Preparation method of freeze-dried natto powder

Abstract

The invention belongs to the field of biological medicine, particularly relates to a preparation method of natto freeze-dried powder, and aims to solve the problems of low viable bacteria rate, unstable enzyme activity and poor raw material adaptability of the existing natto product. The method comprises the following steps: pretreating and weighing small soybeans or broken chickpeas, preparing a fermentation substrate containing a gradient carbon source (3.5% of glucose and 0.5-2% of maltooligosaccharide) and 2% of a nitrogen source, performing steaming sterilization at 115 DEG C, inoculating high-concentration bacillus subtilis, performing constant-temperature fermentation at 37 DEG C, turning over a tray, controlling the temperature, performing after-ripening at 4 DEG C to induce spore formation, and performing quick-freezing at-40 DEG C and vacuum freeze-drying to obtain the chickpeas. And finally performing low-temperature crushing to obtain freeze-dried powder of 80-120 meshes. The water content of the finished product is less than or equal to 3%, the viable count is greater than or equal to 9-power CFU / g of 1 * 10, the NK enzyme activity is greater than or equal to 2000FU / g, and the 12-month stability attenuation is less than 15%. The activity, flavor and industrial feasibility of the product are remarkably improved through carbon source synergy, accurate parameter control and a double-raw-material adaptation process.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and specifically relates to a method for preparing natto freeze-dried powder. Background Technology

[0002] With the deep integration of functional foods and bio-fermentation technology, natto and its derivatives, rich in nattokinase (NK) and probiotic active ingredients, show broad application prospects in areas such as thrombosis prevention and regulation of intestinal microecology. Traditional natto products are mostly distributed in wet or refrigerated forms, which have inherent drawbacks such as short shelf life, high transportation costs, and easy inactivation of active ingredients, making it difficult to meet modern consumers' comprehensive needs for portability, stability, and consistent efficacy. Especially in health intervention and clinical nutritional support scenarios, products need to maintain high bacterial survival rates and stable enzyme activity after long-term storage, which places higher demands on the synergistic optimization of drying processes and fermentation substrates.

[0003] However, existing freeze-dried natto powder preparation processes generally neglect the impact of carbon source ratio on fermentation kinetics, resulting in low Bacillus subtilis proliferation efficiency and significant fluctuations in viable cell count and NK enzyme activity in the final product. Simultaneously, conventional excipient systems lack cryoprotection mechanisms for cell membrane structure and extracellular enzyme conformation, easily leading to cell lysis and protein denaturation during pre-freezing and sublimation stages. Furthermore, fermentation substrates are often limited to a single soybean system, failing to fully explore the synergistic potential of other legume resources such as chickpeas in terms of amino acid composition and polysaccharide matrix, resulting in products with limited functional dimensions and sensory adaptability.

[0004] Therefore, there is a need for a freeze-dried natto powder that combines high viable bacteria retention rate, strong enzyme activity stability, and broad-spectrum raw material compatibility, as well as a precise control method for its preparation. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing freeze-dried natto powder, which can effectively solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for preparing natto freeze-dried powder includes the following specific steps: Step (1) Raw material pretreatment and weighing: Weigh the dry weight of small soybeans or crushed yellow chickpeas and record the material mass 1; Soak the small soybeans in water overnight and then wash and drain them, or soak the crushed yellow chickpeas in 0.5% sodium bicarbonate solution overnight and then wash and drain them, and weigh their wet weight and record the material mass 2; Step (2) Auxiliary material preparation and mixing: Dispense the wet material into containers at a volume of 175 grams per liter, add 2% nitrogen source, 3.5% carbon source such as glucose, and 0.5% to 2% oligomaltose additives, mix well, add 25% of the wet weight of purified water to the soybean system, and add 30% of the wet weight of purified water to the chickpea system; Step (3) Sterilization by cooking: Sterilize the mixture by cooking. After wrapping, steam at 115℃ for 30 minutes, cool to 55 to 60℃, and record the material mass 3; Step (4) Inoculation and fermentation: Inoculate Bacillus subtilis at a ratio of 5% of the wet weight of soybean system or 4% of the wet weight of chickpea system, and culture at 37℃ for 30 hours. When the culture reaches 18 hours, turn the tray and record the material temperature; Step (5) Low-temperature ripening: Place the fermented material in a 4℃ environment for ripening overnight, and determine the total number of colonies of fresh natto; Step (6) Pre-freezing and freeze-drying: Take the ripened material, weigh and record the fresh natto mass, place it in a pre-freezing device for rapid freezing, and then freeze-dry it; Step (7) Finished product collection and testing: Take out the freeze-dried sample, weigh and record the freeze-dried natto mass immediately, and after crushing, determine its total number of colonies and nattokinase NK enzyme activity.

[0007] Preferably, the particle size of the small soybeans in step (1) is 4 to 6 mm, and the particle size of the crushed yellow chickpeas is controlled at 2 to 4 mm, so as to ensure that the water penetrates evenly and the fermentation substrate has a loose structure during the cooking process, which is conducive to the colonization and metabolism of Bacillus subtilis.

[0008] Preferably, the amount of oligomaltose added in step (2) is 0.5%, 1% or 2%, which works synergistically with 3.5% of glucose and other carbon sources to form a gradient carbon source supply system. Oligomaltose acts as a slow-release carbon source to delay the depletion of carbon sources in the later stage of fermentation, while glucose acts as a fast-acting carbon source to promote the rapid proliferation of cells in the early stage. The ratio of the two has been experimentally verified to significantly improve NK enzyme activity and viable cell density.

[0009] Preferably, the nitrogen source in step (2) is yeast extract or soybean protein hydrolysate, and its addition amount is strictly controlled to 2% of the wet weight of the material, so as to provide the amino acid precursor required by Bacillus subtilis to synthesize nattokinase, and avoid excessive nitrogen source leading to the accumulation of by-products or insufficient nitrogen source leading to limited enzyme synthesis.

[0010] Preferably, in step (3), the moisture content of the cooked material is controlled at 60% to 65% for soybeans and 63% to 68% for chickpeas. This moisture content range has been experimentally determined to be the optimal range for fermentation. Too high a moisture content can lead to the growth of anaerobic bacteria, while too low a moisture content can restrict bacterial movement and nutrient diffusion.

[0011] Preferably, in step (4), the concentration of live bacteria in the Bacillus subtilis culture used for inoculation is not less than 1×10⁸ CFU per milliliter, and the inoculation operation is completed within 5 minutes after the material is cooled at 55 to 60°C in an aseptic environment to prevent contamination by other bacteria and ensure that the bacteria start metabolism within the optimal temperature window.

[0012] Preferably, in step (4), the turning operation is carried out by using a sterile stainless steel shovel to turn the material over from top to bottom. The turning depth covers the full thickness of the material layer. After turning, the material temperature rises by no more than 2°C to maintain a constant temperature fermentation environment and promote uniform oxygen distribution, so as to avoid local anaerobic conditions that inhibit NK enzyme synthesis.

[0013] Preferably, the post-ripening time in step (5) is 12 to 16 hours. This low-temperature stage enables nattokinase to complete post-translational modification and stabilize its spatial conformation, while inducing spore formation, laying the foundation for cell protection in the subsequent freeze-drying process.

[0014] Preferably, in step (6), the pre-freezing conditions are rapid freezing at -40°C for 2 hours to quickly reduce the core temperature of the material to below -30°C, forming a fine ice crystal structure and reducing mechanical damage to cell membranes and enzyme proteins; during the freeze-drying stage, the plate temperature is controlled at 25°C, the vacuum degree is 10 Pa, and the drying time is 24 hours to ensure that the water sublimates completely and the heat-sensitive components are not deactivated.

[0015] Preferably, in step (7), the pulverization of the freeze-dried sample is carried out using a low-temperature airflow pulverizer, with the feed temperature controlled at 0 to 5°C and the output particle size at 80 to 120 mesh. This particle size range ensures solubility while avoiding electrostatic agglomeration due to excessive fineness, which may affect subsequent formulation processes.

[0016] Preferably, the final moisture content of the natto freeze-dried powder is not higher than 3%, the total bacterial count is not less than 1×10⁹ CFU per gram, the NK enzyme activity is not less than 2000 FU per gram, and the three indicators are verified by accelerated stability test to have a decay rate of less than 15% within 12 months of sealed storage at 25℃.

[0017] Preferably, the method is applicable to a dual-raw material system of soybeans and chickpeas. The chickpea system is rich in arginine and lysine, which complement the soybean system in amino acids. After processing by the process of the present invention, the NK enzyme activity is increased by 18% to 22%, and the sensory score is improved by 1.5 to 2.0 points (out of 5), which significantly improves the flavor and functional dimensions of the product.

[0018] Compared with the prior art, the present invention has the following beneficial effects: High viable bacteria retention rate and strong enzyme activity stability By precisely controlling the ratio of oligomaltose and glucose as a composite carbon source (0.5% to 2% oligomaltose combined with 3.5% glucose), a gradient energy supply system is constructed, which significantly enhances the metabolic activity of Bacillus subtilis in the later stages of fermentation, ensuring that the total number of colonies in the freeze-dried powder product remains stable at over 1×10⁹ CFU per gram. At the same time, this carbon source combination forms a glassy protective layer during freeze-drying, effectively maintaining the spatial conformation of NK enzymes, with the enzyme activity of the finished product not less than 2000 FU per gram, and the activity decay rate during the 12-month storage period is less than 15%, which is far superior to the volatile performance of a single carbon source system.

[0019] Broad-spectrum raw material compatibility and functional synergistic effect This invention is the first to incorporate chickpeas into the preparation system of freeze-dried natto powder. The pretreatment (soaking in 0.5% sodium bicarbonate) and the water addition ratio (30%) were optimized to take into account the high fiber and high alkaline protein characteristics of chickpeas, and the efficient fermentation of chickpea matrix was successfully achieved. The synergistic effect of arginine in chickpeas and glutamic acid in soybeans not only improves the synthesis efficiency of NK enzyme, but also improves the bitter taste of the product and significantly enhances sensory compatibility, thus expanding the raw material boundaries and application scenarios of functional natto products.

[0020] Precise and controllable process parameters and industrial feasibility From raw material particle size, cooking moisture content, inoculation temperature to freeze-drying curve, this invention establishes a complete parameter control system. Quality recording points are set at each key node (material quality 1 to 3, fresh natto quality, freeze-dried natto quality) to ensure batch-to-batch consistency. All operation steps are completed on conventional food-grade equipment, without the need for special and expensive equipment. The single batch processing capacity can reach more than 100 kg, which has a good foundation for large-scale production and cost control advantages. Attached Figure Description

[0021] Figure 1 This is a flowchart of the overall technical solution of the present invention. Detailed Implementation

[0022] In a method for preparing freeze-dried natto powder, step (1) of raw material pretreatment and weighing includes the following sub-steps: First, weigh the dry weight of small soybeans or crushed yellow chickpeas, which is the material mass 1. The measurement accuracy is not less than 0.01 g. The weighing device is an electronic balance with a maximum weighing range of 10 kg and a minimum graduation value of 0.01 g to ensure data traceability. Then, soak the small soybeans in water overnight for 12 to 16 hours. The water temperature is controlled at 20 to 25°C to avoid protein denaturation caused by high temperature. After soaking, wash the soybeans three times with running water for at least 3 minutes each time to remove surface impurities and soluble salts. After washing, drain the soybeans immediately for 30 minutes. Drain the soybeans by gravity to ensure that there is no free water residue on the surface of the material. Alternatively, soak the crushed yellow chickpeas in a 0.5% sodium bicarbonate solution. Soak overnight in a precisely prepared solution with a pH between 8.0 and 8.5. The soaking time is also 12 to 16 hours. Sodium bicarbonate neutralizes the natural alkaline proteins in chickpeas, reducing their surface charge density and promoting water penetration during subsequent cooking. It also inhibits the activity of some endogenous proteases, preventing excessive degradation. After soaking, the chickpeas are washed using the same process as the soybean system. They are then drained immediately for 30 minutes to ensure no free water remains on the surface. Finally, the wet weight is measured, which is the material mass 2. The weighing accuracy is 0.01 grams. The weighing process must be carried out in a constant temperature and humidity environment, with the ambient temperature controlled between 20 and 25°C and the relative humidity between 50% and 60%, to minimize the impact of environmental factors on the weighing results. The value of material mass 2 is used to calculate the addition ratio of each additive and the amount of water to be added.

[0023] In step (1) above, the particle size range of the small soybeans is 4 to 6 mm. This particle size range is determined by sieving using a standard sieve with apertures of 3.0 mm, 4.0 mm, 5.0 mm, 6.0 mm, and 7.0 mm. The soybean sample is placed on the sieve and sieved by mechanical vibration. The proportion of material in each particle size range is recorded to ensure that more than 95% of the material particle size is within the range of 4 to 6 mm. This particle size design aims to balance cooking efficiency and the looseness of the fermentation substrate structure. If the particle size is too small, water will penetrate quickly during cooking but it is easy to form clumps. Larger particle sizes hinder oxygen diffusion, prolong cooking time, and are not conducive to microbial colonization. The crushed yellow chickpeas are crushed to a particle size of 2 to 4 mm, which is achieved by a hammer crusher with a speed of 1200 rpm and a feed rate of 50 kg / h. The crushed material is then screened by a vibrating screen to ensure that more than 95% of the material particles are within the range of 2 to 4 mm. This particle size design helps to improve cooking uniformity, while increasing the specific surface area, promoting the attachment and metabolism of Bacillus subtilis, and avoiding uneven internal fermentation due to excessively large particles.

[0024] In the above-mentioned method for preparing freeze-dried natto powder, step (2) of preparing and mixing excipients includes the following sub-steps: dispensing the wet material into a container at a rate of 175 grams per liter. The container is a high-temperature resistant glass beaker with a capacity of 1 liter. The material is borosilicate glass, which has good thermal stability and chemical inertness. The filling accuracy is controlled within ±1 gram to ensure batch-to-batch consistency. Adding an additive combination of 2% nitrogen source, 3.5% carbon source such as glucose, and 0.5% to 2% oligomaltose, wherein the nitrogen source is yeast extract or soybean protein hydrolysate, and its addition amount is strictly controlled to be 2% of the wet weight of the material. This provides the amino acid precursors required for Bacillus subtilis to synthesize nattokinase, avoiding excessive nitrogen sources that could lead to byproduct accumulation or insufficient nitrogen sources that could limit enzyme synthesis. The yeast extract has a protein content of no less than 50%, a balanced amino acid composition, and the peptides of soybean protein hydrolysate are mainly distributed between 2 and 10 amino acid residues, making them easily absorbed and utilized by microorganisms. The addition of 3.5% of a carbon source, such as glucose, serves as a readily available carbon source to promote rapid initial cell proliferation. This source is food-grade crystalline glucose with a purity of no less than 99.5% and good solubility. The addition of 0.5%, 1%, or 2% of oligomaltose, along with the 3.5% glucose and other carbon sources, serves as a complementary component. Synergistic effects form a gradient carbon source supply system, in which maltodextrin acts as a slow-release carbon source to delay carbon depletion in the later stages of fermentation. Its molecular weight ranges from 1000 to 5000 Daltons, composed of 2 to 10 glucose units, and has a slow hydrolysis rate, allowing for continuous release of monosaccharides in the mid-to-late stages of fermentation, maintaining the metabolic activity of the microorganisms. All additives are pre-dissolved in a small amount of purified water to form a homogeneous solution before being added to the wet material. The mixing process is carried out manually or with a low-speed magnetic stirrer, with the stirring speed controlled at 100 rpm and the stirring time at least 10 minutes, ensuring thorough mixing of the additives and wet material without clumping. After mixing... 25% of the wet weight of purified water was added to the soybean system, and 30% of the wet weight of purified water was added to the chickpea system. The purified water was deionized water with a conductivity of less than 1 microsiemens per centimeter. The addition process should be carried out slowly with stirring to prevent local concentrations from being too high. The final moisture content of the mixture was controlled at 60% to 65% for the soybean system and 63% to 68% for the chickpea system. This moisture content range was determined to be the optimal fermentation moisture range through experiments. Too high a moisture content can easily lead to the growth of anaerobic bacteria, while too low a moisture content will restrict cell movement and nutrient diffusion. The moisture content was determined by the oven drying method. The samples were dried at 105°C to constant weight, and the percentage of weight loss was calculated.

[0025] In step (2) above, the amount of oligomaltose added is 0.5%, 1%, or 2%, which is determined based on experimental verification results. Experimental groups 1, 2, and 3 correspond to oligomaltose addition amounts of 0.5%, 1%, and 2%, respectively, and are compared with control group 2 (no oligomaltose). The results show that with the increase of oligomaltose addition, the metabolic activity of the cells in the later stage of fermentation is significantly improved, the peak NK enzyme activity increases by 15% to 25%, and the viable cell density remains stable, indicating that oligomaltose plays a key role in the middle and later stages of fermentation, and its slowing effect is significant. The release properties effectively extend the metabolic cycle of the cells, avoiding metabolic stagnation caused by carbon source depletion. The nitrogen source is yeast extract or soybean protein hydrolysate, and its addition is strictly controlled at 2% of the wet weight of the material. This ratio was determined through multiple rounds of optimization experiments. When the nitrogen source addition is less than 1.5%, nattokinase synthesis is limited and enzyme activity decreases significantly. When the nitrogen source addition is more than 2.5%, byproducts such as acetic acid and lactic acid accumulate and increase, causing the pH value of the fermentation broth to drop too quickly and inhibiting cell growth. Therefore, 2% is the optimal balance point, which can meet the enzyme synthesis requirements while avoiding metabolic burden.

[0026] In the above-mentioned method for preparing freeze-dried natto powder, step (3) of steaming and sterilization includes the following sub-steps: wrapping the mixture with a breathable cotton cloth of 1 mm thickness and a porosity of 40% to 50% to ensure steam flow while preventing material leakage; the wrapping method is double-layer wrapping, with each layer wrapped at least three times and tied securely; then placing the wrapped material into a high-pressure steam sterilizer and steaming it at 115°C for 30 minutes. The sterilizer pressure is set to 1.0 bar, and the temperature control accuracy is ±0.5°C. During the steaming process, constant pressure and temperature are maintained to ensure complete sterilization of the material and kill potential bacteria and endogenous microorganisms. Enzymes; After cooking, immediately open the exhaust valve to slowly release the pressure and prevent the material from bursting or deforming due to a sudden drop in pressure; Cool to 55 to 60°C, using a combination of natural cooling and fan-assisted cooling. Initially, allow natural cooling, and once the temperature drops below 70°C, start the fan with a speed of 1 meter per second to ensure uniform cooling and avoid local overheating or undercooling; After cooling, immediately record the material mass 3. This mass is the total mass of the material after cooking, with a weighing accuracy of 0.01 grams. The value of material mass 3 is used for subsequent calculations of moisture changes and gas production during fermentation, and its trend reflects the cooking effect and material stability.

[0027] In step (3) above, the moisture content of the cooked material is controlled at 60% to 65% for soybeans and 63% to 68% for chickpeas. This moisture content is determined by the oven drying method. The sample is dried to constant weight at 105℃ and the weight loss percentage is calculated. The control of moisture content is crucial. Too high a moisture content (>70%) can easily lead to the growth of anaerobic bacteria, especially in the early stage of fermentation. Anaerobic conditions may promote the reproduction of harmful bacteria such as Clostridium, affecting product quality. Too low a moisture content (<55%) restricts cell movement and nutrient diffusion, resulting in uneven fermentation and slow cell growth. Therefore, 60% to 65% (soybeans) and 63% to 68% (chickpeas) are the optimal ranges. This range ensures sufficient moisture to support microbial metabolism and maintains appropriate ventilation, which is conducive to oxygen diffusion and carbon dioxide discharge.

[0028] In the above-mentioned method for preparing natto freeze-dried powder, step (4) inoculation and fermentation includes the following sub-steps: Bacillus subtilis is inoculated at a ratio of 5% wet weight of soybean system or 4% wet weight of chickpea system. The viable concentration of the Bacillus subtilis bacterial solution used for inoculation is not less than 1×10⁸ CFU per milliliter. The bacterial solution is prepared by a three-stage culture process of slant activation, liquid seed culture, and fermenter expansion. The activation stage is cultured at 28℃ for 24 hours, the seed culture stage is cultured at 37℃ for 12 hours, and the expansion stage is cultured at 37℃, pH value of 7.0 to 7.2, and dissolved oxygen concentration of not less than 30% for 18 hours to ensure that the bacteria are in the logarithmic growth phase and the viable concentration meets the standard. The inoculation operation is completed within 5 minutes after the material is cooled at 55 to 60℃ in an aseptic environment. The aseptic environment is verified by a clean bench. Currently, the wind speed is controlled at 0.45 meters per second, and ultraviolet light is used for 30 minutes for spatial sterilization. The inoculation tool is a sterile pipette with a disposable sterile tip to avoid cross-contamination. After inoculation, the material is immediately transferred to a constant temperature incubator. The incubator temperature is set at 37℃, with a fluctuation range not exceeding ±0.5℃, and the incubation time is 30 hours. During this period, the temperature is monitored every 2 hours to ensure stable constant temperature conditions. At 18 hours of incubation, a turning operation is performed. The turning operation is carried out using a sterile stainless steel spatula to turn the material layer from top to bottom, covering the entire thickness of the material layer. After turning, the material temperature rise should not exceed 2℃ to maintain a constant temperature fermentation environment and promote uniform oxygen distribution, avoiding local anaerobic conditions that could inhibit NK enzyme synthesis. The turning process must be completed in a sterile operating table. After turning, the container is immediately resealed to prevent the invasion of contaminating bacteria.

[0029] In step (4) above, the turning operation is carried out by using a sterile stainless steel shovel to turn the material layer from top to bottom. The turning depth covers the full thickness of the material layer. After turning, the material temperature rises by no more than 2°C. This operation aims to break the oxygen gradient inside the material layer, promote the diffusion of oxygen into the material, and avoid the formation of local anaerobic zones caused by oxygen consumption. Anaerobic zones will inhibit the synthesis of nattokinase because the expression of this enzyme is regulated by oxygen concentration. The turning depth needs to cover the full thickness to ensure that the bottom material can also obtain enough oxygen. After turning, the temperature rises by no more than 2°C to prevent abnormal bacterial metabolism or thermal damage caused by temperature rise. The temperature rise is monitored in real time by an infrared thermometer to ensure control accuracy.

[0030] In the above-mentioned method for preparing natto freeze-dried powder, step (5) low-temperature post-ripening includes the following sub-steps: placing the fermented material in a 4°C environment for overnight post-ripening, with a ripening time of 12 to 16 hours. This low-temperature stage promotes the completion of post-translational modification of nattokinase and stabilizes its spatial conformation, while inducing spore formation, laying the foundation for cell protection in the subsequent freeze-drying process; the post-ripening process is carried out in a refrigerator, with the temperature set at 4°C and the fluctuation range not exceeding ±0.5°C, and the humidity controlled at 85% to 90% to prevent water loss from the material surface; after the post-ripening is completed, the total number of colonies of fresh natto is immediately determined. The determination method is the dilution plating method, with the sample dilution factor from 10 to 10 to the power of negative one to 10 to the power of negative six. Three parallel samples are inoculated for each dilution. The culture medium is nutrient agar, and the culture conditions are 37°C for 24 hours. The counting range is 30 to 300 CFU per plate. The formula for calculating the total number of colonies is: in, Total bacterial count (CFU per gram) The number of colonies on the plate. Inoculation volume (ml). This formula, representing the dilution factor, is used to quantify the live bacteria density in fresh natto and is a key indicator for evaluating fermentation effectiveness.

[0031] In step (5) above, the post-ripening time is 12 to 16 hours. This time range is determined based on enzyme kinetic experiments. Nattokinase undergoes post-translational modification processes such as folding, disulfide bond formation, and glycosylation at low temperatures. These processes require a certain amount of time to complete. If the time is too short, the modification will be incomplete and the enzyme activity will be unstable. If the time is too long, it may lead to enzyme degradation or excessive spore maturation, affecting the subsequent freeze-drying effect. Therefore, 12 to 16 hours is the optimal window period. During this period, the enzyme activity reaches its peak and the spore formation rate reaches more than 80%, which provides a guarantee for cell protection during the freeze-drying process.

[0032] In the above-mentioned method for preparing natto freeze-dried powder, step (6) pre-freezing and freeze-drying includes the following sub-steps: weighing and recording the mass of fresh natto after ripening, the mass being the total mass of the material after ripening, with a weighing accuracy of 0.01 g, the mass of fresh natto being used to calculate the moisture loss rate during freeze-drying; then placing the material in a pre-freezing device for rapid freezing, the pre-freezing condition being rapid freezing at -40℃ for 2 hours, so that the core temperature of the material rapidly drops below -30℃, forming a fine ice crystal structure, reducing mechanical damage to cell membranes and enzyme proteins; the pre-freezing device is a low-temperature freezer, the refrigerant is R134a, the temperature control accuracy is ±0.5℃, the material is placed in an aluminum tray, the tray... The dimensions are 200 mm × 200 mm, with a thickness of 5 mm, to ensure uniform heat transfer. After pre-freezing, it is immediately transferred to a freeze dryer for freeze-drying. During the freeze-drying stage, the plate temperature is controlled at 25°C, the vacuum degree is 10 Pa, and the drying time is 24 hours to ensure complete sublimation of water and no deactivation of heat-sensitive components. The drying process is divided into three stages: the primary drying stage (0 to 12 hours), where the plate temperature is gradually increased from 25°C to 30°C and the vacuum degree is maintained at 10 Pa, mainly to remove free water; and the secondary drying stage (12 to 24 hours), where the plate temperature is maintained at 25°C and the vacuum degree is maintained at 10 Pa, mainly to remove bound water to ensure that the final moisture content does not exceed 3%.

[0033] In step (6) above, the pre-freezing condition is to freeze at -40℃ for 2 hours to rapidly reduce the temperature of the material center to below -30℃. This condition is monitored in real time by inserting a thermocouple into the center of the material to ensure that the temperature curve meets the requirements. Rapid cooling helps to form fine ice crystals and avoids mechanical damage to the cell membrane by large ice crystals, thereby improving the retention rate of live bacteria. In the freeze-drying stage, the plate temperature is controlled at 25℃, the vacuum degree is 10 Pa, and the drying time is 24 hours. This combination of parameters is optimized based on a thermodynamic model. Too high a plate temperature will cause enzyme protein denaturation, and too low a vacuum degree will result in a slow sublimation rate and a long drying time. Therefore, 25℃ and 10 Pa are the best balance point to ensure that the water is completely sublimated while retaining the heat-sensitive components to the maximum extent.

[0034] In the above-mentioned method for preparing freeze-dried natto powder, step (7) of finished product collection and testing includes the following sub-steps: taking out the freeze-dried sample and immediately weighing and recording the mass of the freeze-dried natto. This mass is the total mass of the material after freeze-drying, with a weighing accuracy of 0.01 grams. The difference between the mass of the freeze-dried natto and the mass of the fresh natto is the amount of moisture loss. The formula for calculating the moisture loss rate is: in, For moisture loss rate, For the quality of fresh natto, To assess the quality of freeze-dried natto, this formula is used to evaluate freeze-drying efficiency. The freeze-dried sample is then pulverized using a low-temperature airflow pulverizer. The feed temperature is controlled between 0 and 5°C, and the output particle size is 80 to 120 mesh. This particle size range ensures solubility while avoiding electrostatic agglomeration due to excessive fineness, which could affect subsequent formulation processes. After pulverization, the total bacterial count and nattokinase (NK) activity are immediately determined. The total bacterial count is determined using the same method as in step (5). The nattokinase (NK) activity is determined using the fibrin plate method. After sample dilution, fibrinogen solution is added, and after incubation for 30 minutes, the diameter of the hemolysis zone is measured. The enzyme activity unit is defined as the ability of each milligram of sample to hydrolyze 1 microgram of fibrinogen within 1 hour at 37°C. The enzyme activity calculation formula is: in, Enzyme activity (FU per gram). The diameter of the hemolytic zone is in millimeters. Incubation time (hours) The formula is used to quantify the catalytic activity of nattokinase, serving as a calibration coefficient.

[0035] In step (7) above, the pulverization of the freeze-dried sample is carried out using a low-temperature airflow pulverizer. The feed temperature is controlled between 0 and 5°C. This temperature is achieved through a liquid nitrogen cooling system to ensure that the material does not heat up during the pulverization process and to avoid the deactivation of heat-sensitive components. The output particle size is 80 to 120 mesh. This particle size is determined by sieving. Standard sieves are used with apertures of 80 mesh (0.177 mm), 100 mesh (0.149 mm), and 120 mesh (0.125 mm), respectively, to ensure that more than 95% of the material particle size is within this range. This particle size range ensures good solubility and avoids electrostatic agglomeration caused by excessive fineness, which would affect the stability of subsequent formulation processes.

[0036] In the above-mentioned method for preparing natto freeze-dried powder, the final moisture content of the natto freeze-dried powder is no higher than 3%, the total bacterial count is no less than 1×10⁹ CFU per gram, and the NK enzyme activity is no less than 2000 FU per gram. Accelerated stability testing verified that the attenuation rate of these three indicators was less than 15% within 12 months of sealed storage at 25℃. This stability test employed an accelerated aging method, with samples sealed and stored at 25℃ and 50% relative humidity. Samples were taken and tested every 3 months, including moisture content, total bacterial count, and NK enzyme activity. The results showed that the attenuation rate of each indicator was less than 15% within 12 months, demonstrating that the natto freeze-dried powder prepared by this invention has excellent long-term stability.

[0037] In the above-mentioned method for preparing natto freeze-dried powder, the method is applicable to a dual-raw material system of soybeans and chickpeas. The chickpea system, rich in arginine and lysine, forms an amino acid complementarity with the soybean system. After processing with the present invention, its NK enzyme activity increases by 18% to 22%, and its sensory score improves by 1.5 to 2.0 points (out of 5), significantly improving the product's flavor and functional dimensions. This sensory evaluation was conducted using a blind evaluation method, with 10 professional reviewers scoring the samples on aroma, taste, texture, bitterness, etc., on a 5-point scale. The results showed that the chickpea system sample scored significantly higher than the soybean system in bitterness, indicating a superior flavor.

[0038] In the above-mentioned method for preparing freeze-dried natto powder, the industrial feasibility of the method is reflected in the following aspects: from raw material particle size, cooking moisture content, inoculation temperature to freeze-drying curve, this invention establishes a complete parameter control system. Quality recording points (material quality 1 to 3, fresh natto quality, freeze-dried natto quality) are set at each key node to ensure batch-to-batch consistency. All operation steps are completed on conventional food-grade equipment, requiring no special or expensive equipment. Single batch processing capacity can reach over 100 kg, possessing a good foundation for large-scale production and cost control advantages. This process has been successfully applied to a 100 kg-level pilot production line, running continuously for 10 batches with a product qualification rate of 98%, proving its industrial applicability.

[0039] In the above-mentioned method for preparing natto freeze-dried powder, the beneficial effects of the method are reflected in the following: by precisely controlling the composite carbon source ratio of maltodextrin and glucose (0.5% to 2% maltodextrin combined with 3.5% glucose), a gradient energy supply system is constructed, which significantly enhances the metabolic activity of Bacillus subtilis in the middle and late stages of fermentation, so that the total number of colonies in the freeze-dried powder product is stabilized at more than 1×10⁹ CFU per gram; at the same time, this carbon source combination forms a glassy protective layer during the freeze-drying process, effectively maintaining the spatial conformation of NK enzyme, and the enzyme activity of the finished product is not less than 2000 FU per gram, and The activity decay rate during the 12-month storage period is less than 15%, which is far superior to the volatility performance of a single carbon source system. In addition, this invention is the first to incorporate chickpeas into the preparation system of natto freeze-dried powder, and optimizes the pretreatment (soaking in 0.5% sodium bicarbonate) and water addition ratio (30%) for its high fiber and high alkaline protein characteristics, successfully achieving efficient fermentation of chickpea matrix. The synergistic effect of arginine in chickpeas and glutamic acid in soybeans not only improves the synthesis efficiency of NK enzyme, but also improves the bitter taste of the product, significantly enhances sensory compatibility, and expands the raw material boundaries and application scenarios of functional natto products.

[0040] In the above-mentioned method for preparing freeze-dried natto powder, a specific application example of the method is as follows: Taking a soybean system as an example, take 100 grams of dry small soybeans, record material mass 1 as 100 grams, soak overnight, wash and drain, and weigh 150 grams of wet weight, record material mass 2 as 150 grams; dispense the wet material into 1-liter beakers at a rate of 175 grams per liter, add 2% (3 grams) of nitrogen source, 3.5% (5.25 grams) of carbon source such as glucose, and 1% (1.5 grams) of oligomaltose, mix well, add 25% (37.5 grams) of purified water (25% of the wet weight) to the wet material, mix well, and then wrap; after wrapping, place in a high-pressure steam sterilizer, steam at 115°C for 30 minutes, cool to 55-60°C, and record material mass 3 as 190 grams; inoculate with Bacillus subtilis at a ratio of 5% of the wet weight, i.e., inoculate with 7.5 grams of bacterial solution, with a viable bacterial concentration of 1×10⁸ times. The inoculation was performed at 37°C for 30 hours, with each milliliter of CFU added. After inoculation, the mixture was incubated at 37°C for 18 hours, at which point it was turned over, ensuring the temperature rise did not exceed 2°C. After incubation, the mixture was placed in a 4°C environment for overnight ripening (14 hours). The total bacterial count of the fresh natto was measured to be 1.2 × 10¹⁰ CFU per gram. The ripened material was weighed (185 grams of fresh natto) and quickly frozen at -40°C for 2 hours. It was then freeze-dried at 25°C under a vacuum of 10 Pa for 24 hours. The freeze-dried sample was immediately weighed (45 grams of freeze-dried natto). The sample was then pulverized using a low-temperature airflow mill to achieve a particle size of 100 mesh. The total bacterial count was measured to be 1.1 × 10⁹ CFU per gram, and the NK enzyme activity was 2100 FU per gram. The final moisture content was 2.8%, meeting the technical requirements of this invention.

[0041] In the above-mentioned method for preparing freeze-dried natto powder, another specific application example of the method is as follows: Taking the chickpea system as an example, take 100 grams of dry weight of crushed yellow chickpeas, record material mass 1 as 100 grams, soak them in 0.5% sodium bicarbonate solution overnight, wash and drain them, and weigh them to obtain a wet weight of 160 grams, record material mass 2 as 160 grams; dispense the wet material into 1-liter beakers at a rate of 175 grams per liter, and add 2% (i.e., 3.2 grams) of nitrogen source. 3.5% (5.6g) of carbon source such as glucose and 1% (1.6g) of maltodextrin were mixed. Then, 48g of purified water (30% of the wet weight) was added to the wet material, mixed thoroughly, and then wrapped. The wrapped material was placed in a high-pressure steam sterilizer and steamed at 115℃ for 30 minutes. After cooling to 55-60℃, the material mass was recorded as 210g. Bacillus subtilis was inoculated at a ratio of 4% of the wet weight, i.e., 6.4g of bacterial solution, with a viable cell concentration of... The concentration of natto was 1 × 10⁸ CFU per milliliter. After inoculation, the natto was incubated at 37°C for 30 hours. After 18 hours of incubation, the natto was turned over, and the temperature rise after turning should not exceed 2°C. After incubation, the natto was placed in a 4°C environment for overnight ripening for 14 hours. After ripening, the total bacterial count of the fresh natto was measured to be 1.3 × 10¹⁰ CFU per gram. The ripened material was weighed, and the fresh natto weighed 205 grams. It was then placed in a pre-freezing device and quick-frozen at -40°C for 2 hours. Subsequently, it was freeze-dried at a plate temperature of 25°C, a vacuum degree of 10 Pa, and a drying time of 24 hours. The freeze-dried sample was weighed immediately, and the freeze-dried natto weighed 50 grams. It was then pulverized by a low-temperature airflow pulverizer, and the output particle size was 100 mesh. The total bacterial count was measured to be 1.2 × 10⁹ CFU per gram, the NK enzyme activity was 2300 FU per gram, and the final moisture content was 2.5%, which meets the technical requirements of this invention.

[0042] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a freeze-dried powder of natto, characterized by: Comprise the following specific steps: Step (1) raw material pretreatment and weighing: the dry weight of small yellow beans or broken yellow chickpeas is weighed respectively, and the material quality 1 is recorded; the small yellow beans are soaked overnight and then washed and drained, or the broken yellow chickpeas are soaked overnight with 0.5% sodium bicarbonate solution and then washed and drained, and then the wet weight is weighed respectively, and the material quality 2 is recorded; Step (2) preparation and mixing of auxiliary materials: the wet material [1] soaked in step (1) is divided into containers at a loading of 175 grams per liter, and 2% nitrogen source, 3.5% carbon source such as glucose, and 0.5% to 2% oligomaltose additive combination are added, mixed, then 25% purified water of the wet weight of the soybean system is added, and 30% purified water of the wet weight of the chickpea system is added; Step (3) sterilization by boiling: after the mixture is wrapped, it is boiled at 115℃ for 30 minutes, cooled to 55-60℃, and the material quality 3 is recorded; Step (4) inoculation and fermentation: Bacillus subtilis is inoculated at a ratio of 5% of the wet weight of the soybean system or 4% of the wet weight of the chickpea system, incubated at 37℃ for 30 hours, and the material temperature is recorded, wherein the culture is turned over at 18 hours; Step (5) low-temperature ripening: the fermented material is placed in a 4℃ environment for overnight ripening, and the total number of colonies of fresh natto is determined; Step (6) pre-freezing and freeze-drying: the ripened material is weighed to record the fresh natto quality, placed in a pre-freezing device for rapid freezing, and then subjected to freeze-drying treatment; Step (7) product collection and detection: the freeze-dried sample is immediately weighed to record the freeze-dried natto quality, crushed, and then the total number of colonies and natto kinase NK enzyme activity are determined.

2. The method for preparing natto freeze-dried powder according to claim 1, characterized in that: In step (1), the particle size of the small yellow beans is 4-6 mm, and the broken yellow chickpeas are broken to a particle size of 2-4 mm. The particle size and particle size are ensured to meet the range of 95% or more by standard screen sieving to optimize the boiling permeability and fermentation substrate porosity, promote Bacillus subtilis colonization and metabolic efficiency.

3. The method for preparing natto freeze-dried powder according to claim 1, characterized in that: In step (2), the amount of oligomaltose added is 0.5%, 1% or 2%, and 3.5% glucose cooperates to form a gradient carbon source system, wherein oligomaltose as a slow-release carbon source delays carbon source depletion in the late fermentation period, and glucose as an immediate-acting carbon source drives initial bacterial proliferation. The ratio of the two can significantly increase the NK enzyme activity peak by 15-25% and stabilize the viable bacterial density.

4. The method for preparing natto freeze-dried powder according to claim 1, characterized in that: In step (2), the nitrogen source is yeast extract or soybean protein hydrolysate, and the amount added is strictly controlled at 2% of the wet weight of the material. The protein content of the yeast extract is not less than 50%, and the soybean protein hydrolysate has a peptide length distribution of 2-10 amino acid residues, which is used to provide amino acid precursors required for natto kinase synthesis and avoid accumulation of by-products or limited enzyme synthesis.

5. The method for preparing natto freeze-dried powder according to claim 1, characterized in that: In step (3), the moisture content of the boiled material is controlled at 60-65% for the soybean system and 63-68% for the chickpea system. The control range of the moisture content of the material [2] is determined and regulated by oven method. Too high moisture content can easily cause the growth of anaerobic miscellaneous bacteria, and too low moisture content can limit bacterial movement and nutrient diffusion, ensuring the optimal fermentation moisture environment.

6. The method for preparing natto freeze-dried powder according to claim 1, characterized in that: The live bacteria concentration of the Bacillus subtilis used in the inoculation in step (4) is not less than 1×10 8 CFU per milliliter, the inoculation operation is completed within 5 minutes after the material is cooled to 55-60℃ in a sterile environment, the inoculation tool is a sterile pipette gun, and the inoculation is immediately transferred to a constant temperature incubator with a temperature fluctuation of not more than ±0.5℃.

7. The method for preparing natto freeze-dried powder according to claim 1, characterized in that: The turning operation in step (4) uses a sterile stainless steel shovel to turn up and down the full thickness, and the material temperature rises by not more than 2℃ after turning, which breaks the oxygen gradient, promotes internal ventilation, prevents local anaerobic inhibition of NK enzyme synthesis, and is completed in a sterile operation table and monitored by real-time infrared temperature measurement.

8. The method for preparing natto freeze-dried powder according to claim 1, characterized in that: The ripening time in step (5) is 12-16 hours, and step (5) stage [3] is carried out at 4℃ and 85%-90% humidity to induce the formation rate of Bacillus spores to more than 80% for subsequent freeze-drying and provide a cell structure protection basis.

9. The method for preparing natto freeze-dried powder according to claim 1, characterized in that: The pre-freezing condition in step (6) is to freeze the material at -40℃ for 2 hours to reduce the center temperature to below -30℃, and the freeze-drying stage controls the plate temperature at 25℃, the vacuum degree at 10 Pa, and the drying time at 24 hours to remove free water and bound water, ensuring that the final moisture content is not higher than 3% and the heat-sensitive ingredients are not inactivated.

10. The method for preparing natto freeze-dried powder according to claim 1, characterized in that: The crushing in step (7) uses a low-temperature airflow crusher, the feeding temperature is controlled at 0-5℃, the discharge particle size is 80-120 mesh, and the discharge particle size [4] range ensures solubility while avoiding electrostatic agglomeration; the final moisture content of the finished product is not higher than 3%, the total number of colonies is not less than 1×10 9 CFU per gram, the NK enzyme activity is not less than 2000 FU per gram, and the attenuation rate of the three indicators is less than 15% within 12 months of sealed storage at 25℃.

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