Flavor compensation fermentation preparation process of low-salt low-oil fermented soya beans

Abstract

The invention discloses a flavor compensation fermentation preparation process of low-salt and low-oil fermented soya beans, and relates to the technical field of fermentation processing of bean products. According to the process, the core metabolism function of a strain is activated by combining time sequence segmented inoculation of aspergillus oryzae, bacillus subtilis and lactobacillus plantarum with gradient concentration CO2 targeted regulation and control, and efficient hydrolysis of soybean protein and oriented synthesis of ester flavor substances are achieved; according to the present invention, the low-salt-content low-salt-content low-salt-content low-salt-content low-salt-content low-salt-content low-salt-content low-salt-content low-salt-content low-salt-content low-salt-content low-salt-content low-salt-content low-salt-content low-salt-content low-salt-content low-salt-content low-salt-content low-salt-content low-salt-content low-salt-content low-salt-content low- And the flavor of the finished product is retained for a long time by combining the composite aroma locking of the beta-cyclodextrin and the Arabic gum with the damp-proof and antibacterial effects of the octenyl succinic acid modified starch coating. The problems that the low-salt low-oil fermented soybeans are boring in fermentation flavor, infectious microbes are easy to breed and the storage stability is poor are effectively solved, and the finished product is low in salt content and oil content and excellent in amino acid nitrogen and total ester content.

Description

Technical Field

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

[0002] Doubanjiang (fermented black soybeans) is a traditional fermented soybean product in my country. With its unique fermented flavor and rich nutrition, it has become an important condiment in daily cooking. Its traditional fermentation process uses high salt and high oil as the core control method. High salt inhibits the growth of miscellaneous bacteria, and high oil locks in flavor substances. Although this can ensure the quality and storage stability of the finished product, the salt and oil content is generally too high, which goes against the modern consumer's demand for a healthy diet with low salt and low oil.

[0003] Existing technologies attempting to improve the fermentation process of fermented black beans by reducing salt and oil content often involve simply lowering the amount of salt and oil added without systematically optimizing the fermentation system for low-salt, low-oil environments. This leads to a series of technical challenges: First, the metabolic efficiency of microorganisms decreases under low-salt conditions, and there is a lack of targeted microbial regulation methods. Aspergillus oryzae protein hydrolysis and Bacillus subtilis's directional aroma production capabilities are significantly weakened, resulting in insufficient synthesis of core flavor compounds and a bland flavor in the finished product. Second, low salt cannot form an effective antimicrobial barrier, allowing miscellaneous bacteria to proliferate easily, and the acidification stability of the fermentation system is poor, making it difficult to guarantee the microbial safety of the finished product. Third, existing processes lack suitable osmotic pressure regulation strategies, and sudden changes in osmotic pressure can easily cause microbial stress and inactivation, further affecting fermentation efficiency and flavor fusion. Fourth, the design of flavor locking and moisture-proof antimicrobial processes is simplistic, allowing volatile flavor compounds to be rapidly lost during storage, and the dry soybean curds are highly hygroscopic, making them prone to mold and yeast growth, significantly shortening the shelf life.

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

[0005] The purpose of this invention is to provide a flavor-compensating fermentation preparation process for low-salt and low-oil fermented soybeans, in order to solve the technical problems that traditional fermented soybean fermentation processes, which are prone to bland flavor, easy growth of miscellaneous bacteria, and poor storage stability after modification from high-salt and high-oil to low-salt and low-oil, are prone to this issue.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a flavor-compensating fermentation preparation process for low-salt, low-oil fermented soybeans, comprising the following steps: Step 1: Raw material pretreatment Soak soybean raw materials in water containing calcium chloride, then place the soaked soybean raw materials in a high-pressure cooking device for pressure cooking, and obtain cooked soybean curd after cooling. Step 2: Preparing the starter culture The cooked soybean curd obtained in step one was inoculated sequentially with Aspergillus oryzae, Bacillus subtilis and Lactobacillus plantarum for segmented koji-making culture. Step 3: CO2 gradient control As the second step of segmented koji-making and cultivation proceeds, a gradient concentration of sterile CO2 is introduced into the koji-making system to form mature koji blanks; Step 4: Post-fermentation The mature koji obtained in step three is put into a low-salt compound brine to form a fermentation system. The fermentation system is first cultured under the initial osmotic pressure for osmotic adaptation of the strain, and then the osmotic pressure is gradually increased to the target value at a rate of 5-10 mOsm / (kg·12h). During the subsequent fermentation process, sterile nitrogen microbubbles are introduced for anaerobic deodorization. Anaerobic fermentation is carried out in two stages until the pH value of the fermentation system is 4.5-5.0, and the fermentation product is obtained. Step 5: Flavor Locking and Seasoning Add a compound flavor-locking agent of β-cyclodextrin and gum arabic to the fermentation product of step four for flavor encapsulation. After low-temperature spray drying, dry soybean curd is obtained. Spray the dry soybean curd with a modified starch moisture-proof and antibacterial coating, then add a low dose of oil and a low-salt compound seasoning agent for blending. After blending, it is subjected to low-temperature preheating and heat stabilization treatment, sterilized, and then cooled to room temperature by gradient to obtain the low-salt and low-oil fermented soybean product.

[0007] In this invention, raw material pretreatment provides high-quality cooked soybean curd with a loose structure and easily usable substrate for koji making; the synergistic effect of three-strain sequential segmented koji making and CO2 gradient regulation achieves directional hydrolysis of soybean protein, directional synthesis of core flavor substances, and preliminary acidification and antibacterial action; the post-fermentation stage, which combines a low-salt complex salt system with a slow increase in osmotic pressure, achieves inhibition of miscellaneous bacteria and full integration of flavor substances in a low-salt environment; sterile nitrogen microbubbles enhance fermentation stability and flavor purity through multiple functions such as anaerobic deodorization, system homogenization, and the construction of a strict anaerobic environment; and the complex aroma-locking and moisture-proof antibacterial flavoring solidify the flavor and protect the quality of the fermentation products, solving the technical problems of bland flavor, easy growth of miscellaneous bacteria, and poor storage stability in traditional low-salt and low-oil fermented soybeans.

[0008] Preferably, in step one: the amount of calcium chloride added is 0.01-0.05% of the soybean raw material mass, the soaking temperature is 20-35℃, the soaking time is 8-12h, and the amount of soaking water is 1.5-2.0 times the soybean raw material mass; the pressure of the pressurized cooking is 0.08-0.12MPa, the cooking time is 25-35min, and the temperature of the cooked soybean curd after cooling is controlled at 28-35℃.

[0009] In this invention, the addition of trace amounts of calcium chloride loosens the soybean cell wall structure, reducing the osmotic resistance of Aspergillus oryzae hydrolytic enzymes, and simultaneously enhances the metabolic activity of the microorganisms as a mineral element. Appropriate soaking temperature, humidity, time, and water volume ensure adequate water absorption by the soybeans, avoiding over-soaking leading to mushy texture or under-soaking resulting in uneven cooking. Pressure cooking at 0.08-0.12 MPa moderately denatures soybean protein and starch, converting them into small-molecule substrates easily utilized by the microorganisms. Cooling to 28-35°C after cooking matches the optimal initial temperature for Aspergillus oryzae inoculation, improving the colonization rate and initial metabolic activity of the microorganisms. The pretreated cooked soybean curds are highly compatible with the subsequent koji-making process in terms of structure, substrate, and temperature, directly improving the metabolic efficiency of the microorganisms during the koji-making stage.

[0010] Preferably, in step two: the inoculum size of *Aspergillus oryzae* is 0.8-1.2% of the cooked soybean curd mass, the culture temperature is 28-35℃, the relative humidity is 75-80%, and the culture time is 20-28 hours; the inoculum size of *Bacillus subtilis* is 0.4-0.8% of the cooked soybean curd mass, the culture temperature is 32-40℃, the relative humidity is 70-75%, and the culture time is 30-40 hours; the inoculum size of *Lactobacillus plantarum* is 0.1-0.5% of the cooked soybean curd mass, the culture temperature is 30-36℃, the relative humidity is 65-70%, and the culture time is 18-22 hours.

[0011] In this invention, *Aspergillus oryzae* is first inoculated and cultured, secreting a large amount of hydrolytic enzymes under suitable temperature and humidity to hydrolyze soybean macromolecular substrates into amino acids and reducing sugars, providing nutrients for subsequent microbial strains. *Bacillus subtilis* is inoculated after hydrolysis, activating aroma-producing enzyme systems and using small-molecule substrates to directionally synthesize ester flavor compounds. *Lactobacillus plantarum* is inoculated last, producing acid under mild temperature and humidity to achieve preliminary acidification and antibacterial effects, laying a stable pH foundation for post-fermentation. Precise control of the inoculation amount and culture time for each microbial strain avoids nutrient competition caused by metabolic overlap. The gradient adjustment of temperature and humidity is adapted to the metabolic needs of each microbial strain, allowing subsequent CO2 gradient control to act solely on a single strain without interference from multiple microorganisms, amplifying the targeted activation effect of CO2 on microbial metabolism.

[0012] Preferably, in step two: the Aspergillus oryzae, Bacillus subtilis, and Lactobacillus plantarum are activated before inoculation, and the number of spores or colonies in the activated inoculum reaches 10. 7 CFU / g and above.

[0013] In this invention, the specific activation parameters for Aspergillus oryzae, Bacillus subtilis, and Lactobacillus plantarum are as follows: Activation of Aspergillus oryzae: Czapek's medium, with sucrose, sodium nitrate, dipotassium hydrogen phosphate, potassium chloride, magnesium sulfate, ferrous sulfate, and agar as the main components, was used. The activation temperature was 28-30℃, and the culture was carried out in the dark for 48-72 hours. After activation, the number of spores in the culture solution reached 10. 7 CFU / g and above; Activation of Bacillus subtilis: LB medium with tryptone, yeast extract, and sodium chloride as the main components was used, with a pH of 7.0-7.2. The activation temperature was 35-37℃, and the culture was carried out on a shaker for 18-24 hours at a shaking rate of 180-200 r / min. After activation, the colony count of the inoculum reached 10. 7 CFU / g and above; Activation of *Lactobacillus plantarum*: MRS medium was used, with peptone, beef extract, yeast extract, glucose, sodium acetate, diammonium citrate, magnesium sulfate, manganese sulfate, Tween-80, and calcium carbonate as the main components. The pH was 6.2-6.6, and the activation temperature was 30-32℃. Anaerobic static culture was carried out for 24-36 hours until the colony count in the activated culture reached 10-1. 7 CFU / g and above.

[0014] Preferably, in step three: the sterile CO2 is filtered through a 0.2-0.3 μm filter membrane, and a CO2 concentration of 4-6% by volume is introduced during the Aspergillus oryzae culture stage at a rate of 0.3-0.7 m / s. 3 / h; During the Bacillus subtilis culture stage, CO2 with a volume concentration of 6-8% is introduced at a rate of 0.4-0.8m. 3 / h; During the Lactobacillus plantarum culture stage, CO2 with a volume concentration of 8-10% is introduced at a rate of 0.2-0.6m. 3 / h.

[0015] In this invention, sterile CO2 filtered through a 0.2-0.3μm filter membrane not only avoids the introduction of exogenous bacteria but also achieves targeted metabolic regulation of various microorganisms through gradient concentrations: 4-6% CO2 (by volume) in the *Aspergillus oryzae* stage activates hydrolytic enzyme activity, improving protein hydrolysis efficiency; 6-8% CO2 (by volume) in the *Bacillus subtilis* stage relieves product feedback inhibition of aroma-producing enzymes, promoting the synthesis of core ester flavor compounds; and 8-10% CO2 (by volume) in the *Lactobacillus plantarum* stage enhances acid production efficiency and strengthens initial acidification and antibacterial action. The CO2 introduction rate at each stage is matched with the mass transfer characteristics of the koji-making system, ensuring uniform CO2 distribution within the koji mold and avoiding uneven koji-making caused by localized concentration deviations. Sequential koji making provides a single, non-competitive target for CO2 gradient regulation, which in turn feeds back into sequential koji making, amplifying the core metabolic functions of each microbial species. The two work together to achieve the dual effects of directional metabolism and selective antibacterial activity during the koji making stage. Furthermore, the introduction of sterile CO2 and the acid production by Lactobacillus plantarum form a synergistic antibacterial effect, further reducing the risk of contaminating bacteria proliferation during the koji making stage.

[0016] Preferably, in step four: the low-salt composite brine is prepared from NaCl, KCl, and potassium citrate in a mass ratio of 3:1:0.5, wherein the total mass of NaCl, KCl, and potassium citrate accounts for 3.5-4.5% of the total mass of the low-salt composite brine, and the amount of low-salt composite brine added is 10-15% of the mass of the mature koji; the initial osmotic pressure of the fermentation system is 110-130 mOsm / kg, the osmotic pressure adaptation time of the strain is 10-12 h, and the target osmotic pressure is 140-160 mOsm / kg; the diameter of the sterile nitrogen microbubbles is 10-20 μm; the conditions for the two-stage anaerobic fermentation are: the first stage fermentation at 25-32℃ for 50-70 h, and the second stage fermentation at 20-25℃ for 7-9 days.

[0017] In this invention, the composite salt system of NaCl:KCl:potassium citrate = 3:1:0.5 ensures the basic osmotic pressure regulation under low salt conditions of 3.5-4.5%, while potassium citrate moderates the saltiness and reduces the ionic strength, thus alleviating the stimulation of osmotic pressure on the strain and providing a stable carrier for the slow increase of osmotic pressure. The initial osmotic pressure of 110-130 mOsm / kg and the adaptation culture of 10-12h allow the strain to gradually adapt to the low salt environment. The gradient slow increase rate of 5-10 mOsm / (kg·12h) avoids stress inactivation of the strain, while forming a mild osmotic pressure stress, which synergizes with the acidification and antibacterial effect of the previous koji making process to achieve highly efficient antibacterial effect under low salt conditions. The 10-20μm sterile nitrogen microbubbles, combined with a complex salt system, slow osmotic pressure rise, and two-stage fermentation, have the following functions: anaerobic deodorization can adsorb and remove fermentation off-flavors such as butyric acid and propionic acid, ensuring pure flavor; it also constructs a strictly anaerobic environment, replacing oxygen in the system and forming a nitrogen isolation membrane on the surface, forming a synergistic antibacterial effect with osmotic pressure stress and acidification inhibition, reducing the risk of aerobic bacteria growth under low salt conditions; furthermore, the nitrogen microbubbles prevent the settling of the fermented material and the stratification of the brine through gentle bubbling disturbance, ensuring uniform distribution of osmotic pressure, temperature, and nutrients, guaranteeing a slow osmotic pressure rise, and preventing localized microbial stress or uneven fermentation; simultaneously, it can accelerate the solid-liquid two-phase transfer of amino acids, reducing sugars, and ester flavor substances, solving the flavor stratification problem caused by slow molecular diffusion under low salt conditions; it also ensures the uniform distribution of lactic acid produced by *Lactobacillus plantarum*, avoiding taste defects caused by sudden drops in local pH; and it adsorbs gases such as CO2 produced by microbial metabolism and allows them to escape with the bubbling, maintaining a stable fermentation environment.

[0018] Preferably, in step five: the amount of β-cyclodextrin added to the composite aroma-locking agent is 0.3-0.7% of the mass of the mature koji, and the amount of gum arabic added is 0.1-0.2% of the mass of the mature koji. After addition, the mixture is stirred for 20-40 minutes until it is evenly dispersed. The inlet air temperature of the low-temperature spray drying is 45-55℃, the air velocity is 1.5-2.0m / s, the feeding speed is 8-12kg / h, and the moisture content of the dried soybean curd obtained after drying is controlled to be 12-14%.

[0019] In this invention, β-cyclodextrin encapsulates volatile flavor compounds through molecular cavities, while gum arabic forms a gel film on the surface of fermentation products. The two work synergistically to achieve double-layer protection of flavor compounds. The ratio of 0.3-0.7% β-cyclodextrin to 0.1-0.2% gum arabic ensures efficient flavor retention while avoiding excessive addition that could result in a sticky texture. Stirring for 20-40 minutes ensures uniform dispersion of the flavor-locking agent, guaranteeing uniform encapsulation and coating. Low-temperature spray drying at 45-55℃ removes moisture while preventing flavor compound decomposition caused by high temperatures. Controlling the airflow rate, feed rate, and moisture content to 12-14% maintains a loose structure in the dried soybean curd, facilitating subsequent adsorption and film formation of the modified starch coating while preventing excessive moisture absorption and mold growth. This achieves a synergistic process of flavor locking and drying.

[0020] Preferably, in step five: the modified starch moisture-proof and antibacterial coating is an octenyl succinic acid starch ester coating, and the spraying amount is 0.5-1.0% of the mass of the mature koji; the low-salt compound seasoning agent is composed of 0.2-0.6% edible vinegar, 0.1-0.5% white sugar, 0.05-0.1% disodium inosinate and 0.05-0.1% yeast extract by mass of fermentation product; the low-dose oil is soybean oil, and the addition amount is 1.5-2.5% of the mass of fermentation product; the stirring speed during the seasoning process is 25-35 r / min, the stirring time is 15-25 min, and the seasoning temperature is controlled at 20-35℃.

[0021] In this invention, the octenyl succinic acid starch ester coating is a hydrophobic coating. A spraying amount of 0.5-1.0% can form a dense and uniform film on the surface of dried soybean curd, which not only prevents external moisture from entering and avoids the decomposition of β-cyclodextrin inclusion complex due to moisture absorption, thus enhancing the long-lasting effect of the compound aroma, but also forms a physical barrier to inhibit the growth of miscellaneous bacteria. The low dose of soybean oil of 1.5-2.5% enhances the flavor profile of the finished product with low oil content. At the same time, the oil fills the pores of the soybean curd, further preventing the volatilization of flavor substances, thus forming a double guarantee of flavor preservation with the compound aroma. The low-salt compound seasoning agent harmonizes the taste with edible vinegar and white sugar, and the disodium nucleotide and yeast extract synergistically enhance the umami flavor, making up for the flavor shortcomings of low salt and low oil without increasing salt and oil content.

[0022] Preferably, in step five: the temperature of the low-temperature preheating and thermal stabilization treatment is 60-65℃, and the treatment time is 10-15 min; the gradient cooling is 25℃→15℃→5℃, with a cooling rate of 1.5-2.5℃ / min.

[0023] In this invention, a low-temperature preheating and heat stabilization treatment at 60-65℃ allows the flavor substances and seasonings to form a heat-stable complex without damaging the flavor substances and coating structure, thus avoiding flavor degradation caused by subsequent sterilization. At the same time, it makes the modified starch coating adhere more firmly to the surface of the soybean curd, preventing the coating from peeling off. Gradual cooling from 25℃ to 15℃ to 5℃, combined with a cooling rate of 1.5-2.5℃ / min, avoids condensation caused by a sudden drop in temperature in the finished product, prevents condensation moisture from damaging the coating structure and causing the soybean curd to absorb moisture, and ensures uniform internal temperature of the finished product, guaranteeing the stable retention of flavor substances and improving the shelf life stability of the finished product.

[0024] Preferably, in step one: the soybean raw material is yellow soybean or black soybean, and the soybean particle size is 4-10mm.

[0025] In this invention, both soybeans and black beans are high-quality raw materials for fermented soybean paste. Their protein and starch content is suitable for the metabolic needs of the microbial strains. Black beans can also enhance the nutritional value of the finished product through anthocyanins. The particle size range of 4-10mm ensures that the soybeans absorb water evenly during the soaking stage, avoiding the problem of overcooking due to excessively small particle size and undercooking or uneven cooking due to excessively large particle size. At the same time, this particle size ensures that the specific surface area of ​​the cooked soybean curd is moderate, which is conducive to the subsequent inoculation and colonization of microbial strains and the uniform mass transfer of CO2 in the koji-making system.

[0026] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention employs a time-sequential inoculation process using Aspergillus oryzae, Bacillus subtilis, and Lactobacillus plantarum to avoid nutrient competition and metabolic interference among the microbial strains. Combined with targeted regulation using gradient concentrations of CO2, this process activates the hydrolytic enzyme activity of Aspergillus oryzae, relieves the product feedback inhibition of aroma-producing enzymes in Bacillus subtilis, and enhances the acid production efficiency of Lactobacillus plantarum. This strengthens the conversion of soybean protein into amino acids and reducing sugars, as well as the targeted synthesis of ester flavor compounds. Simultaneously, the gentle disturbance of sterile nitrogen microbubbles during the post-fermentation process accelerates the diffusion and transfer of flavor compounds between the solid and liquid phases, resolving the flavor stratification problem caused by slow molecular diffusion under low-salt conditions. Ultimately, while strictly controlling the salt and oil content, this ensures the richness and harmony of the product's flavor.

[0027] 2. In the koji-making stage, the gradient CO2 introduction synergistically with the acid production of Lactobacillus plantarum, selectively inhibiting the proliferation of aerobic bacteria and laying a low-bacterial foundation for subsequent fermentation. In the post-fermentation stage, the low-salt environment constructed by the ternary complex salt system, combined with the gradual increase of the osmotic pressure gradient, forms a mild osmotic pressure stress, inhibiting the growth of osmotically intolerant bacteria. At the same time, the sterile nitrogen microbubble system replaces oxygen, constructing a strictly anaerobic environment, further blocking the metabolism of aerobic bacteria. In the finished product stage, the octenyl succinic acid starch ester coating forms a dense hydrophobic film on the surface of the soybean curd, physically blocking the invasion of external bacteria and the adsorption of moisture.

[0028] 3. In this invention, the ternary composite salt system composed of NaCl, KCl, and potassium citrate reduces the ionic strength of the system through potassium citrate, alleviating the stimulation of osmotic pressure on the microorganisms and providing a stable carrier for the gradual increase of osmotic pressure gradient. The combination of 10-12 hours of osmotic pressure adaptation culture for the microorganisms and a gradual increase rate of 5-10 mOsm / (kg·12h) avoids cell membrane damage and inactivation of metabolic enzymes caused by sudden changes in osmotic pressure, ensuring continuous and efficient fermentation metabolism. The floating disturbance of sterile nitrogen microbubbles prevents the settling of koji cakes and stratification of brine, allowing osmotic pressure, temperature, and nutrients to be evenly distributed within the system, solving the problems of easy fluctuation and low fermentation efficiency in low-salt fermentation systems.

[0029] 4. This invention utilizes β-cyclodextrin to encapsulate volatile flavor compounds within molecular cavities, while gum arabic forms a gel film on the surface of fermentation products, achieving double-layer protection for flavor compounds and reducing volatilization loss during storage. The hydrophobic properties of the octenyl succinic acid starch ester coating effectively block external moisture from entering, preventing the soybean curd from absorbing moisture and increasing water activity, and inhibiting the growth of molds, yeasts, and other spoilage bacteria. Low-temperature preheating and heat stabilization treatment allows flavor compounds and flavorings to form a heat-stable complex, and gradient cooling prevents condensation from damaging the coating structure. These multiple protections work together to delay the quality deterioration of the finished product during storage. Attached Figure Description

[0030] Figure 1 This is a bar chart comparing the amino acid nitrogen content of Examples 1-3 and Comparative Examples 1-6; Figure 2 This is a bar chart comparing the total ester content of Examples 1-3 and Comparative Examples 1-6; Figure 3 The bar chart shows the water activity comparison between Examples 1-3 and Comparative Examples 1-6; Figure 4 Line graphs showing the total ester retention rates of Examples 1-3 and Comparative Examples 1-6. Detailed Implementation

[0031] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] Example 1 This embodiment provides a flavor-compensating fermentation preparation process for low-salt, low-oil fermented soybeans, specifically including the following steps: Step 1: Raw material pretreatment Soybeans with a particle size of 4-6 mm, plump and free from mold and insect infestation were selected as soybean raw materials. The soybeans were soaked in water containing 0.03% calcium chloride by weight of the soybean raw materials. The soaking temperature was controlled at 28℃ and the soaking time was 10 hours. The amount of water used for soaking was 1.8 times the weight of the soybean raw materials. After soaking, the soybeans were placed in a high-pressure cooking device and pressure-cooked at 0.10 MPa for 30 minutes. After cooking, the soybeans were naturally cooled to 30℃ to obtain cooked soybean curd.

[0033] Step 2: Preparing the starter culture The cooked soybean curd obtained in step one was sequentially inoculated with Aspergillus oryzae, Bacillus subtilis, and Lactobacillus plantarum for segmented koji-making culture. All Aspergillus oryzae, Bacillus subtilis, and Lactobacillus plantarum were activated before inoculation, and the spore count or colony count of the activated inoculum reached 10. 7 CFU / g or higher; the inoculation amount of Aspergillus oryzae was 1.0% of the cooked soybean curd mass, and after inoculation, it was cultured at 30℃ and 78% relative humidity for 24h; after the Aspergillus oryzae culture was completed, Bacillus subtilis was inoculated into the system at an inoculation amount of 0.6% of the cooked soybean curd mass, and after inoculation, it was cultured at 35℃ and 72% relative humidity for 36h; after the Bacillus subtilis culture was completed, Lactobacillus plantarum was inoculated into the system at an inoculation amount of 0.3% of the cooked soybean curd mass, and after inoculation, it was cultured at 32℃ and 68% relative humidity for 20h.

[0034] Step 3: CO2 gradient control As the segmented koji-making and cultivation process proceeds in step two, a gradient concentration of sterile CO2 is introduced into the koji-making system. All sterile CO2 is filtered through a 0.25 μm filter membrane before being introduced, ultimately forming mature koji molds. During the Aspergillus oryzae cultivation stage, a 5% (by volume) concentration of CO2 is introduced into the koji-making system at a rate of 0.5 m / s. 3 / h; During the Bacillus subtilis culture stage, CO2 with a volume concentration of 7% was introduced into the koji-making system at a rate of 0.6m. 3 / h; During the Lactobacillus plantarum culture stage, CO2 with a volume concentration of 9% was introduced into the koji-making system at a rate of 0.4m. 3 / h.

[0035] Step 4: Post-fermentation The mature koji obtained in step three is added to a low-salt compound brine and thoroughly mixed to form a fermentation system. The low-salt compound brine is prepared by mixing NaCl, KCl, and potassium citrate in a mass ratio of 3:1:0.5, with the total mass of NaCl, KCl, and potassium citrate accounting for 4.0% of the total mass of the low-salt compound brine. The amount of low-salt compound brine added is 12% of the mass of the mature koji. The fermentation system is first cultured for 11 hours at an initial osmotic pressure of 120 mOsm / kg for osmotic pressure adaptation of the microorganisms. Then, the osmotic pressure of the fermentation system is gradually increased at a rate of 8 mOsm / (kg·12h) until the target osmotic pressure of 150 mOsm / kg is reached. During the entire post-fermentation process, sterile nitrogen microbubbles with a diameter of 15 μm are introduced into the system for anaerobic deodorization. At the same time, anaerobic fermentation is carried out in two stages until the pH value of the fermentation system is 4.8. The first stage is anaerobic fermentation at 28℃ for 60 hours, and the second stage is anaerobic fermentation at 22℃ for 8 days. After the fermentation is completed, the fermentation product is obtained.

[0036] Step 5: Flavor Locking and Seasoning A composite flavor-locking agent of β-cyclodextrin and gum arabic was added to the fermentation product obtained in step four for flavor encapsulation. The amount of β-cyclodextrin added was 0.5% of the mass of the mature koji, and the amount of gum arabic added was 0.15% of the mass of the mature koji. After addition, the mixture was stirred at 25 r / min for 30 min until the composite flavor-locking agent was evenly dispersed. Then, the system was placed in a spray drying device for low-temperature spray drying. The drying inlet air temperature was 50℃, the air velocity was 1.8 m / s, and the feeding rate was 10 kg / h. After drying, dried soybean curd was obtained, and the moisture content of the dried soybean curd was controlled to be 13%. An octenyl succinate starch ester coating was evenly sprayed onto the surface of the dried soybean curd at a spraying amount of 0.8% of the mass of the mature koji, forming a modified starch moisture-proof and antibacterial coating. Subsequently, a low dose of oil and a low-salt compound flavoring agent were added to the system for blending. The low dose of oil was food-grade soybean oil, and the amount added was 2.0% of the mass of the fermentation product. The oil was sterilized at 70℃ for 10 minutes and then cooled to 25℃ before being added. The low-salt compound seasoning consisted of 0.4% edible vinegar, 0.3% white sugar, 0.08% disodium inosinate, and 0.08% yeast extract by weight of the fermentation product. During the seasoning process, the stirring speed was 30 r / min, the stirring time was 20 minutes, and the seasoning temperature was controlled at 28℃. After blending, the system was subjected to low-temperature preheating and heat stabilization treatment at 62℃ for 12 minutes. Subsequently, the system was vacuum-packed with a vacuum degree of -0.090 MPa, a vacuuming time of 30 seconds, a sealing temperature of 120℃, and a sealing time of 5 seconds. After vacuum packaging, the system was sterilized by water bath pasteurization at 88℃ for 22 minutes. After sterilization, the system was gradually cooled in a gradient of 25℃→15℃→5℃ at a cooling rate of 2.0℃ / min until it reached room temperature, thus obtaining the low-salt, low-oil fermented black bean product.

[0037] Example 2 This embodiment provides a flavor-compensating fermentation preparation process for low-salt, low-oil fermented soybeans, specifically including the following steps: Step 1: Raw material pretreatment Black soybeans with a particle size of 6-8 mm, plump and free from mold and insect infestation were selected as soybean raw materials. The black soybeans were soaked in water containing 0.01% calcium chloride by weight of the soybean raw materials. The soaking temperature was controlled at 20℃ and the soaking time was 12 hours. The amount of water used for soaking was 1.5 times the weight of the soybean raw materials. After soaking, the black soybeans were placed in a high-pressure cooking device and pressure-cooked at 0.08 MPa for 35 minutes. After cooking, the soybeans were naturally cooled to 28℃ to obtain cooked soybean curd.

[0038] Step 2: Preparing the starter culture The cooked soybean curd obtained in step one was sequentially inoculated with Aspergillus oryzae, Bacillus subtilis, and Lactobacillus plantarum for segmented koji-making culture. All Aspergillus oryzae, Bacillus subtilis, and Lactobacillus plantarum were activated before inoculation, and the spore count or colony count of the activated inoculum reached 10. 7 CFU / g or higher; the inoculation amount of Aspergillus oryzae was 0.8% of the cooked soybean curd mass, and after inoculation, it was cultured at 28℃ and 75% relative humidity for 28h; after the Aspergillus oryzae culture was completed, Bacillus subtilis was inoculated into the system at an inoculation amount of 0.4% of the cooked soybean curd mass, and after inoculation, it was cultured at 32℃ and 70% relative humidity for 40h; after the Bacillus subtilis culture was completed, Lactobacillus plantarum was inoculated into the system at an inoculation amount of 0.1% of the cooked soybean curd mass, and after inoculation, it was cultured at 30℃ and 65% relative humidity for 22h.

[0039] Step 3: CO2 gradient control As the segmented koji-making and cultivation process proceeds in step two, a gradient concentration of sterile CO2 is introduced into the koji-making system. All sterile CO2 is filtered through a 0.20 μm filter before being introduced, ultimately forming mature koji molds. During the Aspergillus oryzae cultivation stage, a 4% (by volume) concentration of CO2 is introduced into the koji-making system at a rate of 0.3 m / s. 3 / h; During the Bacillus subtilis culture stage, CO2 with a volume concentration of 6% was introduced into the koji-making system at a rate of 0.4m. 3 / h; During the Lactobacillus plantarum culture stage, CO2 with a volume concentration of 8% was introduced into the koji-making system at a rate of 0.2m. 3 / h.

[0040] Step 4: Post-fermentation The mature koji obtained in step three is added to a low-salt compound brine and thoroughly mixed to form a fermentation system. The low-salt compound brine is prepared by mixing NaCl, KCl, and potassium citrate in a mass ratio of 3:1:0.5, with the total mass of NaCl, KCl, and potassium citrate accounting for 3.5% of the total mass of the low-salt compound brine. The amount of low-salt compound brine added is 10% of the mass of the mature koji. The fermentation system is first cultured for 10 hours at an initial osmotic pressure of 110 mOsm / kg for osmotic pressure adaptation of the microorganisms. Then, the osmotic pressure of the fermentation system is gradually increased at a rate of 5 mOsm / (kg·12h) until the target osmotic pressure of 140 mOsm / kg is reached. During the entire post-fermentation process, sterile nitrogen microbubbles with a diameter of 10 μm are introduced into the system for anaerobic deodorization. At the same time, anaerobic fermentation is carried out in two stages until the pH value of the fermentation system is 4.5. The first stage is anaerobic fermentation at 25℃ for 70 hours, and the second stage is anaerobic fermentation at 20℃ for 9 days. After the fermentation is completed, the fermentation product is obtained.

[0041] Step 5: Flavor Locking and Seasoning A composite flavor-locking agent of β-cyclodextrin and gum arabic was added to the fermentation product obtained in step four for flavor encapsulation. The amount of β-cyclodextrin added was 0.3% of the mass of the mature koji, and the amount of gum arabic added was 0.1% of the mass of the mature koji. After addition, the mixture was stirred at 20 r / min for 20 min until the composite flavor-locking agent was evenly dispersed. Then, the system was placed in a spray drying device for low-temperature spray drying. The drying inlet air temperature was 45℃, the air velocity was 1.5 m / s, and the feeding rate was 8 kg / h. After drying, dried soybean curd was obtained, and the moisture content of the dried soybean curd was controlled to be 12%. Octenyl succinate starch ester coating was evenly sprayed onto the surface of the dried soybean curd, with a spray amount of 0.5% of the mass of the mature koji, to form a modified starch moisture-proof and antibacterial coating. Subsequently, a low dose of oil and a low-salt compound flavoring agent were added to the system for blending. The low dose of oil was food-grade soybean oil, with an addition amount of 1.5% of the mass of the fermentation product. After sterilization at 65℃ for 12 minutes, the mixture was cooled to 20℃ and added. The low-salt compound seasoning consisted of 0.2% edible vinegar, 0.1% white sugar, 0.05% disodium inosinate, and 0.05% yeast extract by weight of the fermentation product. During the seasoning process, the stirring speed was 25 r / min, the stirring time was 15 minutes, and the seasoning temperature was controlled at 20℃. After mixing, the system was subjected to low-temperature preheating and heat stabilization treatment at 60℃ for 10 minutes. Subsequently, the system was vacuum-packed with a vacuum degree of -0.095 MPa, a vacuuming time of 25 seconds, a sealing temperature of 115℃, and a sealing time of 3 seconds. After vacuum packaging, the system was sterilized by water bath pasteurization at 85℃ for 20 minutes. After sterilization, the system was gradually cooled in a gradient of 25℃→15℃→5℃ at a cooling rate of 1.5℃ / min until it reached room temperature, thus obtaining the low-salt, low-oil fermented black bean product.

[0042] Example 3 This embodiment provides a flavor-compensating fermentation preparation process for low-salt, low-oil fermented soybeans, specifically including the following steps: Step 1: Raw material pretreatment Soybeans with a particle size of 8-10 mm, plump and free from mold and insect infestation are selected as soybean raw materials. The soybeans are soaked in water containing 0.05% calcium chloride by weight of the soybean raw materials. The soaking temperature is controlled at 35℃ and the soaking time is 8 hours. The amount of water used for soaking is 2.0 times the weight of the soybean raw materials. Then, the soaked soybeans are placed in a high-pressure cooking device and pressure-cooked at 0.12 MPa for 25 minutes. After cooking, the soybeans are naturally cooled to 35℃ to obtain cooked soybean curd.

[0043] Step 2: Preparing the starter culture The cooked soybean curd obtained in step one was sequentially inoculated with Aspergillus oryzae, Bacillus subtilis, and Lactobacillus plantarum for segmented koji-making culture. All Aspergillus oryzae, Bacillus subtilis, and Lactobacillus plantarum were activated before inoculation, and the spore count or colony count of the activated inoculum reached 10. 7 CFU / g or higher; the inoculation amount of Aspergillus oryzae was 1.2% of the cooked soybean curd mass, and after inoculation, it was cultured at 35℃ and 80% relative humidity for 20h; after the Aspergillus oryzae culture was completed, Bacillus subtilis was inoculated into the system at an inoculation amount of 0.8% of the cooked soybean curd mass, and after inoculation, it was cultured at 40℃ and 75% relative humidity for 30h; after the Bacillus subtilis culture was completed, Lactobacillus plantarum was inoculated into the system at an inoculation amount of 0.5% of the cooked soybean curd mass, and after inoculation, it was cultured at 36℃ and 70% relative humidity for 18h.

[0044] Step 3: CO2 gradient control As the segmented koji-making and cultivation process proceeds in step two, a gradient concentration of sterile CO2 is introduced into the koji-making system. All sterile CO2 is filtered through a 0.30 μm filter before being introduced, ultimately forming mature koji molds. During the Aspergillus oryzae cultivation stage, a 6% (by volume) concentration of CO2 is introduced into the koji-making system at a rate of 0.7 m / s. 3 / h; During the Bacillus subtilis culture stage, CO2 with a volume concentration of 8% was introduced into the koji-making system at a rate of 0.8m. 3 / h; During the Lactobacillus plantarum culture stage, CO2 with a volume concentration of 10% was introduced into the koji-making system at a rate of 0.6m. 3 / h.

[0045] Step 4: Post-fermentation The mature koji obtained in step three is added to a low-salt compound brine and thoroughly mixed to form a fermentation system. The low-salt compound brine is prepared by mixing NaCl, KCl, and potassium citrate in a mass ratio of 3:1:0.5, with the total mass of NaCl, KCl, and potassium citrate accounting for 4.5% of the total mass of the low-salt compound brine. The amount of low-salt compound brine added is 15% of the mass of the mature koji. The fermentation system is first cultured for 12 hours at an initial osmotic pressure of 130 mOsm / kg for osmotic pressure adaptation. Then, the osmotic pressure of the fermentation system is gradually increased at a rate of 10 mOsm / (kg·12h) until the target osmotic pressure of 160 mOsm / kg is reached. During the entire post-fermentation process, sterile nitrogen microbubbles with a diameter of 20 μm are introduced into the system for anaerobic deodorization. At the same time, anaerobic fermentation is carried out in two stages until the pH value of the fermentation system is 5.0. The first stage is anaerobic fermentation at 32℃ for 50 hours, and the second stage is anaerobic fermentation at 25℃ for 7 days. After the fermentation is completed, the fermentation product is obtained.

[0046] Step 5: Flavor Locking and Seasoning A composite flavor-locking agent of β-cyclodextrin and gum arabic was added to the fermentation product obtained in step four for flavor encapsulation. The amount of β-cyclodextrin added was 0.7% of the mass of the mature koji, and the amount of gum arabic added was 0.2% of the mass of the mature koji. After addition, the mixture was stirred at 30 r / min for 40 min until the composite flavor-locking agent was evenly dispersed. Then, the system was placed in a spray drying device for low-temperature spray drying. The drying inlet air temperature was 55℃, the air velocity was 2.0 m / s, and the feeding rate was 12 kg / h. After drying, dried soybean curd was obtained, and the moisture content of the dried soybean curd was controlled to be 14%. An octenyl succinate starch ester coating was evenly sprayed onto the surface of the dried soybean curd, with a spray amount of 1.0% of the mass of the mature koji, forming a modified starch moisture-proof and antibacterial coating. Subsequently, a low dose of oil and a low-salt compound flavoring agent were added to the system for blending. The low dose of oil was food-grade soybean oil, with an addition amount of 2.5% of the mass of the fermentation product. Soybean oil was sterilized at 75℃ for 8 minutes and then cooled to 30℃ before being added. The low-salt compound seasoning consisted of 0.6% edible vinegar, 0.5% white sugar, 0.1% disodium inosinate, and 0.1% yeast extract by weight of fermentation product. During the seasoning process, the stirring speed was 35 r / min, the stirring time was 25 min, and the seasoning temperature was controlled at 35℃. After blending, the system was subjected to low-temperature preheating and heat stabilization treatment at 65℃ for 15 min. Subsequently, the system was vacuum-packed with a vacuum degree of -0.075 MPa, a vacuuming time of 35 s, a sealing temperature of 125℃, and a sealing time of 7 s. After vacuum packaging, the system was sterilized by water bath pasteurization at 90℃ for 25 min. After sterilization, the system was gradually cooled in a gradient of 25℃→15℃→5℃ at a cooling rate of 2.5℃ / min until it reached room temperature, thus obtaining the low-salt, low-oil fermented soybean product.

[0047] Comparative Example 1 The only difference between this comparative example and Example 1 is that: in step two, the segmented sequential inoculation method was not used. Instead, Aspergillus oryzae, Bacillus subtilis, and Lactobacillus plantarum were mixed according to the inoculation amount in Example 1 and then inoculated into cooked soybean curd for joint culture. The culture time was the sum of the culture times of the three strains in Example 1.

[0048] Expected performance: Due to the intense competition for nutrients and ecological niches in the mixed culture of the three strains, the core metabolic functions of each strain cannot be fully utilized. Aspergillus oryzae has a decreased protein hydrolysis efficiency, Bacillus subtilis has a weakened directional aroma production ability, and Lactobacillus plantarum has a poor acidification and antibacterial effect. The risk of contaminant proliferation increases during the koji-making stage, the content of hydrolyzed substrates and flavor substances in the koji blanks is significantly reduced, the flavor integration of the subsequent fermentation system is insufficient, the final product has a weak soy sauce aroma, poor antibacterial effect, and is prone to contaminant exceedance.

[0049] Comparative Example 2 The only difference between this comparative example and Example 1 is that: no gradient CO2 control was performed in step three, and no CO2 was introduced into the system throughout the entire koji-making process.

[0050] Expected performance: Without the targeted regulation and selective antibacterial effect of CO2 during the koji-making stage, the activity of Aspergillus oryzae hydrolase cannot be activated, the product feedback inhibition of Bacillus subtilis aroma-producing enzyme cannot be relieved, and the acid production efficiency of Lactobacillus plantarum is reduced. At the same time, a large number of miscellaneous bacteria proliferate in the koji-making system, interfering with the metabolism of functional bacteria, significantly reducing the amount of flavor substances synthesized in the koji blank, and easily producing off-flavors from miscellaneous bacteria. The off-flavors cannot be effectively eliminated in the subsequent post-fermentation stage, resulting in a bland flavor and obvious off-flavors in the final product, and a significant decrease in the inhibition rate of miscellaneous bacteria.

[0051] Comparative Example 3 The only difference between this comparative example and Example 1 is that the low-salt composite brine in step four is a binary salt system prepared by mixing NaCl and KCl in a mass ratio of 3:1, without the addition of potassium citrate, and the total salt content is still 4.0%.

[0052] Expected performance: Without the harmonizing and complexing effect of potassium citrate, the saltiness of the fermentation system is monotonous, easily resulting in a bland saltiness and a bitter taste. At the same time, the increased ionic strength of the system stimulates the metabolism of the microorganisms, reduces the activity of residual enzymes, and decreases the efficiency of flavor fusion. The lack of complexation by potassium citrate also leads to a decrease in the stability of flavor substances, with some easily oxidized flavor substances decomposing, resulting in poor taste harmony in the final product, reduced retention of soy sauce aroma, and decreased stability of pH control in the fermentation system, which easily leads to uneven local acidification.

[0053] Comparative Example 4 The only difference between this comparative example and Example 1 is that: in step four, the osmotic pressure adaptation culture of the strain was not carried out, and the osmotic pressure of the fermentation system was increased from 120 mOsm / kg to the target value of 150 mOsm / kg in one go, without using a gradual increase method.

[0054] Expected performance: The sudden increase in osmotic pressure causes a strong stress response in the microorganisms, resulting in cell membrane dehydration and damage. The metabolic enzyme activity of the remaining functional bacteria is significantly deactivated, and even the microorganisms die. The subsequent fusion and synthesis of flavor substances almost stops, resulting in a weak flavor and lack of complexity in the finished product. At the same time, after the microorganisms are deactivated, the autonomous antibacterial ability of the fermentation system decreases, and miscellaneous bacteria can easily multiply, leading to problems such as mold and off-odors in the fermentation products, and reducing the microbial safety of the finished product.

[0055] Comparative Example 5 The only difference between this comparative example and Example 1 is that gum arabic was not added in step five, and only β-cyclodextrin was used as a single aroma-locking agent, with the amount added still being 0.5% of the mass of the mature koji.

[0056] Expected performance: Without the surface gel film coating effect of gum arabic, volatile flavor substances that rely solely on the cavity encapsulation of β-cyclodextrin molecules are easily released and volatilized from the inclusion complex during drying, sterilization, and subsequent storage, resulting in a significant decrease in flavor retention. The dried soybean curd surface, lacking gel film protection, also easily absorbs moisture from the air, causing the soybean curd to become soft and lose its texture, while accelerating the oxidative decomposition of flavor substances. Ultimately, the finished product suffers from rapid flavor loss during its shelf life, poor storage stability, and a significant reduction in the intensity of the soy sauce aroma.

[0057] Comparative Example 6 The only difference between this comparative example and Example 1 is that: in step five, the dried soybean curd was not sprayed with an octenyl succinic acid starch ester coating, and a modified starch moisture-proof and antibacterial coating was not formed.

[0058] Expected performance: The dried soybean curds lack a hydrophobic and dense coating for protection, resulting in significantly increased hygroscopicity. During storage, they easily absorb moisture from the air, leading to increased moisture content and water activity. This makes them more susceptible to the proliferation of bacteria, significantly increasing the risk of mold growth in the finished product and drastically shortening the shelf life. Furthermore, without a coating, the flavor compounds on the soybean curds are easily exposed to air and undergo oxidative decomposition, further exacerbating flavor loss. In addition, the soybean curds are prone to crusting and breakage during packaging and transportation, resulting in a decrease in the integrity of the finished product's shape and the firmness of its texture.

[0059] To compare the performance differences of the flavor-compensating fermentation preparation processes for low-salt, low-oil fermented soybeans provided in Examples 1-3 and Comparative Examples 1-6, the present invention provides the following experimental methods: For the fermented black soybean products prepared in Examples 1-3 and Comparative Examples 1-6, performance testing was conducted from three core dimensions: physicochemical indicators, microbiological indicators, and storage stability. All basic tests were performed on the third day after the finished product was prepared. The storage stability test was conducted again after the finished product was stored at room temperature in a sealed container for three months. The details are as follows: (I) Physicochemical index testing 1. Salt content: Referring to GB 5009.44-2022 National Food Safety Standard for Determination of Chloride in Food, the chloride content in the finished product was determined by silver nitrate titration and converted into salt content as NaCl to reflect the effect of the low-salt process. 2. Oil content: Referencing GB 5009.6-2025 National Food Safety Standard - Determination of fat in food, the first method Soxhlet extraction was used to determine the oil content, reflecting the effectiveness of the low-oil process. 3. Amino acid nitrogen: Determined by potentiometric titration according to GB 5009.235-2016 National Food Safety Standard - Determination of amino acid nitrogen in food, in g / 100g, reflecting the hydrolysis efficiency of soybean protein and indirectly reflecting the metabolic effect of the microorganisms during the koji-making stage; 4. Total ester content: Determined by titration according to GB / T 10345-2022 "Analytical Methods for Baijiu" (a type of Chinese liquor), unit mg / kg, reflecting the amount of synthesis of core flavor substances such as esters in fermented black soybeans; 5. pH value: A portable digital pH meter was used. The finished product and deionized water were mixed and homogenized at a mass ratio of 1:10. After standing, the supernatant was taken for measurement to reflect the acidification stability of the fermentation system.

[0060] (II) Microbial Indicator Detection The microbiological safety of the finished product was assessed according to GB 4789.23-2024, National Food Safety Standard for Microbiological Examination of Food - Sampling and Sample Processing of Soy Products. Specific indicators and methods are as follows: 1. Total bacterial count: Plate count method, unit CFU / g; 2. Coliform bacteria: Plate count method, unit MPN / g; 3. Molds and yeasts: Plate count method, unit CFU / g.

[0061] (iii) Storage stability testing All finished products were sealed and stored for 3 months under standard storage conditions of room temperature (25±2℃) and relative humidity (60±5%). Key indicators were then retested to evaluate storage performance. Testing items and methods: 1. Total bacterial count, molds and yeasts: The detection methods are the same as before, to assess microbial proliferation; 2. Water activity (Aw): Measured directly using a water activity meter. The lower the value, the lower the risk of moisture absorption and mold growth. ≤0.65 is the acceptable standard for storing soy products. 3. Total ester retention rate: Total ester content after storage / Total ester content before storage × 100%, reflecting the storage retention effect of core flavor compounds. Experimental data are as follows: Table 1. Results of Physicochemical Indicators

[0062] Table 2. Microbiological index test results (3 days after finished product preparation)

[0063] Table 3. Storage stability test results (after 3 months of sealed storage at room temperature)

[0064] Based on the experimental data in Tables 1-3 and Figures 1-4, it can be seen that the salt content of the finished products in Examples 1-3 was controlled at 3.5-4.0% and the oil content at 1.5-2.4%, which not only achieved low salt and low oil content, but also maintained excellent protein hydrolysis and flavor substance synthesis efficiency under these conditions. The amino acid nitrogen content reached 0.55-0.62 g / 100 g, the total ester content was 88.6-95.8 mg / kg, and the pH value was stable at 4.5-4.9, which highly matched the pH requirement at the fermentation endpoint, and the acidification stability of the fermentation system was good. The lack of a single core technology directly leads to significant deterioration of relevant physicochemical indicators. Specifically, Comparative Example 1 (mixed inoculation), Comparative Example 2 (no CO2 gradient control), and Comparative Example 4 (sudden increase in osmotic pressure) all severely inhibited soybean protein hydrolysis and the synthesis of ester flavor compounds, resulting in a significant decrease in amino acid nitrogen and total ester content. Comparative Example 3 (no potassium citrate added) not only reduced the total ester content but also caused the pH value to deviate to 5.0, affecting the acidification stability of the fermentation system and the retention of flavor compounds. Comparative Example 5 (no gum arabic) and Comparative Example 6 (no modified starch coating) had no significant impact on the physicochemical indicators of the early fermentation stage, indicating that the role of these two technologies is concentrated in the subsequent flavor lock-in and storage stability stages.

[0065] Microbiological testing results on the third day after product preparation showed that the microbiological safety of Examples 1-3 was far superior to that of the comparative examples. The total bacterial count was only 280-350 CFU / g, coliform bacteria <0.3 MPN / g, and mold and yeast <10 CFU / g, all far below the microbiological safety limits for soy products. This indicates that the time-sequential koji-making + gradient CO2 regulation of the present invention forms a synergistic effect with the fermentation inhibition achieved by the slow increase of osmotic pressure, thus inhibiting the proliferation of miscellaneous bacteria from both the source and the fermentation stage. In contrast, the lack of core processes in the comparative examples directly led to a large proliferation of miscellaneous bacteria, with the total bacterial count of Comparative Examples 1, 2, and 4 all exceeding 10 CFU / g. 4The CFU / g levels of mold and yeast were significantly elevated, and the coliform count exceeded the standard, indicating a substantial loss of the system's self-inhibiting ability. Comparative Example 3, which did not add potassium citrate, showed that although the total bacterial count and mold and yeast counts did not exceed the standard, they were significantly higher than those in the Example, demonstrating the stabilizing and auxiliary antibacterial effect of potassium citrate on the microenvironment of the fermentation system. The microbial indicators of Comparative Example 5, which did not have gum arabic, were basically the same as those in the Example, indicating that it had no direct antibacterial effect. However, Comparative Example 6, which did not have the modified starch coating sprayed, showed a significant increase in the total bacterial count and mold and yeast counts, indicating that the physical moisture-proof effect of the coating could initially inhibit the growth of miscellaneous bacteria in the early stages of preparation.

[0066] After 3 months of sealed storage at room temperature, the test results for Examples 1-3 showed no significant deterioration in any of the indicators. The total bacterial count only slightly increased to 350-420 CFU / g, and the mold and yeast counts remained below 10 CFU / g, indicating no risk of bacterial proliferation. The water activity remained stable at 0.61-0.63, below the acceptable storage limit of 0.65 for soy products, indicating an extremely low risk of moisture absorption and mold growth. The total ester retention rate reached 93-96%, with almost no loss of core flavor compounds, proving that the dual protection of the compound aroma-locking agent and modified starch coating effectively prevents the volatilization and oxidation of flavor compounds. In contrast, all comparative examples showed significant deterioration in their indicators during the storage period, with the total bacterial count of Comparative Examples 1, 2, 4, and 6 exceeding 10 CFU / g. 4 CFU / g, mold and yeast levels increased significantly, with Comparative Example 6 showing a water activity of 0.75. Moisture absorption led to the proliferation of miscellaneous bacteria, completely compromising microbial safety. The total ester retention rates of all comparative examples were below 82%, with Comparative Examples 2 and 6 at only 52% and 55% respectively. Comparative Example 5, without gum arabic, had a total ester retention rate of only 68%, indicating that the gel film coating of gum arabic is key to flavor locking, and the modified starch coating can further prevent the oxidation of flavor substances. The absence of both will cause rapid flavor loss during storage. Comparative Example 3 showed relatively mild deterioration in various indicators, but its total ester retention rate was only 82%, and its water activity reached 0.65, which was at the critical limit for storage compliance, resulting in a significantly shortened shelf life of the finished product.

[0067] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. A process for the preparation of a low salt, low oil tempeh flavor compensated fermentation, characterized by, Includes the following steps: Step 1: Raw material pretreatment Soak soybean raw materials in water containing calcium chloride, then place the soaked soybean raw materials in a high-pressure cooking device for pressure cooking, and obtain cooked soybean curd after cooling. Step 2: Preparing the starter culture The cooked soybean curd obtained in step one was inoculated sequentially with Aspergillus oryzae, Bacillus subtilis and Lactobacillus plantarum for segmented koji-making culture. Step 3: CO2 gradient control As the second step of segmented koji-making and cultivation proceeds, a gradient concentration of sterile CO2 is introduced into the koji-making system to form mature koji blanks; Step 4: Post-fermentation The mature koji obtained in step three is put into a low-salt compound brine to form a fermentation system. The fermentation system is first cultured under the initial osmotic pressure for osmotic adaptation of the strain, and then the osmotic pressure is gradually increased to the target value at a rate of 5-10 mOsm / (kg·12h). During the subsequent fermentation process, sterile nitrogen microbubbles are introduced for anaerobic deodorization. Anaerobic fermentation is carried out in two stages until the pH value of the fermentation system is 4.5-5.0, and the fermentation product is obtained. Step 5: Flavor Locking and Seasoning Add a compound flavor-locking agent of β-cyclodextrin and gum arabic to the fermentation product of step four for flavor encapsulation. After low-temperature spray drying, dry soybean curd is obtained. Spray the dry soybean curd with a modified starch moisture-proof and antibacterial coating, then add a low dose of oil and a low-salt compound seasoning agent for blending. After blending, it is subjected to low-temperature preheating and heat stabilization treatment, sterilized, and then cooled to room temperature by gradient to obtain the low-salt and low-oil fermented soybean product.

2. The flavor-compensating fermentation preparation process for low-salt, low-oil fermented soybeans according to claim 1, characterized in that, In step one: the amount of calcium chloride added is 0.01-0.05% of the soybean raw material mass, the soaking temperature is 20-35℃, the soaking time is 8-12h, and the amount of soaking water is 1.5-2.0 times the soybean raw material mass; the pressure of the pressurized cooking is 0.08-0.12MPa, the cooking time is 25-35min, and the temperature of the cooked soybean curd after cooling is controlled at 28-35℃.

3. The flavor-compensating fermentation preparation process for low-salt, low-oil fermented soybeans according to claim 1, characterized in that, In step two: the inoculation amount of *Aspergillus oryzae* is 0.8-1.2% of the cooked soybean curd mass, the culture temperature is 28-35℃, the relative humidity is 75-80%, and the culture time is 20-28h; the inoculation amount of *Bacillus subtilis* is 0.4-0.8% of the cooked soybean curd mass, the culture temperature is 32-40℃, the relative humidity is 70-75%, and the culture time is 30-40h; the inoculation amount of *Lactobacillus plantarum* is 0.1-0.5% of the cooked soybean curd mass, the culture temperature is 30-36℃, the relative humidity is 65-70%, and the culture time is 18-22h.

4. The flavor-compensating fermentation preparation process for low-salt, low-oil fermented soybeans according to claim 3, characterized in that, In step two, the Aspergillus oryzae, Bacillus subtilis and Lactobacillus plantarum are activated before inoculation, and the spore count or colony count of the activated strain liquid reaches 10 7 CFU / g or above.

5. The flavor-compensating fermentation preparation process for low-salt, low-oil fermented soybeans according to claim 1, characterized in that, In step three: the sterile CO2 is filtered through a 0.2-0.3 μm filter membrane, the Aspergillus oryzae culture stage is supplied with CO2 at a volume concentration of 4-6%, at a rate of 0.3-0.7 m 3 / h; the Bacillus subtilis culture stage is supplied with CO2 at a volume concentration of 6-8%, at a rate of 0.4-0.8 m 3 / h; the Lactobacillus plantarum culture stage is supplied with CO2 at a volume concentration of 8-10%, at a rate of 0.2-0.6 m 3 / h.

6. The flavor-compensating fermentation preparation process for low-salt, low-oil fermented soybeans according to claim 1, characterized in that, In step four: the low-salt compound brine is prepared by mixing NaCl, KCl, and potassium citrate in a mass ratio of 3:1:0.5, with the total mass of NaCl, KCl, and potassium citrate accounting for 3.5-4.5% of the total mass of the low-salt compound brine. The amount of low-salt compound brine added is 10-15% of the mass of the mature koji. The initial osmotic pressure of the fermentation system is 110-130 mOsm / kg, the osmotic pressure adaptation time of the inoculum is 10-12 h, and the target osmotic pressure is 140-160 mOsm / kg. The diameter of the sterile nitrogen microbubbles is 10-20 μm. The conditions for the two-stage anaerobic fermentation are: the first stage is fermentation at 25-32℃ for 50-70 h, and the second stage is fermentation at 20-25℃ for 7-9 days.

7. The flavor-compensating fermentation preparation process for low-salt, low-oil fermented soybeans according to claim 1, characterized in that, In step five: the amount of β-cyclodextrin added to the compound aroma-locking agent is 0.3-0.7% of the mass of the mature koji cake, and the amount of gum arabic added is 0.1-0.2% of the mass of the mature koji cake. After addition, stir for 20-40 minutes until uniformly dispersed; the inlet air temperature of the low-temperature spray drying is 45-55℃, the air velocity is 1.5-2.0m / s, the feeding speed is 8-12kg / h, and the moisture content of the dried soybean cake obtained after drying is controlled to be 12-14%.

8. The flavor-compensating fermentation preparation process for low-salt, low-oil fermented soybeans according to claim 7, characterized in that, In step five: the modified starch moisture-proof and antibacterial coating is an octenyl succinic acid starch ester coating, and the spraying amount is 0.5-1.0% of the mass of the mature koji; the low-salt compound seasoning agent is composed of 0.2-0.6% edible vinegar, 0.1-0.5% white sugar, 0.05-0.1% disodium inosinate and 0.05-0.1% yeast extract by mass of fermentation product; the low-dose oil is soybean oil, and the addition amount is 1.5-2.5% of the mass of fermentation product; the stirring speed during the seasoning process is 25-35 r / min, the stirring time is 15-25 min, and the seasoning temperature is controlled at 20-35℃.

9. The flavor-compensating fermentation preparation process for low-salt, low-oil fermented soybeans according to claim 8, characterized in that, In step five: the temperature of the low-temperature preheating and thermal stabilization treatment is 60-65℃, and the treatment time is 10-15 min; the gradient cooling is 25℃→15℃→5℃, with a cooling rate of 1.5-2.5℃ / min.

10. The flavor-compensating fermentation preparation process for low-salt, low-oil fermented soybeans according to claim 1, characterized in that, In step one: the raw material is yellow soybeans or black soybeans, and the soybean particle size is 4-10mm.

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