Method for preparing walnut soy sauce by using segmented wet heat pretreatment combined with multi-strain fermentation

Abstract

The application relates to the technical field of food brewing processing, and discloses a method for preparing walnut soy sauce by using segmented wet heat pretreatment combined with multi-strain fermentation, which comprises the following steps: subjecting walnut meal to dry heat treatment, adjusting humidity, transferring into a high-temperature steam explosion equipment for steam explosion treatment and air cooling; inoculating the air-cooled walnut meal with mixed strains composed of aspergillus oryzae, saccharomyces rouxii and lactobacillus plantarum to prepare koji; adding brine to the koji and carrying out segmented fermentation including three stages to obtain soy sauce mash; adding oil to the soy sauce mash, precipitating the original solution, filtering the clear liquid, cooling the sterilized product to obtain a finished product. The present application combines wet heat pretreatment with multi-strain segmented fermentation, destroys the dense structure of the walnut meal, improves the substrate hydrolysis rate, meets the metabolic conditions suitable for different strains, reduces the substrate competition, shortens the fermentation period, and improves the content of free amino acids and ester substances.

Description

Technical Field

[0001] This invention relates to the field of food brewing and processing technology, specifically a method for preparing walnut soy sauce using segmented wet heat pretreatment combined with multi-strain fermentation. Background Technology

[0002] Walnuts produce a large amount of walnut meal after walnut oil extraction, which retains rich plant protein, carbohydrates, and minerals. Currently, walnut meal is mostly used as animal feed or discarded directly, resulting in low economic value. Utilizing walnut meal as a substrate for brewing walnut soy sauce is an effective way to achieve high-value utilization of walnut by-products.

[0003] In traditional walnut soy sauce brewing, walnut meal, which has been simply crushed or steamed, is usually directly mixed with brewing microorganisms for fermentation. However, walnut meal has a relatively dense physical structure, with nutrients such as proteins and polysaccharides tightly embedded in cell walls and a network of lignocellulose. Conventional pretreatment methods are insufficient to effectively break down this encapsulation structure, preventing the proteases and amylases secreted by microorganisms during fermentation from fully penetrating the material and limiting enzyme-substrate contact. This results in incomplete hydrolysis of macromolecules, leading to low protein conversion and utilization rates in the raw materials, directly affecting the amino acid nitrogen content in the soy sauce.

[0004] Meanwhile, existing brewing processes mostly employ single-strain Aspergillus oryzae fermentation or simultaneous constant-temperature fermentation of multiple microorganisms. Because different microorganisms have significantly different requirements for temperature and environment during acid production, enzyme production, and aroma production stages, simultaneous mixed fermentation easily leads to competition for nutrient substrates among different strains, and even antagonistic effects. This fermentation mode limits the metabolic activity of yeasts and lactic acid bacteria, hindering the transformation of flavor precursors such as alcohols and esters in the fermentation broth. This not only prolongs the overall brewing cycle of soy sauce but also results in insufficient accumulation of volatile aroma compounds in the final product, leading to a relatively monotonous flavor. Therefore, it is necessary to improve existing pretreatment methods and fermentation processes to increase the hydrolysis efficiency of walnut meal and improve the flavor quality of soy sauce. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for preparing walnut soy sauce using segmented wet heat pretreatment combined with multi-strain fermentation. This method solves the problems of insufficient substrate hydrolysis due to the difficulty in breaking down the dense structure of walnut meal during pretreatment, and the long fermentation cycle and monotonous product flavor caused by competition among strains in conventional fermentation modes.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A method for preparing walnut soy sauce using segmented wet heat pretreatment combined with multi-strain fermentation includes the following steps: Walnut meal is placed in a dry heat equipment for dry heat treatment, the walnut meal after dry heat treatment is humidified, the humidified walnut meal is transferred to a high temperature steam explosion equipment for high temperature steam explosion treatment, and the walnut meal after high temperature steam explosion treatment is cooled by air cooling for later use. A mixed microbial culture consisting of Aspergillus oryzae, Saccharomyces rouxii, and Lactobacillus plantarum is inoculated into the air-cooled walnut meal. The walnut meal inoculated with the mixed microbial culture is placed in a koji-making machine for koji-making. During the koji-making process, the koji is turned over. After the koji-making is completed, fermentation koji is obtained. Salt water is added to the fermentation starter, and the fermentation starter with added salt water is placed in a fermentation container for segmented fermentation. The segmented fermentation includes a first stage fermentation, a second stage fermentation and a third stage fermentation. After the segmented fermentation is completed, soy sauce mash is obtained. The soy sauce mash is subjected to an oil-draining operation. The soy sauce concentrate obtained after the oil-draining operation is collected and allowed to settle to obtain a clear liquid. The clear liquid is filtered using a precision filtration device. The filtered liquid is then heated and sterilized. After cooling the heated and sterilized liquid, the walnut soy sauce product is obtained.

[0007] By adopting the above technical solution, which combines pretreatment with multi-strain staged fermentation, the utilization rate of walnut meal and the quality of soy sauce are improved. The specific mechanism of action is as follows: During dry heat treatment, the proteins in walnut meal undergo moderate denaturation, and anti-nutritional factors are destroyed. Humidification treatment allows the material to absorb moisture, providing a mass transfer medium for steam explosion. During high-temperature steam explosion, high-temperature, high-pressure steam penetrates the interior of the walnut meal and generates mechanical shear force upon pressure release. This shear force disrupts the network structure of the walnut meal cell walls, exposing the embedded proteins and polysaccharides, increasing the specific surface area of ​​the material, and providing a physical channel for the invasion of microbial enzymes.

[0008] In the first stage of fermentation, Lactobacillus plantarum metabolizes the free sugars in walnut meal to produce lactic acid, which causes the pH of the mash to drop, inhibits the growth of miscellaneous bacteria, and establishes a slightly acidic basic environment for subsequent fermentation.

[0009] In the second stage of fermentation, the temperature was adjusted to 32℃, allowing Aspergillus oryzae to reach a suitable metabolic phase and secrete proteases. With the participation of water molecules, the proteases hydrolyzed the exposed macromolecular proteins of the walnut into polypeptides and free amino acids; simultaneously, amylase hydrolyzed the polysaccharide structure into reducing sugars with the participation of water molecules. These amino acids and reducing sugars constitute the basis of the umami and sweetness of the fermentation broth.

[0010] In the third stage of fermentation, the temperature is raised to 40℃, and *Saccharomyces rouxii* utilizes the reducing sugars and free amino acids accumulated in the previous stage to undergo aldol condensation and esterification reactions. During this process, alcohols and carboxylic acids undergo esterification, removing water molecules to generate volatile esters such as ethyl acetate and ethyl lactate, which impart an ester aroma to the walnut soy sauce. Segmented temperature control ensures that the acid production, enzymatic hydrolysis, and esterification fermentation processes proceed independently and progressively, reducing substrate competition between microbial strains.

[0011] Preferably, the temperature of the dry heat treatment is 105°C to 120°C, and the time of the dry heat treatment is 5 minutes to 10 minutes; the moisture content of the walnut meal after the moisture conditioning treatment is 38% to 42%; the pressure of the high-temperature steam explosion treatment is 0.6 MPa to 1.8 MPa; and the walnut meal after the high-temperature steam explosion treatment is cooled to room temperature by air cooling for later use.

[0012] By employing the above technical solutions, the limited dry heat temperature and time prevent the walnut protein from losing its hydrolytic activity due to caramelization. Limiting the moisture content and steam explosion pressure allows water vapor to create expansion pressure within the material, achieving cell wall disruption and preventing thermal degradation of nutrients due to excessive pressure. It also maintains the material's supporting structure and permeability during the koji-making process.

[0013] Preferably, the mass ratio of Aspergillus oryzae, Saccharomyces rouxii, and Lactobacillus plantarum is 1:0.5:0.3, and the total inoculation amount of the mixed strain is 0.5% of the total mass of walnut meal; the koji-making temperature is 30℃ to 34℃, the koji-making time is 40h to 50h, and the koji-turning operation is performed every 12h during the koji-making time.

[0014] By adopting the above technical solution, a stable fermentation system was constructed using a limited ratio of mixed microorganisms. *Aspergillus oryzae* was the main component providing enzyme production, while *Saccharomyces rouxii* and *Lactobacillus plantarum* were added in specific proportions to avoid antagonistic effects. The controlled koji-making temperature and timed turning operations effectively dissipated the bio-heat generated by metabolism, maintained the oxygen content inside the koji material, prevented koji burning, and promoted normal mycelial reproduction.

[0015] Preferably, the added brine has a mass fraction of 20%, and the mass ratio of the fermentation starter to the added brine is 1 kg:1.0 L to 1 kg:2.0 L; the first stage fermentation temperature is 25°C, and the first stage fermentation time is 5 days; the second stage fermentation temperature is 32°C, and the second stage fermentation time is 8 days; the third stage fermentation temperature is 40°C, and the third stage fermentation time is 6 to 14 days.

[0016] By employing the above technical solution, a high osmotic pressure environment is established using a 20% salt solution with a specific material-to-liquid ratio, inhibiting the growth of unwanted microorganisms and maintaining the metabolism of halophilic fermentation strains. Staged temperature control satisfies the temperature requirements for lactic acid bacteria to produce acid, Aspergillus to secrete proteases, and yeast to carry out esterification reactions, accelerating the degradation of macromolecules and shortening the overall fermentation time while ensuring the stability of the fermentation base solution.

[0017] Preferably, the oil-spraying operation is performed three times consecutively; the heating sterilization is carried out at 90°C for 15 minutes, and the heat-sterilized filtrate is cooled to room temperature to obtain the finished walnut soy sauce.

[0018] By employing the above technical solution, continuous oil extraction can wash away soluble solids and amino acid nitrogen from the soy sauce mash. Sterilization at 90℃ can inactivate residual enzymes and kill microorganisms, while the heat treatment process triggers the Maillard reaction, deepening the color of the soy sauce liquid and enhancing its caramel flavor.

[0019] Preferably, the dry heat treatment temperature is 110℃, and the dry heat treatment time is 10 minutes; the moisture content of the walnut meal after the moisture conditioning treatment is 40%; the pressure of the high-temperature steam explosion treatment is 1.2 MPa; the koji-making temperature is 32℃, and the koji-making time is 45 hours; the mass ratio of the fermentation koji material to the volume ratio of the added brine is 1 kg: 1.25 L; and the third-stage fermentation time is 8 days.

[0020] By adopting the above technical solution, a set of process parameters within a moderate range is provided. These parameters are set to achieve a balance between pretreatment energy consumption and structural damage, coordinating the substrate hydrolysis rate with the aroma production process, and producing a soy sauce product with balanced physicochemical properties.

[0021] Preferably, the dry heat treatment temperature is 105℃, the dry heat treatment time is 5 minutes; the moisture content of the walnut meal after the moisture conditioning treatment is 38%; the pressure of the high-temperature steam explosion treatment is 0.6 MPa; the koji-making temperature is 30℃, the koji-making time is 40 hours; the mass ratio of the fermentation koji material to the volume ratio of the added brine is 1 kg: 1.0 L; and the third-stage fermentation time is 6 days.

[0022] By adopting the above technical solution and applying the lower limit process parameters, the requirements for high temperature and high pressure conditions are reduced and the time consumed in each stage is shortened, the heat energy consumption in the preparation process is reduced, and the overall preparation cycle is shortened while maintaining the basic fermentation efficiency.

[0023] Preferably, the dry heat treatment temperature is 120℃, and the dry heat treatment time is 10 minutes; the moisture content of the walnut meal after the moisture conditioning treatment is 42%; the pressure of the high-temperature steam explosion treatment is 1.8 MPa; the koji-making temperature is 34℃, and the koji-making time is 50 hours; the mass ratio of the koji material used for fermentation to the volume ratio of the added brine is 1 kg: 2.0 L; and the third-stage fermentation time is 14 days.

[0024] By adopting the above technical solution and applying the upper limit process parameters, the high-intensity wet heat pretreatment combined with a longer koji-making and fermentation time further dissociates the cross-linked structure in the material. The increased esterification time promotes the reaction of alcohol compounds and increases the content of soluble nitrogen and ester substances in the finished product.

[0025] This invention provides a method for preparing walnut soy sauce using segmented wet heat pretreatment combined with multi-strain fermentation. It has the following beneficial effects: 1. This invention utilizes a wet heat pretreatment process that combines dry heat treatment, humidity conditioning, and high-temperature steam explosion treatment. By using the mechanical shear force generated during steam explosion depressurization, the network structure of the walnut meal cell wall is destroyed, exposing the embedded proteins, polysaccharides, and other macromolecules. This increases the porosity and specific surface area of ​​the material, providing a physical channel for the invasion of microorganisms and enzyme systems, thereby improving the hydrolysis rate and raw material utilization rate of the walnut meal substrate.

[0026] 2. This invention uses a mixed inoculation of Aspergillus oryzae, Saccharomyces rouxii, and Lactobacillus plantarum, combined with a segmented fermentation mode of 25°C in the first stage, 32°C in the second stage, and 40°C in the third stage. This allows the processes of lactic acid bacteria producing acid, Aspergillus secreting enzymes to hydrolyze macromolecules, and yeast carrying out esterification reactions to proceed independently and progressively under their respective suitable temperature conditions. This reduces substrate competition between strains, increases the content of free amino acids and volatile esters in the fermentation broth, and shortens the overall fermentation cycle.

[0027] 3. This invention establishes a high osmotic pressure environment by controlling the specific ratio of fermentation starter and 20% salt solution. Combined with the acid production of Lactobacillus plantarum in the first stage to lower the pH value, it effectively inhibits the growth and reproduction of environmental bacteria and maintains the metabolic stability of the target microorganisms in the fermentation system. Combined with the subsequent heat sterilization operation, it not only inactivates residual enzyme activity and ensures food safety, but also promotes the Maillard reaction between amino acids and reducing sugars, deepening the color of walnut soy sauce and enhancing the overall flavor. Attached Figure Description

[0028] Figure 1 This is a process diagram for preparing walnut soy sauce according to the present invention; Figure 2 This is a diagram showing the effect of the humidity-regulating high-temperature gas explosion treatment of the present invention on the protein conformation in walnut meal; Figure 3 This is a graph showing the effect of different bacterial strain ratios on protease and amylase in this invention. Figure 4 Radar graph showing the effect of different material-to-liquid ratios on walnut soy sauce according to the present invention; Figure 5 This is a diagram showing the effect of fermentation temperature and fermentation time on fermentation in this invention; Figure 6 Radar graph showing the effect of different fermentation cycles on walnut soy sauce according to the present invention; Figure 7 This is a response surface methodology diagram of the present invention; Figure 8 This is a schematic diagram showing the results of total protein determination in walnut meal according to the present invention; Figure 9 This is a schematic diagram showing the results of the determination of soluble protein content in walnut meal according to the present invention. Figure 10 This is a schematic diagram showing the results of the determination of total sugar content in walnut meal according to the present invention; Figure 11 This is a schematic diagram showing the results of the determination of reducing sugar content in walnut meal according to the present invention; Figure 12 This is a graph showing the dynamic changes in pH value of the system during the fermentation cycle of this invention; Figure 13 This is a diagram showing the dynamic changes in enzyme activity during the fermentation cycle of this invention. Figure 14 This is a schematic diagram showing the determination results of amino acid nitrogen content in the finished walnut soy sauce products of each group according to the present invention; Figure 15 This is a schematic diagram showing the results of measuring the total nitrogen utilization rate and walnut meal raw material utilization rate of each group of processes in this invention; Figure 16 This is a comparison chart of the comprehensive sensory quality evaluation results of the walnut soy sauce products of each group according to the present invention; Figure 17 This is a comparison chart of the total fermentation cycle for industrial production of each group of the present invention; Figure 18 This is a dynamic distribution diagram of the contamination rate of miscellaneous bacteria and the product qualification rate in the industrial production of this invention. Detailed Implementation

[0029] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings, preparation examples, embodiments, comparative examples, and test examples. 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.

[0030] Examples 1-3: Example 1: This embodiment provides a method for preparing walnut soy sauce using segmented wet heat pretreatment combined with multi-strain fermentation, including the following steps: (1) Pretreatment: Weigh a certain amount of walnut meal and place it in a dry heat equipment. Perform dry heat treatment at 105℃ for 5 minutes. After the dry heat treatment is completed, perform moisture conditioning treatment to adjust the moisture content of the material to 38%. Then transfer the moisture-conditioned walnut meal into a high-temperature steam explosion equipment and perform high-temperature steam explosion treatment at a pressure of 0.6MPa. After the treatment is completed, air-cool it to room temperature for later use. (2) Mixed koji making: Inoculate the cooled walnut meal after pretreatment in step (1) with a mixed strain of Aspergillus oryzae, Saccharomyces roximatee and Lactobacillus plantarum, wherein the mass ratio of the three strains is 1:0.5:0.3, and the total inoculation amount of the mixed strain is 0.5% of the total mass of the walnut meal; place the material in a koji making machine, control the koji making temperature at 30℃, and the koji making time at 40h, and perform a turning operation every 12h during the period. After the koji making is completed, the fermentation koji material is obtained. (3) Segmented fermentation: Add 20% brine to the koji material obtained in step (2), and control the ratio of brine to koji material to liquid to be 1:1.0 (m / v, kg / L). Place the koji material in a fermentation container for segmented fermentation: In the first stage, Lactobacillus plantarum is the main fermenter to lower the pH, and the temperature is controlled at 25℃ for 5 days; in the second stage, Aspergillus oryzae is the main protease to secrete hydrolyzed protein, and the temperature is controlled at 32℃ for 8 days; in the third stage, Saccharomyces roxburghii is the main ester to produce aroma, and the temperature is controlled at 40℃ for 6 days. The total fermentation cycle is 19 days. (4) Post-processing: After fermentation, the mash is subjected to three consecutive oil rinsing operations, the obtained soy sauce original liquid is collected and allowed to settle; then, the clear liquid after sedimentation is filtered to remove impurities using a precision filtration device, the filtrate is heated and sterilized at 90°C for 15 minutes, and after cooling to room temperature, the walnut soy sauce product is obtained.

[0031] Example 2: This embodiment provides a method for preparing walnut soy sauce using segmented wet heat pretreatment combined with multi-strain fermentation, including the following steps: (1) Pretreatment: Weigh a certain amount of walnut meal and place it in a dry heat equipment. Dry heat treatment is carried out at 110℃ for 10 minutes. After the dry heat treatment is completed, moisture conditioning treatment is carried out to adjust the moisture content of the material to 40%. Then the moisture-conditioned walnut meal is transferred to a high-temperature steam explosion equipment and high-temperature steam explosion treatment is carried out at a pressure of 1.2MPa. After the treatment is completed, it is cooled to room temperature by air cooling for later use. (2) Mixed koji making: Inoculate the cooled walnut meal after pretreatment in step (1) with a mixed strain of Aspergillus oryzae, Saccharomyces roximatee and Lactobacillus plantarum, wherein the mass ratio of the three strains is 1:0.5:0.3, and the total inoculation amount of the mixed strain is 0.5% of the total mass of the walnut meal; place the material in a koji making machine, control the koji making temperature at 32℃, and the koji making time at 45h, and perform a turning operation every 12h during the period. After the koji making is completed, the fermentation koji material is obtained. (3) Segmented fermentation: Add 20% brine to the koji material obtained in step (2), and control the ratio of brine to koji material to liquid to be 1:1.25 (m / v, kg / L). Place the koji material in a fermentation container for segmented fermentation: In the first stage, Lactobacillus plantarum is the main fermenter to lower the pH, and the temperature is controlled at 25℃ for 5 days; in the second stage, Aspergillus oryzae is the main protease to secrete hydrolyzed protein, and the temperature is controlled at 32℃ for 8 days; in the third stage, Saccharomyces roxburghii is the main ester to enhance the aroma, and the temperature is controlled at 40℃ for 8 days. The total fermentation cycle is 21 days. (4) Post-processing: After fermentation, the mash is subjected to three consecutive oil rinsing operations, the obtained soy sauce original liquid is collected and allowed to settle; then, the clear liquid after sedimentation is filtered to remove impurities using a precision filtration device, the filtrate is heated and sterilized at 90°C for 15 minutes, and after cooling to room temperature, the walnut soy sauce product is obtained.

[0032] Example 3: This embodiment provides a method for preparing walnut soy sauce using segmented wet heat pretreatment combined with multi-strain fermentation, including the following steps: (1) Pretreatment: Weigh a certain amount of walnut meal and place it in a dry heat equipment. Perform dry heat treatment at 120℃ for 10 minutes. After the dry heat treatment is completed, perform moisture conditioning treatment to adjust the moisture content of the material to 42%. Then transfer the moisture-conditioned walnut meal into a high-temperature steam explosion equipment and perform high-temperature steam explosion treatment at a pressure of 1.8MPa. After the treatment is completed, air-cool it to room temperature for later use. (2) Mixed koji making: Inoculate the cooled walnut meal after pretreatment in step (1) with a mixed strain of Aspergillus oryzae, Saccharomyces roximatee and Lactobacillus plantarum, wherein the mass ratio of the three strains is 1:0.5:0.3, and the total inoculation amount of the mixed strain is 0.5% of the total mass of the walnut meal; place the material in a koji making machine, control the koji making temperature at 34℃, and the koji making time is 50h, and turn the koji once every 12h during the period. After the koji making is completed, the fermentation koji material is obtained. (3) Segmented fermentation: Add 20% brine to the koji material obtained in step (2), and control the ratio of brine to koji material to liquid to be 1:2.0 (m / v, kg / L). Place the koji material in a fermentation container for segmented fermentation: In the first stage, Lactobacillus plantarum is the main fermenter to lower the pH, and the temperature is controlled at 25℃ for 5 days; in the second stage, Aspergillus oryzae is the main protease to secrete hydrolyzed protein, and the temperature is controlled at 32℃ for 8 days; in the third stage, Saccharomyces roxburghii is the main ester to produce aroma, and the temperature is controlled at 40℃ for 14 days. The total fermentation cycle is 27 days. (4) Post-processing: After fermentation, the mash is subjected to three consecutive oil rinsing operations, the obtained soy sauce original liquid is collected and allowed to settle; then, the clear liquid after sedimentation is filtered to remove impurities using a precision filtration device, the filtrate is heated and sterilized at 90°C for 15 minutes, and after cooling to room temperature, the walnut soy sauce product is obtained.

[0033] Comparative Examples 1-4: Comparative Example 1: Compared with Example 2, the difference lies in the pretreatment method of step (1): the walnut meal is not subjected to dry heat, humidity adjustment and high temperature steam explosion treatment, but only conventional single high temperature steaming is used for pretreatment, and the rest are the same.

[0034] Comparative Example 2: Compared with Example 2, the difference lies in the strains and fermentation cycles used in steps (2) and (3): only Aspergillus oryzae is inoculated during koji making (the inoculation amount is still 0.5%), and the segmented fermentation is changed to a single-strain fermentation at 32°C for 32 days (the conventional fermentation cycle of the prior art), while the rest are the same.

[0035] Comparative Example 3: Compared with Example 2, the difference lies in the fermentation temperature control method in step (3): after adding brine, the mixture is fermented for 21 days under constant temperature (32°C) conditions instead of undergoing multi-temperature variable temperature fermentation. The rest are the same.

[0036] Comparative Example 4: Compared with Example 2, the difference lies in the pretreatment method of step (1): the conventional single compound enzymatic hydrolysis method (adding cellulase and protease) is used instead of the dry heat, humidity adjustment and steam explosion segmented humid heat pretreatment of the present invention, while the rest are the same.

[0037] Test Examples 1-5: Test Example 1: This test aims to verify the destructive effect of segmented moist heat pretreatment on the tissue structure of walnut meal and its effect on the release of intracellular nutrients. Specifically, it measures various basic nutritional indicators of the blank group (untreated walnut meal) and the products from the pretreatment stages of Examples 1-3; combined with... Figure 1As can be seen from the process diagram for preparing walnut soy sauce of this invention, the pretreatment steps such as dry heat and humidity-regulating high-temperature steam explosion are the leading steps in the entire fermentation production chain, and their treatment effect directly determines the accessibility of microbial substrates in the subsequent koji-making and fermentation steps.

[0038] Sample preparation: Walnut meal from the blank group and materials that have undergone high-temperature steam explosion treatment and cooling in Examples 1 to 3 were collected, placed in an electric heating blast drying oven and dried at a constant temperature of 60°C to constant weight, pulverized using a cyclone pulverizer, passed through a 60-mesh standard sieve, and the sieve-underfill material was collected and stored in a desiccator for later use.

[0039] Total protein determination: Weigh 1.00 g of dry sample into a digestion tube, add concentrated sulfuric acid and a catalyst mixture, and digest at high temperature in a digestion furnace until the solution turns transparent blue-green. After cooling, perform distillation titration using a fully automated Kjeldahl nitrogen analyzer, record the volume of standard acid consumed, and calculate the total protein content.

[0040] Determination of soluble protein content: Weigh 5.00 g of dry sample, add deionized water at a solid-liquid ratio of 1:10 (m / v), and extract at room temperature for 2 h on a magnetic stirrer. Centrifuge the suspension at 4000 r / min for 15 min and collect the supernatant. Using the biuret method, add biuret reagent to a specific volume of supernatant, react in the dark for 30 min, and then measure the absorbance at 540 nm using a spectrophotometer. Calculate the soluble protein content by referring to the standard curve.

[0041] Total sugar content determination: The extraction procedure is the same as above. Take the supernatant and add a certain proportion of phenol solution and concentrated sulfuric acid. Heat in a boiling water bath for 15 min for color development. After cooling to room temperature, measure the color at a wavelength of 490 nm. Quantitatively assess the total sugar content using a glucose standard curve.

[0042] Determination of reducing sugar content: The supernatant from the centrifugation was analyzed using the 3,5-dinitrosalicylic acid (DNS) colorimetric method. After adding DNS reagent, the solution was heated in boiling water for 5 min, rapidly cooled in an ice bath, and brought to volume. The absorbance was measured at 540 nm, and the reducing sugar concentration was calculated.

[0043] Each test was performed in triplicate, and the results were taken as the average and standard deviation.

[0044] Table 1. Results of the determination of the release of basic nutrients from walnut meal by different pretreatment parameters in conclusion: According to Table 1 and Figure 8The results of the total protein determination in walnut meal (the height of the bars in the figure represents the protein content calculated from the total nitrogen after sample digestion, and the error bars represent the standard deviation of three parallel determinations) show that the segmented wet heat pretreatment did not cause a significant loss of total nitrogen in the walnut meal, and the total protein content remained relatively stable, but the internal nutrient composition distribution of the material shifted. Combined with... Figure 9 Results of soluble protein content determination in walnut meal Figure 10 The results of the determination of total sugar content in walnut meal and Figure 11 The results of the walnut meal reducing sugar content determination showed that the soluble protein, total sugar, and reducing sugar contents of the blank group were all at low levels, indicating that the plant cell wall and cellulose-lignin network structure of natural walnut meal have physical resistance, hindering the dissolution of intracellular substances. After combined treatment with dry heat, humidity regulation, and high-temperature steam explosion in Examples 1 to 3, the soluble protein and reducing sugar contents showed an increase. Example 2 showed the highest conversion rate, with soluble protein reaching 16.08 g / 100 g and reducing sugar reaching 2.48 g / 100 g.

[0045] The aforementioned data changes validate the physicochemical effectiveness of the pretreatment mechanism. Dry heat treatment induces depolymerization of the cellulose crystals in walnut meal, causing internal free water to vaporize upon heating, thus loosening the cell wall microstructure. Subsequently, during high-temperature steam explosion under moisture-controlled conditions, high-pressure steam penetrates the material's internal structure, generating mechanical shear force and volume expansion work upon pressure release, tearing apart the dense cell walls. This structural disintegration leads to the release of proteins that were originally bound to or encapsulated by polysaccharides. Simultaneously, the high-temperature, high-pressure environment breaks the secondary bonds within the proteins, inducing conformational unfolding and denaturation of the protein molecules, exposing hydrophilic residues, manifested as an increase in water-soluble proteins, such as... Figure 9 As shown, this microscopic physicochemical change is achieved through... Figure 2 This has been further confirmed. Figure 2 This is the UV absorption spectrum of the effect of the humidification and high-temperature steam explosion treatment of the present invention on the protein conformation in walnut meal; where the horizontal axis represents wavelength (nm), the vertical axis represents absorbance, and the legend "Control" represents the blank group. Figure 2 It can be seen that the characteristic absorption peak near 300 nm changes after treatment, indicating the unfolding of the protein's three-dimensional conformation and the exposure of aromatic amino acid residues; when the water content is 40% and the pressure is 1.2 MPa (i.e., Figure 2 When the green line (corresponding to the parameters in Example 2) is in the range, the absorbance peak reaches its highest value, indicating that the protein conformation is most fully unfolded under this condition, which supports the test data of increased soluble protein content at the molecular level.

[0046] Furthermore, macromolecular cellulose and hemicellulose degrade under the influence of thermomechanical forces, such as... Figure 10As shown. These substances are further hydrolyzed into soluble oligosaccharides and monosaccharides, contributing to the increase in reducing sugar content, such as... Figure 11 As shown, the released small-molecule free sugars and water-soluble peptides provide carbon and nitrogen sources for the growth of microorganisms in the subsequent inoculation stage, demonstrating the feasibility of this pretreatment process in improving fermentation efficiency.

[0047] Test Example 2: This test aims to examine the physicochemical environment and enzyme secretion patterns during the fermentation process of Example 2.

[0048] Sampling points were set for day 0, day 5, day 13, and day 21 of the fermentation cycle. At each sampling point, 10.0 g of fermented mash was taken from the middle of the fermentation vessel, placed in a homogenizing cup, and 90 mL of 0.85% sterile physiological saline was added. The mixture was homogenized at 8000 rpm for 2 min. The homogenized liquid was transferred to a centrifuge tube and centrifuged at 4℃ and 5000 rpm for 15 min. The supernatant was collected. This supernatant was used as the pH determination solution and crude enzyme solution.

[0049] Using a precision pH meter calibrated with a standard buffer solution, immerse the electrode in the supernatant at room temperature and record the pH value of the system after the reading stabilizes.

[0050] The Folin-Ciocalteu method was used to determine protease activity. 1.0 mL of diluted crude enzyme solution was added to 1.0 mL of 1% casein solution (prepared with phosphate buffer at pH 7.2). After reacting precisely for 10 min in a 40°C water bath, 2.0 mL of 0.4 mol / L trichloroacetic acid solution was quickly added to terminate the reaction. After standing for 15 min, the supernatant was collected by centrifugation. 1.0 mL of the supernatant was then added sequentially to 5.0 mL of sodium carbonate solution and 1.0 mL of diluted Folin-Ciocalteu reagent, and the mixture was incubated at 40°C for 20 min. The absorbance was measured at 680 nm using a spectrophotometer. Enzyme activity was calculated based on the tyrosine standard curve. One unit of enzyme activity (U / g) was defined as the amount of enzyme that hydrolyzes casein to produce 1 microgram of tyrosine per minute.

[0051] Amylase activity was determined using the 3,5-dinitrosalicylic acid method. 1.0 mL of crude enzyme solution was mixed with 1.0 mL of a 1% (w / w) soluble starch solution and reacted at 40 °C for 15 min. After the reaction was complete, 2.0 mL of 3,5-dinitrosalicylic acid reagent was added to terminate the reaction, and the mixture was heated in a boiling water bath for 5 min for color development. After cooling to room temperature under running water, the mixture was diluted to volume, and the absorbance was measured at 540 nm. Quantification was performed using a maltose standard curve. One unit of amylase activity (U / g) was defined as the amount of enzyme that catalyzes the production of 1 mg of maltose from starch per minute.

[0052] Each test was performed in triplicate, and the average value of the results was taken.

[0053] Table 2. Dynamic changes in pH and enzyme activity during the fermentation cycle of Example 2 in conclusion: According to Table 2 and Figure 12 Dynamic changes in pH value of the system during the fermentation cycle Figure 13 Data from the dynamic changes in enzyme activity during the fermentation cycle. Figure 12 The figure shows the decreasing trend of pH value in the fermentation mash system over a total fermentation period of 21 days. The fermentation process is divided into three stages using vertical dashed lines: the lactic acid bacteria-dominated stage (days 0-5), the Aspergillus oryzae-dominated stage (days 5-13), and the yeast-dominated stage (days 13-21). On day 0 of fermentation, the system pH is near neutral, and the baseline enzyme activity is derived from the accumulation during the koji-making stage; good koji-making quality is a prerequisite for establishing high levels of initial enzyme activity. Figure 5 The effect of koji-making temperature and time on koji-making is shown in the figure. Under suitable temperature and time parameters (e.g., 32℃, 46h), spores germinate evenly on the surface of the koji material and the mycelium grows fully, avoiding the phenomenon of low-temperature greening or high-temperature burning of koji, and ensuring sufficient primary enzyme reserves before fermentation in the fermentation tank. The first 5 days of fermentation are dominated by Lactobacillus plantarum, and the temperature is controlled at 25℃. At this temperature, lactic acid bacteria produce a large amount of acid through metabolism, and the pH of the system decreases from 6.53 to 4.87. Figure 12 The solid black dots in the diagram visually reflect the rapid pH drop caused by lactic acid bacteria production in the early stages, followed by a stable acidic environment maintained in the later stages. This stage establishes a slightly acidic environment, inhibiting the growth of less acid-tolerant bacteria and providing a pH basis for the subsequent metabolism of *Aspergillus oryzae* and *Rhodotorula glutinis*. During this period, *Aspergillus oryzae* is in its growth adaptation phase, with a slow increase in protease and amylase activity.

[0054] Figure 13 The graph illustrates the rise and fall of protease and amylase activities within the same fermentation cycle. Solid lines and squares represent protease activity, while dashed lines and triangles represent amylase activity. The graph also marks the three temperature-controlled stages of fermentation. Days 5 to 13 are the Aspergillus oryzae-dominant period, with the temperature rising to 32°C, which is within the optimal temperature range for Aspergillus oryzae enzyme production. Figure 13 It was clearly revealed that under suitable temperature conditions (32℃) during the mid-term growth of Aspergillus oryzae, the secretion of both enzyme systems reached its peak. Aspergillus oryzae secreted extracellular enzymes, and the protease activity in the system reached a peak of 1845.3 U / g on day 13, while the amylase activity reached 689.7 U / g. The release of such high levels of enzyme activity depended on the scientific initial bacterial population ratio. Figure 3The effect of different strain ratios on protease and amylase in this invention shows that, under a specific inoculation ratio of the compound strains, the activities of protease and amylase can be synergistically maximized; excessive or insufficient yeast and lactic acid bacteria both antagonize and interfere with the enzyme production efficiency of Aspergillus oryzae. This parameter optimization provides an inoculation benchmark for the large-scale secretion of enzymes in this embodiment. Enzyme secretion promotes the conversion of macromolecular proteins in the walnut meal matrix into peptides and free amino acids, while starch is degraded into reducing sugars. The material completes the main hydrolysis process at this stage, the pH decrease slows down, and it is maintained at 4.62. The free amino acids and organic acids in the system form a buffer system.

[0055] From day 13 to 21, the yeast enters its dominant phase, and the temperature is adjusted to 40℃. Figure 13 It can be seen that during the later stage of yeast dominance (40℃), the fermentation mechanism exhibited a moderate decline in enzyme activity while maintaining a high catalytic level. High temperature limited the continued proliferation of *Aspergillus oryzae*, leading to a decrease in enzyme activity; protease and amylase levels dropped to 1532.8 U / g and 512.4 U / g, respectively. However, the system still maintained a high catalytic level to sustain the hydrolysis reaction. Under high temperature and the acidic environment accumulated in the early stages, *Saccharomyces rouxii* utilized small-molecule amino acids and reducing sugars for metabolism, producing alcohols, organic acids, and ester flavor compounds.

[0056] Experimental data confirmed the scientific validity of the segmented temperature-controlled fermentation logic. Segmented temperature control achieved an orderly alternation of pH reduction in the early stage of lactic acid bacteria, protein hydrolysis via enzyme secretion in the middle stage of Aspergillus oryzae, and flavor synthesis by yeast using hydrolysis products in the later stage. The variable temperature operation avoided the phenomenon of mixed strains competing for substrates or inhibiting each other's metabolites at a single temperature, proving the feasibility of coordinating the metabolic pathways of multiple strains through physical temperature control.

[0057] Test Example 3: This test measured the final product and fermentation residue of Example 2 and Comparative Examples 1, 2, and 4 to evaluate the impact of different process routes on the core nutritional indicators and substrate conversion rate of the target product.

[0058] Collect the raw walnut soy sauce products prepared in Examples 2, 1, 2, and 4, as well as the fermented soy sauce residue after pressing and separation. Record the initial dry weight of the walnut meal added to each group and the total volume of soy sauce produced. Wash the fermented soy sauce residue multiple times with water to remove soluble substances, and dry it in an electric heating forced-air drying oven at 105℃ until constant weight. Weigh the dry weight of the residue.

[0059] The amino acid nitrogen content was determined by formaldehyde titration. 5.0 mL of each group of soy sauce product was pipetted into a 100 mL volumetric flask and diluted to volume with deionized water. 20.0 mL of the diluted solution was transferred to a beaker, 60 mL of deionized water was added, and the pH of the system was titrated to 8.2 using a 0.05 mol / L sodium hydroxide standard titration solution. 10.0 mL of neutral formaldehyde solution was added, mixed well, and titrated further with the same sodium hydroxide standard titration solution until the pH reached 9.2. The volume of sodium hydroxide solution consumed after adding formaldehyde was recorded, and the amino acid nitrogen content (g / 100 mL) in the sample was calculated using the formula.

[0060] The Kjeldahl method was used to evaluate total nitrogen utilization. Walnut meal raw materials and finished soy sauce products from each group were digested at high temperature with sulfuric acid and a catalyst, followed by distillation and titration. The total nitrogen mass of the walnut meal and the total nitrogen mass of the finished soy sauce were calculated. Total nitrogen utilization is the ratio of the total nitrogen mass of the finished soy sauce to the total nitrogen mass of the raw materials.

[0061] The raw material utilization rate was calculated using the dry weight difference method. Based on the initial dry weight of walnut meal and the dry weight of drying residue obtained in step 1, the raw material utilization rate was defined as the ratio of the difference between the initial dry weight and the dry weight of the residue to the initial dry weight.

[0062] Each test was performed in triplicate, and the mean and standard deviation of the data were recorded.

[0063] Table 3. Results of determination of core nutritional indicators and raw material utilization rate of walnut soy sauce in the examples and different comparative examples in conclusion: According to Table 3, Figure 14 Results of amino acid nitrogen content determination in walnut soy sauce products of each group and Figure 15 The data on the total nitrogen utilization rate and walnut meal raw material utilization rate of each group of processes show that there are physicochemical differences in the nutritional conversion and raw material consumption of walnut soy sauce due to different process treatments. Figure 14 The figure shows a comparison of the core flavor and nutritional evaluation indicators of the final products of Example 2 and the comparative examples. The horizontal dashed line in the figure marks the lower limit benchmark of 1.25 g / 100 mL set in the claims. The height of the bar chart shows that only Example 2, which underwent segmented wet heat pretreatment and multi-strain synergistic fermentation, broke through this technical threshold. Comparative Example 1 used a single high-temperature cooking pretreatment, and its amino acid nitrogen content was 0.82 g / 100 mL, with a raw material utilization rate of 54.6%. Conventional cooking did not break through the dense cross-linked structure of cellulose and lignin in the cell wall of walnut meal. Nitrogenous substances such as proteins were encapsulated inside the cells, and the enzymes in the fermentation stage failed to form effective contact with the substrate, resulting in inhibited protein hydrolysis and the solidification of excess substrate in the residue.

[0064] Comparative Example 4 used a compound enzymatic hydrolysis method instead of moist heat pretreatment, achieving an amino acid nitrogen content of 1.14 g / 100 mL. The addition of cellulase and protease disrupted part of the cell wall and degraded surface proteins, but the penetration and diffusion of free enzyme macromolecules in the solid walnut meal matrix was limited, and the disruption of the internal structure was physically restricted, resulting in a total nitrogen utilization rate of only 76.2%. Example 2 employed a segmented moist heat pretreatment, where humidity adjustment combined with the physical and mechanical shear force generated by high-temperature steam explosion physically tore the cell wall at the structural level. This process reduced the crystallinity of cellulose, forcing endogenous macromolecular proteins to undergo spatial conformational unfolding and denaturation, exposing internal peptide bond interaction sites, thus increasing the substrate's sensitivity to enzymes, and achieving a raw material utilization rate of 83.2%.

[0065] Figure 15 The bar chart is a dual-index grouped bar chart. Dark gray bars represent the overall utilization of nitrogen sources by the fermentation system, while light gray bars represent the consumption and degradation of solid walnut meal raw materials. The horizontal dashed line marks the 80% process target red line. Data comparison shows that the composite modification technology of this scheme is significantly superior to the traditional single cooking (Comparative Example 1), single-strain fermentation (Comparative Example 2), and conventional compound enzymatic hydrolysis (Comparative Example 4) processes in overcoming the physical barriers of cell walls and improving substrate conversion efficiency. Data comparison of the fermentation stage shows that Comparative Example 2, using single Aspergillus oryzae fermentation, achieved an amino acid nitrogen content of 1.05 g / 100 mL. In the single-strain fermentation system, the conversion of proteins into peptides and amino acids depends on a single enzyme system, lacking dynamic buffering of environmental pH and positive synergy among metabolites of multiple strains. The hydrolysis reaction, accompanied by the accumulation of free amino acids, exhibits a negative feedback inhibition effect. Example 2, using multi-strain segmented temperature-controlled fermentation, increased the amino acid nitrogen content to 1.36 g / 100 mL, achieving a total nitrogen utilization rate of 86.1%. The early-stage acid-reducing effect of the microenvironment in *Lactobacillus plantarum*, the large-scale secretion of enzymes in the middle stage of *Aspergillus oryzae*, and the late-stage substrate consumption and ester synthesis in yeast form a progressive metabolic chain. The metabolic consumption in the later stages relieves the inhibitory effect of the products on hydrolytic enzymes, prolonging the overall system's efficient hydrolysis period. Physical structural modification combined with a multi-species segmented metabolic synergistic mechanism promotes the complete degradation of macromolecules in walnut meal, meeting the data requirements of ≥1.25% amino acid nitrogen and ≥80% raw material utilization in this application. To maximize the generation of amino acid nitrogen in actual preparation, precise control of the interaction relationships of various process variables is necessary. For example... Figure 7 As shown in the response surface methodology diagram of this invention, there are significant nonlinear interactions among parameters such as liquid-to-material ratio, fermentation time, koji-making time, and koji-making temperature. By locking the peak region of the three-dimensional surface through the response surface model, the optimal parameter domain for maximizing the conversion rate of amino acid nitrogen is precisely defined, providing systematic mathematical model support for achieving the aforementioned nutritional indicators.

[0066] Test Example 4: This test aims to examine the physicochemical characterization of the finished soy sauce prepared by the processes of Example 2 and Comparative Examples 2 and 3 in terms of sensory dimensions, and to quantitatively evaluate the influence of different fermentation systems on the flavor composition and macroscopic physical state of the target product.

[0067] A panel of 12 qualified food sensory evaluation professionals was selected. Blind tests were conducted in a standardized sensory evaluation laboratory with a constant temperature of 25°C and a relative humidity of 60%.

[0068] The finished product stock solutions prepared in Example 2, Comparative Example 2, and Comparative Example 3 were allowed to stand at room temperature for 24 hours. 30.0 mL of soy sauce samples from each group were measured and placed in identical transparent glass tasting cups. Each sample was coded with a three-digit random number, shuffled, and presented to the evaluators.

[0069] The evaluators independently scored the samples based on a 100-point quantitative scoring sheet. The scoring dimensions and weights were as follows: appearance (15 points, mainly assessing gloss and transparency), aroma (30 points, mainly assessing the fullness of soy sauce aroma and ester aroma and the absence of off-odors), taste (30 points, mainly assessing freshness, mellowness and residual astringency), body (15 points, mainly assessing fluid viscosity and suspended matter state), and color uniformity (10 points, mainly assessing pigment distribution and the presence or absence of layering).

[0070] During the test, evaluators rinsed their mouths with 35°C warm water and plain soda crackers between tasting different samples, resting for 3 minutes between each rinse. All rating sheets were collected, and the highest and lowest scores in each group were removed. The average and standard deviation of the remaining scores were used as the final evaluation result.

[0071] Table 4. Quantitative evaluation results of the overall sensory quality of the finished walnut soy sauce products in the examples and comparative examples. in conclusion: According to Table 4 and Figure 16 The data in the comparative chart of the overall sensory quality evaluation results of walnut soy sauce products in each group show that the differences in fermentation process parameters directly led to the differentiation of sensory dimensions of the finished walnut soy sauce products. Figure 16 A multi-index grouped bar chart is used to visually present the differences in scores between Example 2 (multi-strain segmented temperature control) and Comparative Example 2 (single-strain fermentation) and Comparative Example 3 (multi-strain constant temperature) in five core sensory dimensions: appearance, aroma, taste, texture, and color uniformity. In the legend, the dark gray bars represent Example 2, and standard deviation error bars are superimposed above the data to reflect the stability of the evaluation.

[0072] Based on the graphic comparison, Comparative Example 2, which uses a single Aspergillus oryzae fermentation, scored only 19.4 points for aroma and 22.3 points for taste, ranking last with a total score of 72.9 points. The single-strain fermentation system lacks the metabolic intervention of aroma-producing yeasts, preventing the free amino acids and sugars within the system from being further converted into complex alcohols and esters through biochemical pathways. This results in a thin soy sauce flavor, and the original beany flavor of walnut meal and the astringency produced by natural tannins are not masked or degraded. Macroscopically, this manifests as a lack of aroma complexity and a bland taste.

[0073] Comparative Example 3 employed a multi-strain, constant-temperature, non-segmented fermentation method. Although lactic acid bacteria and yeast were introduced, the total score only increased to 78.8 points. In a single-temperature environment lacking temperature gradient control, Aspergillus oryzae, lactic acid bacteria, and yeast engaged in substrate competition and metabolic antagonism. The disordered acid production of lactic acid bacteria prematurely lowered the pH of the system, inhibiting the release of neutral protease activity from Aspergillus oryzae, leading to incomplete protein hydrolysis. Simultaneously, yeast metabolism was slowed at suboptimal temperatures, even producing byproducts such as fusel oils. This was reflected in the data as the aroma (21.7 points) and appearance indicators not reaching ideal levels, with the system exhibiting a risk of turbidity and residual off-odors.

[0074] Example 2 demonstrated a significant advantage across all evaluation dimensions, particularly in aroma and flavor, which received the highest weighting. Example 2 employed multi-strain, staged, temperature-controlled fermentation, achieving high scores across all five sensory indicators, with a total score of 92.6. Furthermore, the proportion of the liquid medium was also a crucial physical factor determining the final product's texture. Figure 4 The radar chart showing the effect of different material-to-liquid ratios on walnut soy sauce in this invention demonstrates that a suitable material-to-liquid ratio can achieve a balance between the exchange of substances and the enrichment of flavor compounds in the fermentation mash. At a specific material-to-liquid ratio, indicators such as appearance, aroma, and taste achieve maximum radar coverage, providing a fundamental textural guarantee for obtaining a high overall sensory score in Example 2. *Lactobacillus plantarum* rapidly produces acid in the early low-temperature stage to create an antibacterial environment. *Aspergillus oryzae* secretes a large amount of protease in the middle suitable-temperature stage, hydrolyzing the large molecular proteins of walnut meal into flavor peptides and amino acids, providing the umami substrate required for the flavor dimension (flavor score 28.1). In the later stage, the temperature is raised to 40℃. At this temperature, *Saccharomyces roximatee* is metabolically active, utilizing the monosaccharides and amino acids accumulated in the earlier stage to conduct efficient esterification reactions, synthesizing abundant multi-pathway aromatic compounds, endowing the product with a rich soy sauce aroma and complex ester aroma (aroma score 27.8). Simultaneously, the Maillard reaction during fermentation is fully evolved in the segmented temperature system, resulting in a uniform distribution of melanoidins in the product, improving the appearance and color of the soy sauce. The orderly alternation of the above-mentioned physicochemical metabolic processes eliminated the deteriorated flavor of the walnut meal substrate, successfully demonstrating the outstanding technological value of the segmented temperature change process in reshaping the product flavor, removing the off-flavor of the walnut meal substrate, and enhancing the aroma of esters.

[0075] Test Example 5: This test examines the fermentation cycle, pollution resistance, and final product qualification rate of the processes in Examples 1-3 and Comparative Example 2 when scaled up to industrial scale.

[0076] The workshop is equipped with an effective volume of 5.0m³. 3 Temperature-controlled stainless steel sealed fermentation tanks were used. The feeding and inoculation were carried out on a scale-up basis according to the process parameters of Examples 1, 2, 3, and Comparative Example 2, with 10 batches of trial production conducted continuously for each process.

[0077] During the fermentation process, the amino acid nitrogen content of the fermented mash is sampled and tested at regular intervals every day. When the increase of two consecutive test results is less than 0.02g / 100mL and the absolute value reaches the preset terminal standard, it is determined that the fermentation endpoint has been reached, and the actual fermentation days of each batch are recorded.

[0078] Samples were taken at different stages of fermentation, and the plate count method was used to isolate and determine the contaminating microorganisms. The main focus was on detecting coliform bacteria and non-target yeasts and molds. If the total number of contaminating microorganisms at any sampling point exceeded the limit specified in the national standard, the fermentation batch was considered contaminated. The contamination rate was calculated for 10 batches.

[0079] By combining core physicochemical and sensory indicators such as amino acid nitrogen content, total acid, and sensory scores, the final product is comprehensively judged, the number of batches meeting the superior grade standard is counted, and the product qualification rate is calculated. The average value of the data from each batch is then analyzed.

[0080] Table 5. Results of Industrial Production Cycle and Stability Inhibition of Bacteria in conclusion: According to Table 5, Figure 17 Comparison chart of total fermentation cycle for industrial production of each group and Figure 18 Data from the dynamic distribution chart of contamination rate and product qualification rate in industrial production show that the multi-strain segmented temperature control process demonstrates significant efficiency in periodic control and microbial environment stability during industrial-scale production.

[0081] Figure 17 The differences in time spans required to reach the same fermentation physicochemical endpoints between Examples 1-3 and Comparative Example 2 are visually presented using single-index bar charts. Standard deviation error bars from multiple batches are superimposed above the bars. Combined with... Figure 17Comparing the graphs and data in Table 5, Comparative Example 2, using a single Aspergillus oryzae fermentation, showed an average fermentation cycle extended to 32.7 days. In the single-strain isothermal fermentation system, the hydrolysis rate of the walnut meal substrate by a single enzyme system was limited, and the slow substrate degradation prolonged the overall fermentation cycle. In contrast, the fermentation cycles of Examples 1 to 3 were stably controlled between 20.6 and 21.8 days, effectively reducing the conventional fermentation time of over 32 days to approximately 21 days, demonstrating the significant advantages of this technology in accelerating substrate hydrolysis and shortening industrial production line turnaround time. Figure 6 The radar chart illustrating the impact of different fermentation cycles on walnut soy sauce provided multi-dimensional quality verification for this production cycle setting. The radar chart data shows that when the fermentation cycle is controlled at around 21 days, key indicators such as amino acid nitrogen, umami flavor, and richness of the finished product all reach their peak values. A cycle that is too short results in incomplete biochemical substrate conversion, while a longer cycle easily leads to a decline in umami flavor or the accumulation of off-flavors. This chart establishes approximately 21 days as the optimal fermentation endpoint that balances biochemical quality and production turnover rate.

[0082] Figure 18 A dual Y-axis line chart structure is used to compare highly correlated proportional data. The solid line (marked with hollow squares) on the left side of the chart represents the contamination rate of miscellaneous bacteria during fermentation; the dashed line (marked with dark gray triangles) on the right side of the chart represents the pass rate of the final product. Figure 18 The data points trend is compared with the data in Table 5. The extended fermentation time in Example 2 further increased the probability of system exposure and environmental contamination. The lack of a rapid acid reduction mechanism in the early stage caused the pH value of the mash to remain in the slightly acidic to neutral range for a long time. This environment is very easy to breed putrefactive bacteria and wild yeast, resulting in a contamination rate of up to 14.2%, and the final product qualification rate was only 81.3%.

[0083] The data points and trends in Examples 1 to 3 clearly demonstrate that the examples, through the antimicrobial barrier constructed by lowering the pH in the early stages, successfully controlled the risk of contamination by miscellaneous bacteria in large-scale production within a safe threshold of 3%, while simultaneously ensuring a high-quality product yield of over 95%. The introduction of *Lactobacillus plantarum* at the initial stage of fermentation, combined with the 25°C low-temperature stage, promoted the rapid occupation of the ecological niche by lactic acid bacteria and the large-scale production of lactic acid. The biochemical process rapidly lowered the pH value of the system's microenvironment, forming a physicochemical barrier against the proliferation of miscellaneous bacteria. The acidic antimicrobial environment established in the early stages reduced the competitive pressure from miscellaneous bacteria in the later high-temperature fermentation stages, ensuring the purity of enzyme production by *Aspergillus oryzae* and aroma production by *Saccharomyces rouxii*.

[0084] Under the synergistic effect of the three microorganisms, a relay-style metabolic pathway is constructed within the fermentation system. The initial acidification alters the biochemical environment of the raw materials; the large-scale enzymatic hydrolysis by *Aspergillus oryzae* at suitable temperatures in the middle stage accelerates protein degradation and deamination; and the rapid consumption of hydrolysis intermediates by *Saccharomyces roxburghii* in the later stage breaks the product inhibition effect of the enzymatic reaction. The coupling of physical temperature control and microbial metabolism improves the conversion efficiency of the walnut meal substrate, enabling the biochemical reaction to reach dynamic equilibrium in a relatively short time. Experimental data confirms the technical benefits of this process in shortening production time, suppressing large-scale contamination losses, and improving the quality of the final product. From an engineering data perspective, the anti-interference stability and high economic efficiency of this scheme under mass production conditions are verified.

Claims

1. A method for preparing walnut soy sauce using segmented wet heat pretreatment combined with multi-strain fermentation, characterized in that, Includes the following steps: Walnut meal is placed in a dry heat equipment for dry heat treatment, the walnut meal after dry heat treatment is humidified, the humidified walnut meal is transferred to a high temperature steam explosion equipment for high temperature steam explosion treatment, and the walnut meal after high temperature steam explosion treatment is cooled by air cooling for later use. A mixed microbial culture consisting of Aspergillus oryzae, Saccharomyces rouxii, and Lactobacillus plantarum is inoculated into the air-cooled walnut meal. The walnut meal inoculated with the mixed microbial culture is placed in a koji-making machine for koji-making. During the koji-making process, the koji is turned over. After the koji-making is completed, fermentation koji is obtained. Salt water is added to the fermentation starter, and the fermentation starter with added salt water is placed in a fermentation container for segmented fermentation. The segmented fermentation includes a first stage fermentation, a second stage fermentation and a third stage fermentation. After the segmented fermentation is completed, soy sauce mash is obtained. The soy sauce mash is subjected to an oil-draining operation. The soy sauce concentrate obtained after the oil-draining operation is collected and allowed to settle to obtain a clear liquid. The clear liquid is filtered using a precision filtration device. The filtered liquid is then heated and sterilized. After cooling the heated and sterilized liquid, the walnut soy sauce product is obtained.

2. The method for preparing walnut soy sauce using segmented wet heat pretreatment combined with multi-strain fermentation according to claim 1, characterized in that, The temperature of the dry heat treatment is 105°C to 120°C, and the time of the dry heat treatment is 5 minutes to 10 minutes; The moisture content of the walnut meal after the humidification treatment is 38% to 42%. The pressure of the high-temperature steam explosion treatment is 0.6 MPa to 1.8 MPa; The walnut meal that has undergone high-temperature steam explosion treatment is cooled to room temperature by air cooling for later use.

3. The method for preparing walnut soy sauce using segmented wet heat pretreatment combined with multi-strain fermentation according to claim 2, characterized in that, The mass ratio of Aspergillus oryzae, Saccharomyces rouxii, and Lactobacillus plantarum is 1:0.5:0.3, and the total inoculum amount of the mixed strain is 0.5% of the total mass of walnut meal. The koji-making temperature is 30℃ to 34℃, the koji-making time is 40h to 50h, and the koji-turning operation is performed every 12h during the koji-making time.

4. The method for preparing walnut soy sauce using segmented wet heat pretreatment combined with multi-strain fermentation according to claim 3, characterized in that, The added brine has a mass fraction of 20%, and the mass ratio of the fermentation starter to the volume of the added brine is from 1 kg:1.0 L to 1 kg:2.0 L. The temperature for the first stage of fermentation is 25℃, and the fermentation time for the first stage is 5 days. The second stage of fermentation was carried out at a temperature of 32°C for 8 days. The temperature for the third stage of fermentation is 40°C, and the fermentation time for the third stage is 6 to 14 days.

5. The method for preparing walnut soy sauce using segmented wet heat pretreatment combined with multi-strain fermentation according to claim 4, characterized in that, The oil spraying operation was performed three times consecutively. The heat sterilization is carried out at 90°C for 15 minutes, and the filtered liquid after heat sterilization is cooled to room temperature to obtain the finished walnut soy sauce.

6. The method for preparing walnut soy sauce using segmented wet heat pretreatment combined with multi-strain fermentation according to claim 5, characterized in that, The dry heat treatment temperature is 110°C, and the dry heat treatment time is 10 minutes; The moisture content of the walnut meal after the humidification treatment is 40%. The pressure of the high-temperature steam explosion treatment is 1.2 MPa.

7. The method for preparing walnut soy sauce using segmented wet heat pretreatment combined with multi-strain fermentation according to claim 6, characterized in that, The koji-making temperature is 32℃, and the koji-making time is 45 hours. The mass ratio of the fermentation starter to the volume ratio of the added brine is 1 kg: 1.25 L; The third stage of fermentation takes 8 days.

8. The method for preparing walnut soy sauce using segmented wet heat pretreatment combined with multi-strain fermentation according to claim 5, characterized in that, The dry heat treatment temperature is 105°C, and the dry heat treatment time is 5 minutes; The moisture content of the walnut meal after the humidification treatment is 38%. The pressure of the high-temperature steam explosion treatment is 0.6 MPa.

9. The method for preparing walnut soy sauce using segmented wet heat pretreatment combined with multi-strain fermentation according to claim 8, characterized in that, The temperature for making the koji is 30℃, and the time for making the koji is 40 hours. The mass ratio of the fermentation starter to the volume ratio of the added brine is 1 kg: 1.0 L; The third stage of fermentation takes 6 days.

10. The method for preparing walnut soy sauce using segmented wet heat pretreatment combined with multi-strain fermentation according to claim 5, characterized in that, The dry heat treatment temperature is 120°C, and the dry heat treatment time is 10 minutes; The moisture content of the walnut meal after the humidification treatment is 42%. The pressure of the high-temperature steam explosion treatment is 1.8 MPa; The temperature for making the koji is 34℃, and the time for making the koji is 50 hours. The mass ratio of the fermentation starter to the volume ratio of the added brine is 1 kg: 2.0 L; The third stage of fermentation takes 14 days.

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