphosphate (SHMP), nisin, edible alcohol, coarse calcium carbonate and GDL were added;

[0103] (3) No artificial temperature control is carried out during the fermentation process. Natural temperature fermentation is used, and GDL is not added. The fermentation cycle is extended to 90 days to reach the maturity standard.

[0104] The pretreatment of other raw materials and the steps for making koji are the same.

[0105] Comparative Example 2: The difference from Example 1 is that sodium hexametaphosphate (SHMP) was not added; otherwise, they are the same.

[0106] Comparative Example 3: Compared with Example 1, the difference is that nisin and edible alcohol were not added, but everything else is the same.

[0107] Comparative Example 4: Compared with Example 1, the difference is that no granulated coarse calcium carbonate was added, and no GDL solution was added. In order to simulate an acidic environment, L-lactic acid solution was continuously added dropwise by a peristaltic pump during the 40th to 64th hour of fermentation to control the final pH of the system to be consistent with that of Example 1 (about 4.9). However, due to the lack of a chemical buffer system of calcium carbonate, the smooth pH decrease curve of Example 1 could not be reproduced (the pH showed a faster decreasing trend). Everything else was the same.

[0108] Comparative Example 5: Compared with Example 1, the difference is that the 40-60 mesh granulated coarse calcium carbonate prepared according to the method of Preparation Example 1 was replaced with an equal mass of 200 mesh or finer powder calcium carbonate prepared according to the method of Preparation Example 2, and all other aspects are the same.

[0109] Comparative Example 6: Compared with Example 1, the difference is that the granulated coarse calcium carbonate prepared according to the method of Preparation Example 1 was replaced with soluble calcium chloride (CaCl2) with an equimolar calcium content, and GDL was not added (in order to avoid uncontrollable acidity, L-lactic acid was added dropwise to adjust the pH in this group at the 40th hour), and everything else was the same.

[0110] Test Example 1: Monitoring pH buffering kinetics and thermostability of neutral protease during fermentation

[0111] The experimental steps are as follows:

[0112] Fermented mash from Examples 1, 4, 5, and 6 were selected as test subjects. The sampling time point was set at the 40th hour of the fermentation process (the starting point of GDL feeding / acid adjustment), and then samples were taken every 4 hours until the 64th hour (the end point of acidification).

[0113] The specific steps are as follows:

[0114] At a preset time point, a multi-point sampler was used to extract approximately 100g of the fermented mash sample from the middle layer of the fermentation tank and quickly placed it in an ice bath to pause the biochemical reaction.

[0115] Take 50g of sample and add 5 times the volume of distilled water. Stir magnetically at 35℃ for 20 minutes, then centrifuge at 4000r / min for 10 minutes and collect the supernatant.

[0116] The pH value of the supernatant was directly measured using a calibrated precision pH meter (accuracy 0.01). Each sample was measured in triplicate and the average value was taken.

[0117] Neutral protease activity was determined using the Folin-phenol method. Casein was used as a substrate, and the reaction was carried out at pH 7.0 and 40℃ for 10 minutes, and the absorbance value was measured. One unit of enzyme activity (U / g) was defined as 1 μg of tyrosine produced per minute by hydrolyzing casein. To eliminate the interference of pH difference on enzyme activity measurement, the pH of all sample buffers was uniformly adjusted to 7.0 before the measurement. The focus was on investigating the degree of irreversible denaturation of enzyme protein by the acid and heat environment during fermentation.

[0118] The experimental data are shown in Table 1:

[0119] Table 1: Monitoring data on pH changes and neutral protease activity retention during the fermentation acidification stage

[0120]

[0121] Note: Comparative Examples 4 and 6 were adjusted by artificial addition of lactic acid. The rate of pH decrease was mainly controlled by the rate of addition, but the local pH fluctuations were large. In Comparative Example 5, the pH was too high in the early stage (6.58) due to the rapid reaction of the micro powder, and the buffering capacity was exhausted in the later stage.

[0122] Conclusion: Based on Table 1 and the appendix Figure 1 According to the data, Example 1 exhibited significant chemical buffering properties and enzyme activity protection effects during the acidification stage.

[0123] In terms of pH kinetics, the pH value of Example 1 decreased from 6.41 at 40 hours to 4.93 at 64 hours, exhibiting a gentle decreasing gradient. This is attributed to the stoichiometric equilibrium reaction between granulated coarse-grained calcium carbonate and GDL hydrolysate. Within the critical range of 44 to 52 hours, the pH of Example 1 remained between 5.72 and 6.28, indicating that the coarse-grained calcium carbonate, acting as a solid-phase alkali reservoir, effectively neutralized the hydrogen ions generated by the rapid hydrolysis of GDL through the gradual disintegration and release of carbonate ions, thus mitigating the acid shock. In contrast, Comparative Example 4 used direct acid dripping without a buffer medium, and the pH dropped to 5.64 after 44 hours and was close to the final pH after 48 hours. This rapid acidification led to a sudden change in the environment. Although Comparative Example 5 added calcium carbonate, the micronized particles were too small (<75μm) and had too large a specific surface area, which caused it to be consumed by trace amounts of acid or SHMP in the early stage of fermentation (manifested as an abnormal increase in the initial pH to 6.58 after 40 hours). It could not provide a continuous buffering capacity in the subsequent acidification stage, and the pH curve was similar to that of the calcium-free group.

[0124] Regarding the thermostability of enzyme activity, Example 1 showed an absolute advantage in enzyme activity retention under a moderate temperature (36-37°C) acidification environment; by the end of 64 hours, the neutral protease activity of Example 1 was still 842.5 U / g (approximately 65.5% of the initial value), while the residual enzyme activities of Comparative Examples 4, 5, and 6 were only 265.3 U / g, 386.6 U / g, and 334.2 U / g, respectively.

[0125] The data discrepancies confirm the effectiveness of phase isolation and the in-situ calcium release mechanism:

[0126] Calcium ion thermal protection mechanism: In Example 1, coarse-grained calcium carbonate slowly releases Ca during the acidolysis process. 2+ These new ecosystems Ca 2+ It rapidly binds to the calcium-binding site of neutral protease, maintaining the three-dimensional conformational stability of the enzyme molecule and resisting denaturation caused by acid and heat. Although Comparative Example 6 contains equimolar amounts of calcium ions, because it is added in the form of dissolved calcium chloride, the calcium ions are chelated by sodium hexametaphosphate (SHMP) or non-specifically bound to soybean protein in the early stage of fermentation, resulting in a lack of free calcium during the acidification stage. 2+ Protect enzyme activity.

[0127] Micro-acidity control: The enzyme activities of Comparative Examples 4 and 6 showed a sharp drop in the 44-48 hour range, indicating that the rapid pH crossing of the enzyme's isoelectric point or stable range was the main cause of enzyme inactivation. Example 1 extended the effective enzymatic hydrolysis window by delaying the time it took for the pH to cross the enzyme inactivation threshold.

[0128] In summary, the coarse-particle calcium carbonate-GDL coupling system resolves the contradiction between acidification and enzymatic hydrolysis in low-salt fermentation by constructing a slow-release buffer environment and providing continuous calcium ion protection, ensuring that the protease still has high catalytic activity in the later stages of fermentation.

[0129] Test Example 2: Protein Solubility and Substrate Transformation Kinetics Test in the Early Stage of Fermentation

[0130] The experimental steps are as follows:

[0131] Fermented mash from Example 1, Comparative Example 2, and Comparative Example 6 were selected as test subjects. The changes in substrate state during the first 40 hours of fermentation (medium-temperature enzymatic hydrolysis period) were investigated. The sampling time point was set at the 4th hour after the start of fermentation, and then samples were taken every 8 hours until the 40th hour (before GDL addition).

[0132] Approximately 200g of mixed mash samples were collected from different depths of each fermentation tank and homogenized using a tissue homogenizer for 1 minute. 10.00g of the homogenized sample was weighed and placed in a 250mL Erlenmeyer flask with a stopper. 100mL of distilled water (40℃) was added, and the sample was stirred at a constant temperature on a magnetic stirrer for 30 minutes to extract water-soluble components.

[0133] Centrifuge the extract at 5000 r / min for 15 minutes, filter to remove floating oil and precipitate, collect the supernatant and make up to 200 mL as the test solution; retain the precipitate and the original fermented soybean paste sample for total nitrogen determination.

[0134] The Kjeldahl method (GB-5009.5) was used to determine the total nitrogen content (TN) in the original sample and the water-soluble nitrogen content (WSN) in the test solution. The nitrogen solubility index (NSI) was calculated using the formula: NSI = (WSN / TN) × 100%. This index is used to quantify the physical solubility and dispersion state of favone in the system.

[0135] A portion of the test solution was taken, and the amino acid nitrogen (AAN) content was determined by the formaldehyde value method (GB / T-5009.39). This index is used to characterize the hydrolysis efficiency of proteases on dissolved substrates. All determinations were performed in triplicate, and the results were taken as the arithmetic mean.

[0136] The experimental data are shown in Table 2:

[0137] Table 2: Monitoring data of nitrogen solubility index (NSI) and amino acid nitrogen production rate during the initial stage of fermentation

[0138]

[0139] Note: The increase in NSI value includes the dual contribution of physical dissolution and enzymatic hydrolysis to generate small molecule peptides; amino acid nitrogen data has been converted to wet basis of soy sauce mash.

[0140] Conclusion: Based on Table 2 and the appendix Figure 2 The data showed that Example 1 exhibited better substrate dissolution behavior and enzymatic hydrolysis efficiency in the early stage of fermentation than the comparative example, verifying the ionic strength regulation and solubilization mechanism of sodium hexametaphosphate (SHMP) under low-salt conditions.

[0141] Regarding the nitrogen solubility index (NSI), Example 1 showed a rapid upward trend; at the 4th hour of fermentation, the NSI of Example 1 reached 18.42%, higher than the 12.15% of Comparative Example 2 and 13.04% of Comparative Example 6; by the 40th hour, the NSI of Example 1 reached 54.12%, while that of Comparative Example 2 was only 34.65%. The data indicate that in a low-salt environment of 7%, the solubility of isolated fava bean globulin (Comparative Example 2) is limited, exhibiting a tight coiled conformation; the SHMP added in Example 1 increased the ionic strength of the microenvironment through the polyelectrolyte effect, inducing the "salt-dissolving" phenomenon of the protein, causing the quaternary structure of the protein to unfold, thereby increasing the release ratio of water-soluble nitrogen.

[0142] It is noteworthy that although Comparative Example 6 added the same amount of SHMP, its NSI value (35.81%) was not significantly different from that of the blank control Comparative Example 2 (34.65%); this confirms the interference effect of calcium source solubility on the solubilization mechanism: the calcium chloride added to Comparative Example 6 is a readily soluble calcium salt, and the dissociated Ca... 2+ Rapid complexation or precipitation reactions with SHMP phosphate groups consume the effective SHMP in the system, preventing it from exerting its solubilizing effect on proteins. In contrast, Example 1 uses coarse-grained calcium carbonate, which utilizes its poor solubility to achieve phase isolation, ensuring that SHMP can focus on substrate solubilization in the early stages.

[0143] Regarding the amino acid nitrogen generation rate, Example 1 showed a high positive correlation with the NSI data. At 40 hours, the amino acid nitrogen content in Example 1 reached 0.84 g / 100 g, which was 1.71 times and 1.64 times that of Comparative Example 2 (0.49 g / 100 g) and Comparative Example 6 (0.51 g / 100 g), respectively. This result confirms that SHMP-induced substrate conformational unfolding exposes more hydrophobic cleavage sites, eliminating steric hindrance to protease action, allowing Aspergillus oryzae protease to more efficiently cleave and hydrolyze peptide bonds. In Comparative Examples 2 and 6, because the substrate mainly existed in an aggregated state, the enzymatic reaction was limited by mass transfer resistance at the phase interface, resulting in a slow amino acid generation rate.

[0144] In summary, by combining the ion regulation of SHMP with a poorly soluble calcium source, this invention successfully constructed a highly efficient conversion pathway of first solubilization followed by enzymatic hydrolysis, overcoming the problem of incomplete enzymatic hydrolysis caused by low substrate solubility in low-salt solid-state fermentation.

[0145] Test Example 3: Microbial Safety Challenges and Putrefaction Kinetics Tests in the Mesophilic Fermentation Stage

[0146] The experimental steps are as follows:

[0147] Fermented sauce mash from Examples 1, 4, and 3 were selected as test subjects. Given that 35-37℃ is the optimal temperature for mesophilic enzymatic hydrolysis and also the outbreak temperature range for common spoilage bacteria such as Bacillus, this test focused on monitoring the microbial growth and decline and chemical spoilage indicators within the first 48 hours of fermentation.

[0148] Sampling and pretreatment: Samples were collected from different sites in the fermenter using a sterile sampler at the fermentation start times of 0h, 12h, 24h, 36h, and 48h. 25g of sample was placed in a homogenizing bag containing 225mL of sterile physiological saline and homogenized for 2 minutes using a tapping homogenizer to prepare a 1:10 sample homogenate.

[0149] Bacillus spp. count: 1 mL of homogenate was serially diluted; the appropriate dilutions were plated onto MYP (mannitol-egg yolk-polymyxin) agar plates; the plates were incubated at 36±1℃ for 24±2 hours; typical pink colonies (turbid background with a halo) were counted, and Gram-positive Bacillus spp. were confirmed by microscopic examination. The results are expressed as 1g CFU / g. This indicator is directly related to the target inhibitory component of nisin.

[0150] Determination of volatile basic nitrogen (TVB-N): Refer to the steam distillation method in the National Food Safety Standard - Determination of Volatile Basic Nitrogen in Food (GB-5009.228-2016). Take 10 mL of the clarified filtrate and inject it into a distillation tube. Add 10 mL of 10 g / L magnesium oxide (MgO) suspension to create a weakly alkaline environment and perform steam distillation. Absorb the distillate with 20 g / L boric acid absorption solution (containing methyl red-bromocresol green mixed indicator) and titrate to the endpoint with 0.01 mol / L hydrochloric acid standard solution. Simultaneously, perform a blank test and calculate the TVB-N content based on the amount of hydrochloric acid consumed. TVB-N reflects the total amount of ammonia and amines produced by the deamination process of spoilage bacteria and is a core specific chemical indicator for determining whether "pseudo-fermentation" (spoilage) has occurred in the fermented bean curd.

[0151] The experimental data are shown in Table 3:

[0152] Table 3: Monitoring data on Bacillus growth and TVB-N content during the mesophilic enzymatic hydrolysis period

[0153]

[0154] Note: Bacillus count data are logarithmically processed; TVB-N exceeding 20mg / 100g usually indicates an initial risk of spoilage, while exceeding 50mg / 100g is considered severe spoilage.

[0155] Conclusion: Based on Table 3 and the appendix Figure 3 Based on the data, Examples 1 and 4 successfully achieved effective control of spoilage microorganisms in the high-risk temperature range of 36-37℃, verifying the reliability of the "biological-chemical synergistic anti-corrosion" mechanism.

[0156] Microbial growth kinetics data showed that in Comparative Example 3 without preservatives, Bacillus exhibited typical logarithmic growth phase characteristics; the concentration surged from 2.88 lg CFU / g at 0 hours to 6.54 lg CFU / g at 24 hours, and reached a putrefaction level of 8.65 lg CFU / g at 48 hours. This explosive growth led to abnormal decomposition of proteins in the system by miscellaneous bacteria, as evidenced by the TVB-N value exceeding the 50 mg / 100g spoilage threshold at 36 hours, eventually reaching 94.27 mg / 100g, indicating that the fermented mash had undergone irreversible rancidity and ammonia production.

[0157] In contrast, Example 1, through the combination of Nisin (0.02%) and ethanol (1.5%), strictly limited the growth of Bacillus to the lag phase. After 48 hours, the total bacterial count was only 3.381g CFU / g, and the TVB-N content was controlled at an extremely low level of 13.52mg / 100g. This confirms that Nisin molecules, through the bactericidal mechanism of specifically binding to lipid II of Gram-positive bacterial cell membranes to form pores, combined with the non-specific disruption of cell membrane permeability by ethanol, construct a dense antibacterial barrier.

[0158] The data from Example 4 further revealed the flexibility of this preservative system; under the condition of halving the Nisin dosage (0.01%) but increasing the ethanol concentration to 2.0%, its antibacterial effect (3.45lg CFU / g after 48 hours) was not statistically significantly different from that in Example 1; this result supports the argument that there is a non-linear synergistic effect between ethanol and Nisin: although the higher concentration of ethanol is not enough to completely sterilize, the cell membrane damage it causes lowers the tolerance threshold of bacteria to Nisin, so that even a low dose of Nisin can exert a sufficient lethal effect.

[0159] Furthermore, the slow increase in TVB-N in Examples 1 and 4 was mainly due to the normal enzymatic release of amino acids from the substrate by Aspergillus oryzae protease, rather than bacterial putrefaction and deamination. The high concentration of broad bean protein substrate played a competitive protective role in the system, delaying the hydrolysis of Nisin peptide chains by proteases in the fermentation matrix, and ensuring that the preservative maintained an effective antimicrobial concentration during the critical 48-hour start-up period.

[0160] Test Example 4: Comprehensive Evaluation of Finished Product Physicochemical Indicators and Fermentation Efficiency

[0161] The experimental steps are as follows:

[0162] The fermented soybean paste products from Examples 1-4 and Comparative Examples 1-6 after fermentation termination were selected as test subjects. All samples were taken from the lower mixing zone of the fermentation tank, and were tested after grinding and homogenization. Quantitative analysis was mainly conducted on core quality indicators such as amino acid nitrogen, reducing sugar, total acid, and amino acid conversion rate.

[0163] Sample pretreatment: Accurately weigh 5.00 g of the uniformly pulverized sample and place it in a 250 mL volumetric flask. Add water to make up to the mark. Shake and extract in a 45 °C water bath for 30 minutes. Filter, discard the initial filtrate, and collect the subsequent filtrate as the reserve solution for testing.

[0164] Amino acid nitrogen determination: Refer to the first method (potential titration) in the National Food Safety Standard - Determination of Amino Acid Nitrogen in Food (GB-5009.235-2016). Formaldehyde is used to fix the amino groups of amino acids, making the carboxyl groups acidic. The solution is titrated with sodium hydroxide standard to the pH endpoint of 9.2, and the amino acid nitrogen content is calculated. This indicator is the core basis for the grading of broad bean paste. Referring to the industry benchmark standard "Geographical Indication Product - Pixian Broad Bean Paste" (GB / T-20560-2006), the limit for Grade 1 products should be ≥0.50g / 100g.

[0165] Determination of reducing sugars: The direct titration method in the National Food Safety Standard - Determination of Reducing Sugars in Food (GB-5009.7-2016) was used; methylene blue was used as a redox indicator, Fehling's reagent was calibrated, and the reducing sugar content in the sample was calculated; this index reflects the effect of the amylase system and the accumulation of flavor precursor substances during the post-ripening stage.

[0166] Total acid determination: Refer to the National Food Safety Standard - Determination of Total Acid in Food (GB-12456-2021), and calculate the total acid content (calculated as lactic acid) with pH 8.2 as the titration endpoint. This indicator reflects the acidity control and buffer system stability during the fermentation process.

[0167] Raw material utilization rate calculation: Based on the total amount of protein in the feed and the total amount of amino acid nitrogen in the finished product, calculate the protein hydrolysis rate. Formula: Protein hydrolysis rate (%) = [(Amino acid nitrogen content of finished product × Total weight of finished product × 6.25) / (Total protein content of raw materials)] × 100%.

[0168] The experimental data are shown in Table 4:

[0169] Table 4: Comparison of Physicochemical Indicators and Fermentation Cycles of Fermented Bean Paste Products from Each Group

[0170]

[0171] Conclusion: Based on Table 4 and the appendix Figure 4The data shows that the process of this invention, while significantly shortening the fermentation cycle, exhibits significant advantages in all core physicochemical indicators, verifying the decisive role of the stoichiometric buffer system in improving enzymatic hydrolysis efficiency and controlling fermentation quality.

[0172] Regarding the depth of protein hydrolysis and amino acid nitrogen: In Example 1, the amino acid nitrogen content reached 0.96 g / 100 g within a fermentation cycle of only 28 days, and the protein hydrolysis rate was as high as 58.4%. This value not only far exceeds that of the traditional process in Comparative Example 1 (90 days, 0.52 g / 100 g), but also significantly surpasses the first-grade limit (≥0.50 g / 100 g) specified in the benchmark standard for soybean paste, "Geographical Indication Product - Pixian Douban" (GB / T-20560-2006). Even compared with the more stringent physicochemical requirements of premium soybean paste (0.80 g / 100 g), this product still has a significant advantage. A horizontal comparison with Comparative Example 2 (0.58 g / 100 g) confirmed that the introduction of sodium hexametaphosphate (SHMP) played a crucial role. In Comparative Example 2, due to the lack of ionic strength regulation and polyelectrolyte effect of SHMP, broad bean protein remained in a tightly aggregated state, resulting in hidden enzymatic hydrolysis sites. Even under the same process conditions, the substrate conversion rate could not be effectively improved.

[0173] Regarding the role of the buffer system in maintaining enzyme efficiency: The amino acid nitrogen content in Example 1 (0.96 g / 100 g) was higher than that in Comparative Example 4 (0.65 g / 100 g) and Comparative Example 5 (0.71 g / 100 g). Comparative Example 4 used direct acid addition for adjustment, lacking the calcium carbonate-GDL coupled buffering mechanism, resulting in drastic pH fluctuations in the middle of fermentation, causing irreversible inactivation of neutral proteases and insufficient enzymatic kinetics in the later stages. Although Comparative Example 5 added calcium carbonate, the micronized particle size caused it to be depleted prematurely, failing to provide a continuous supply of Ca during the acidification stage. 2+ Protection and pH buffering limited further increases in protein hydrolysis rate. Data from Comparative Example 6 (0.55 g / 100 g) further corroborated the interference of soluble calcium sources on the solubilization effect of SHMP, leading to a dual negative effect of "solubilization failure" and "enzyme protection failure".

[0174] Regarding the preservation system and flavor compounds: Comparative Example 3 showed extremely low reducing sugar content (2.15 g / 100 g) and abnormally high total acid (2.64 g / 100 g), indicating that in the absence of a Nisin-ethanol synergistic preservation system, excessive microbial growth consumed reducing sugar and produced acid, resulting in severe spoilage. In contrast, Examples 1 and 4, while ensuring food safety, retained higher levels of reducing sugar (>8.3 g / 100 g). This not only endowed the product with a mellow, sweet aftertaste but also provided sufficient precursors for the Maillard reaction during the post-ripening stage, ensuring the final product's bright red color and harmonious flavor.

[0175] In summary, the process of this invention achieves high-quality fermented soybean paste production under low-salt and short-cycle conditions through the coupling of three technical dimensions: substrate solubilization, enzyme activity protection, and synergistic preservation. This overcomes the technical bottlenecks of traditional low-salt fermentation, such as bland flavor, incomplete enzymatic hydrolysis, and susceptibility to bacterial contamination.

[0176] Test Example 5: Characterization of the Textural Properties (TPA) and Microscopic Water-Retention Mechanism of Sauce Particles

[0177] The experimental steps are as follows:

[0178] Fermentation endpoint samples from Examples 1, 4, and 5 were selected. This test aims to verify the formation of the "in-situ micropore" structure within the fermentation system through macroscopic physical and mechanical indicators.

[0179] After fermentation, the mash samples from each group were gently stirred to ensure uniformity without damaging the overall structure. They were then carefully packed into a standard cylindrical test container (50mm in diameter, 20mm in height) using a wide-mouth packer. Strict control of the packing force was required to avoid over-compacting and damaging the internal pores. Large macroscopic air bubbles introduced during packing were removed by three gentle vertical vibrations. The surface was then smoothed using a scraper.

[0180] Total texture analysis (TPA) was performed using a texture analyzer (TA.XTPlus) equipped with a P / 36R cylindrical probe. The test mode was set to "compression-springback," with a pre-test velocity of 2.0 mm / s, a test velocity of 1.0 mm / s, a compression ratio of 40%, an interval of 5.0 s between compressions, and a trigger force of 5.0 g. The system collected force-time curves and automatically calculated hardness, adhesion, and adhesiveness. Each sample was prepared in parallel and tested six times, discarding the maximum and minimum values.

[0181] Simultaneously, water-holding capacity (WHC) was measured to characterize the physical water retention capacity. 20.00 g of the fermented soybean paste sample (M1) was accurately weighed and placed in a 50 mL centrifuge tube lined with double-layered filter paper. The tube was centrifuged at 4000 r / min for 20 minutes. After centrifugation, the filter paper and the free water precipitated from the top layer were carefully removed, and the weight of the remaining wet fermented soybean paste in the centrifuge tube (M2) was accurately measured. Water-holding capacity was calculated using the formula: WHC(%) = (M2 / M1) × 100%.

[0182] The experimental data are shown in Table 5:

[0183] Table 5: Textural properties and water-holding capacity data of the sauce

[0184]

[0185] Note: Hardness value reflects the sauce's ability to resist deformation; a higher value indicates a more compacted texture. Adhesion reflects the work done when the sauce separates from the probe and is positively correlated with the smoothness and oiliness of the texture. WHC reflects the physical binding efficiency of the gel network for water.

[0186] Conclusion: Based on Table 5 and the appendix Figure 5 Based on the data, Example 1 successfully regulated the physical and rheological properties of the fermented soybean paste matrix through the delayed coupling reaction of coarse-particle calcium carbonate and GDL. Its texture characteristics and water-holding efficiency were superior to those of the comparative examples, which proved the effectiveness of the "in-situ micropore formation" mechanism in solving the problem of caking in low-salt solid-state fermentation.

[0187] Regarding the decrease in hardness and the formation of steric hindrance: The average hardness of Example 1 remained at around 345g, only 28% of that of Comparative Example 4 (approximately 1216g). This difference reveals two distinct protein aggregation pathways: In Comparative Example 4, simple acid regulation led to intense hydrophobic aggregation of proteins near their isoelectric points, forming a dense and continuous blocky gel network, macroscopically characterized by extremely high hardness and compaction. In Example 1, however, coarse-particle calcium carbonate slowly released carbon dioxide microbubbles in the GDL acid hydrolysis environment. These bubbles acted as physical spacers embedded in the forming protein gel network, preventing excessively tight packing of protein micelles. This in-situ generated porous structure reduced the compressive strength of the paste, giving the finished product a soft, easily dispersible texture.

[0188] Regarding water retention capacity and capillary trapping effect: The centrifugal water retention capacity of Example 1 remained stable at over 89%, while that of Comparative Example 4 was only around 62%, with significant water separation at the bottom of the centrifuge tube. This phenomenon is directly related to the microporous structure. The dense structure of Comparative Example 4, lacking internal space, caused water molecules to be "squeezed out" of the network by strong interactions between protein molecules, resulting in severe dehydration and shrinkage. Conversely, in Example 1, the micropores left by air bubbles formed a rich capillary network, using capillary pressure to lock free water inside the matrix. Furthermore, the microporous structure increased the specific surface area of ​​the matrix, exposing more hydrophilic groups and further enhancing the proportion of physically adsorbed water.

[0189] Regarding the matching of gas release rate and structural stability: Data from Comparative Example 5 (hardness approximately 961g, WHC approximately 68%) demonstrates the criticality of "pore-forming timing." Although calcium carbonate was also added, the reaction rate of the micronized raw material was too fast, and carbon dioxide escaped in large quantities before the protein gel network gained mechanical strength, failing to form effective residual pores. This led to the eventual collapse of the system, reverting to a dense state close to that of Comparative Example 4. Example 1, through control of coarse particle size, achieved a kinetic match between gas production rate and gelation rate, ensuring that pores were effectively solidified in the final finished structure.

[0190] In summary, this test case, from the perspectives of rheology and physical mechanics, confirms that the process of this invention, by constructing a "protein-pore" composite network, fundamentally overcomes the defects of low-salt fermented soybean paste, such as crusting and poor water retention caused by high protein density.

[0191] Test Example 6: Taste Fingerprint and Color Analysis Based on Intelligent Sensing Technology

[0192] The experimental steps are as follows:

[0193] The final products of Examples 1, 4, Comparative Examples 1 and 3 were selected as test subjects. This test aims to objectively characterize the taste profile and color quality of the products by utilizing modern sensor technology, while eliminating the interference of human sensory fatigue and subjective preferences.

[0194] Sample extraction solution preparation: Accurately weigh 30.0 g of each group of fermented soybean paste samples, add deionized water at a ratio of 1:5 (w / v), and magnetically stir at 45℃ for 30 minutes to fully extract the flavor substances. Centrifuge the mixture at 8000 r / min for 15 minutes, and pass the supernatant through a 0.45 μm aqueous microporous membrane. The filtrate is used as the test sample for electronic tongue testing. Four replicates of each sample are prepared.

[0195] Electronic tongue taste analysis: The TS-5000Z electronic tongue system (equipped with a lipid membrane sensor array) was used for detection. The system is equipped with five independent sensor probes for umami, saltiness, sourness, bitterness, and astringency. Before testing, activation and zero-point calibration were performed using a reference solution (composed of 30mM potassium chloride + 0.3mM tartaric acid solution). The sample sampling time was set to 120 seconds, and the washing time to 300 seconds. The system outputs the relative intensity values ​​of each taste index based on changes in membrane potential. This value is based on the Weber-Fechner law, where each unit change represents a perceptible sensory difference.

[0196] Precision color difference analysis: The original sauce sample (excluding extract) was spread evenly in an optical glass dish, with a thickness controlled at more than 15 mm to avoid light transmission interference; using a CR-400 handheld colorimeter, selecting a D65 standard light source and a 10° field of view, the CIEL of the sample was measured. ∗ a ∗ b ∗ Color space parameters. Pay special attention to recording L. ∗ Value (brightness, 0-100) and a ∗ Value (redness-greenness, positive value represents red), each sample was measured 6 times at different sites and the average value was taken.

[0197] Off-flavor / spoilage feature identification: The response values ​​of the "sourness" and "off-flavor (bitter aftertaste)" sensors of the electronic tongue are used as objective criteria for determining spoilage. In the fermentation of soy sauce mash, an abnormally high acidity value accompanied by an increased bitter aftertaste is a typical signal of acid production by miscellaneous bacteria and protein spoilage.

[0198] The experimental data are shown in Table 6:

[0199] Table 6: Intelligent Sensory Evaluation Indicators: Taste Intensity Value and Color Difference Parameter Measurement Data

[0200]

[0201] Note: In the electronic tongue data, the "tasteless point" is usually set to 0 or -X (referencing liquid standard). The larger the positive value, the higher the strength. Comparative Example 1 is the traditional high-salt process. Comparative Example 3 is the group without preservatives.

[0202] Conclusion: Based on Table 6 and the appendix Figure 6 The data from Examples 1 and 4 show that while maintaining the low-salt characteristics, they established a high-quality flavor fingerprint with high umami as the core, and the color performance is better than that of traditional processes. The objective data and the aforementioned physicochemical indicators form a rigorous logical mutual verification.

[0203] Regarding umami enhancement and saltiness perception: Electronic tongue data showed that the umami intensity of Example 1 (average 14.83) was higher than that of Comparative Example 1, which used a traditional high-salt process (average 9.20). This significant sensory difference is directly attributed to the enzymatic hydrolysis system assisted by sodium hexametaphosphate in this invention; the aforementioned Test Example 4 has confirmed that Example 1 has an extremely high amino acid nitrogen content, and these free amino acids (especially glutamic acid) are the main response substrates for the umami sensor. Notably, although the actual salt content of Example 1 was much lower than that of Comparative Example 1, its saltiness intensity value (approximately 11.24) did not drop drastically, but remained at a moderate level. This aligns with the taste interaction mechanism of "umami suppressing bitterness and enhancing saltiness," where the high concentration of umami substances produced a synergistic effect in the oral cavity (or sensor), compensating for the bland taste caused by low salt and solving the "flavor deficiency" problem commonly faced by low-salt foods.

[0204] Regarding spoilage identification and preservation effectiveness: The taste fingerprint of Comparative Example 3 exhibited typical spoilage characteristics. Its acidity intensity value surged to the 8.9-9.1 range (while the normal group had negative values), accompanied by an abnormally elevated bitter aftertaste (approximately 4.97). This indicates that in the absence of the Nisin-ethanol synergistic preservation system, carbohydrates in the fermentation substrate were metabolized by miscellaneous bacteria into organic acids such as lactic acid and acetic acid, while proteins were non-specifically degraded by spoilage bacteria (such as Bacillus) into bitter peptides and amines. This quantitative taste data is highly consistent with the chemical results of excessive TVB-N in Test Example 3, confirming that Comparative Example 3 had completely lost its edible value. In contrast, after adjusting the preservative ratio, Example 4 showed no significant difference in acidity and bitterness indices compared to Example 1, demonstrating the safety of the adjusted preservation system.

[0205] Regarding color quality and Maillard reaction: In terms of color, the redness value (a) of Example 1... ∗ The concentration of α16.84 was higher than that of Comparative Example 1 (12.12) and Comparative Example 3 (8.34). Comparative Example 1, due to its long fermentation period (90 days), tended to have a darker color due to prolonged oxidation (L). ∗ The value was lower in Example 1, while in Comparative Example 3 the substrate was cloudy and grayish due to microbial decay. Example 1 exhibited an attractive bright red color because, on the one hand, the shorter fermentation cycle reduced the oxidative browning of pigments, and on the other hand, the microporous structure (confirmed in Test Example 5) promoted adequate oxygen exchange. Combined with well-retained reducing sugars (data from Test Example 4), a controlled and sufficient Maillard reaction occurred during the post-ripening stage, generating ideal melanoidins and reddish-brown pigments, giving the product an excellent commercial appearance.

[0206] In summary, the results of the intelligent sensory test show that the fermented soybean paste prepared by the process of this invention achieves a significant increase in umami intensity and optimization of color while greatly reducing salt content (with controllable saltiness), and completely eliminates the unpleasant flavor caused by spoilage, thus achieving the technical goal of "reducing salt without reducing flavor".

Claims

1. A composite substrate composition for fermenting low-salt fermented soybean paste, characterized in that, It is made from the following components in parts by weight: 550 to 650 portions of broad beans were used to make koji (fermented rice wine). 65 to 75 parts table salt; Sodium hexametaphosphate 0.8 to 1.2 parts; Nisin 0.1 to 0.25 parts; 12 to 20 parts of edible alcohol; 0.5 to 0.8 parts of granulated calcium carbonate with a particle size of 40 to 60 mesh; 8 to 12 parts of glucono-δ-lactone; 250 to 400 parts water; The granulated calcium carbonate with a particle size of 40-60 mesh is prepared by a method including the following steps: weighing light calcium carbonate powder, adding deionized water to mix and form a soft material; granulating the soft material through a 20-mesh sieve, drying it until the moisture content is less than 1 wt%; and sieving to retain particles between 40-60 mesh.

2. The composite substrate composition for low-salt fermented soybean paste according to claim 1, characterized in that, The proportions of the components are as follows: 580 portions of broad bean koji were produced. 70 parts table salt; Sodium hexametaphosphate 1.0 part; Nisin 0.15 to 0.2 parts; 15 portions of edible alcohol; 0.6 parts of granulated calcium carbonate with a particle size of 40-60 mesh; 10 parts of glucono-δ-lactone; 320 portions of water.

3. The composite substrate composition for low-salt fermented soybean paste according to claim 1, characterized in that, The lactic acid nisin is added in the form of a pre-dissolved solution; the pre-dissolved solution is prepared by: Add the lactic acid nisin to sterile water, add citric acid or dilute hydrochloric acid dropwise to adjust the pH of the system to 3.0-3.5, and stir until the active ingredient dissolves or forms a uniform suspension.

4. A low-salt fermented soybean paste production process based on segmented temperature-controlled fermentation, comprising fermentation using the composite substrate composition for low-salt fermented soybean paste as described in any one of claims 1-3, characterized in that, Includes the following steps: S1. Mix broad bean curd, water, edible salt, sodium hexametaphosphate, nisin, edible alcohol and granulated calcium carbonate evenly, and adjust the initial pH of the system to 6.2-6.

4. S2. Control the temperature of the fermented soybean paste at 35℃~37℃ and ferment at a constant temperature for 36h~42h; utilize the solubilizing effect of the sodium hexametaphosphate and the solid-phase buffering properties of the granulated calcium carbonate to maintain the enzymatic hydrolysis environment. S3. When fermentation is in progress for 36 to 42 hours, start adding an aqueous solution of gluconate-δ-lactone to the system, and control the addition time to be completed within 20 to 30 hours. Utilize the carbon dioxide gas generated in situ to construct internal micropores and reduce the pH of the system to 4.8 to 5.

0. S4. Lower the temperature of the product to 28℃~30℃, inoculate with Lutein's yeast, and ferment at a constant temperature for 20d~30d; S5. After fermentation, the product is sterilized and packaged to obtain the final product.

5. The low-salt fermented soybean paste production process based on segmented temperature-controlled fermentation according to claim 4, characterized in that, In step S1, the method for preparing the broad bean koji includes: Peel, soak, drain, and steam dried broad beans, then cool to 38°C; mix with flour, and inoculate with Aspergillus oryzae spore powder (3.042); incubate at 32°C–35°C for 40–44 hours to obtain the broad bean koji. The dried broad beans have a protein content of 24.0 wt% to 25.0 wt%, and the flour has a protein content of 11.0 wt%.

6. The low-salt fermented soybean paste production process based on segmented temperature-controlled fermentation according to claim 4, characterized in that, In step S2, the specific conditions for constant temperature fermentation are as follows: the temperature is controlled at 36℃~37℃, and the fermentation is allowed to stand for 40 hours; during this period, the mixture is stirred at low speed for 5 minutes every 12 hours, and the stirring speed is 10 rpm.

7. The low-salt fermented soybean paste production process based on segmented temperature-controlled fermentation according to claim 4, characterized in that, In step S3, the concentration of the aqueous solution of glucono-δ-lactone is 30 wt%. The specific method of adding an aqueous solution of gluconate-δ-lactone to the system is as follows: the solution is continuously added at a constant rate so that the hydrolysis acid production rate of the gluconate-δ-lactone matches the disintegration and neutralization rate of the granulated calcium carbonate, so that the pH value of the system stabilizes between 4.8 and 5.0 at the end of the addition.

8. The low-salt fermented soybean paste production process based on segmented temperature-controlled fermentation according to claim 4, characterized in that, In step S4, the amount of *Gnaphalium rouxiense* added is 2v / v% to 4v / v% of the total system volume. The constant temperature fermentation time is 25 days.

9. The low-salt fermented soybean paste production process based on segmented temperature-controlled fermentation according to claim 4, characterized in that, In step S5, the sterilization conditions are heating to 85°C and holding at that temperature for 20 minutes; The resulting low-salt fermented soybean paste has an amino acid nitrogen content of ≥0.95g / 100g and a protein hydrolysis rate of ≥57.0wt%.

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