Microbial fermentation process for food processing

Abstract

The invention relates to the technical field of microbial fermentation, and particularly discloses a microbial fermentation process for food processing. The composition used in the process comprises lactobacillus plantarum, aspergillus oryzae and a compound enzyme preparation containing neutral protease, cellulase and phytase. The preparation method comprises the following steps: firstly, pretreating the raw materials, then compounding lactobacillus plantarum and aspergillus oryzae into a fermentation starter according to a specific proportion, fermenting in a liquid environment, and implementing dynamic stepped oxygen control; adding a complex enzyme preparation in a hypha growth period; then transferring into solid matrix conversion to construct a porous structure; and finally, carrying out two-stage temperature-controlled vacuum drying to obtain the product. The composition disclosed by the invention can be used for fermenting plant-based raw materials such as beans and grains, and has the advantage that the availability of a substrate can be improved through the synergistic interaction of bacteria and enzymes; in addition, according to the process, through stepped oxygen control, the double-bacterium respiratory rhythm can be precisely adapted, and the problem of metabolism mutual exclusion in a constant oxygen mode is thoroughly solved.

Description

Technical Field

[0001] This application relates to the field of microbial fermentation technology, and more specifically, to a microbial fermentation process for food processing. Background Technology

[0002] Microbial fermentation is a bioprocessing technique that utilizes the metabolic activities of microorganisms to transform macromolecules in food raw materials into specific functional products. This technology can significantly improve the nutritional value, flavor characteristics, and functional activity of food and is widely used in the production of condiments, fermented beverages, and functional foods.

[0003] The current mainstream process uses a constant dissolved oxygen fermentation mode, which is convenient for process control, but it has the following key drawbacks: this single oxygen concentration cannot simultaneously match the differentiated respiratory requirements of the two types of microorganisms. For Aspergillus oryzae, low dissolved oxygen limits its growth rate, while for Lactobacillus plantarum, high dissolved oxygen inhibits its metabolic activity. As a result, the microbial community interaction becomes unbalanced, substrate conversion efficiency decreases, and the survival rate of Lactobacillus plantarum is difficult to guarantee, ultimately limiting the product yield and quality stability. Summary of the Invention

[0004] To address the problem of mutual exclusion of microbial metabolism and reduced synergistic efficiency caused by constant dissolved oxygen fermentation mode in the prior art, this application provides a microbial fermentation process for food processing.

[0005] This application provides a microbial fermentation process for food processing, which employs the following technical solution:

[0006] A microbial fermentation process for food processing includes the following steps:

[0007] S1. Raw material pretreatment: Crush the grain raw material to a particle size of 0.5-2mm, mix it with water at a mass ratio of 1:1.5-1:3, and cook it at a temperature of 98-102℃ for 35-50 minutes.

[0008] S2, Liquid fermentation: Cool the cooked material to 35-38℃, then inoculate with compound microbial agent, and ferment for 36-48 hours at pH 5.5-6.0 and dissolved oxygen saturation of 30-50%.

[0009] S3, Solid-state conversion: Add auxiliary materials to the liquid fermentation product, adjust the moisture content to 45-55%, and spread the material to a thickness of 5-8cm. Incubate at 30-32℃.

[0010] S4. Dynamic fermentation: Stir every 6-8 hours, control the material temperature to not exceed 40℃, and continue for 72-96 hours;

[0011] S5. Post-ripening treatment: Heat to 35-37℃ and maintain for 12-18 hours, then cool down to 10-15℃ at a rate of 0.5-1℃ / minute.

[0012] S6. Vacuum drying: Dry at a vacuum of -0.08 to -0.095 MPa and a temperature of 45-50℃ until the moisture content is ≤8%.

[0013] By adopting the above technical solutions, the pretreatment of raw materials thoroughly gelatinizes starch, kills miscellaneous bacteria, and softens fibers, thereby achieving rapid utilization by subsequent microorganisms, reducing the risk of contamination, and improving substrate uniformity. Liquid fermentation precisely matches the respiratory needs of *Lactobacillus plantarum* and *Aspergillus oryzae*, maintaining microbial activity and metabolic balance, resulting in the simultaneous accumulation of high viable cell counts and high enzyme activity. Solid-state conversion rapidly constructs a solid microenvironment, provides space for mycelial colonization, and slow-releases nutrients, achieving a smooth transition from liquid to solid phase and initiating solid-state metabolism. Dynamic fermentation continuously supplies oxygen, evenly dissipates heat, and prevents localized overheating, resulting in deep substrate decomposition, enrichment of metabolites, and uniform quality. Post-ripening promotes the conversion of flavor precursors, stabilizes enzyme activity, and rapidly terminates metabolism, leading to flavor enhancement, functional component locking, and ease of subsequent processing. Vacuum drying dehydrates at low temperatures, prevents the inactivation of heat-sensitive substances, and inhibits secondary fermentation, resulting in high product activity, long shelf life, and ease of storage and transportation.

[0014] Preferably, in step S1, the grain raw material includes at least two of broken rice, wheat, and oats, and the proportion of particles passing through a 40-60 mesh sieve after crushing is ≥85%.

[0015] By adopting the above technical solution, it can simultaneously provide easily gelatinized starch, high-quality gluten skeleton and β-glucan slow-release carbon source, thereby achieving the effects of nutritional complementarity, stable fermentation substrate structure and significantly improved subsequent enzymatic hydrolysis efficiency.

[0016] Preferably, in step S2, the inoculation amount of the compound microbial agent is 1.5-2.5% of the total mass of the material, wherein the viable count of *Lactobacillus plantarum* is ≥1×10⁻⁶. 9 CFU / g, Aspergillus oryzae spore count ≥5×10 8 per g.

[0017] By adopting the above technical solutions, the dominant microbial community can be established in the early stage of fermentation, quickly occupy the ecological niche, and simultaneously activate the lactic acid and protease metabolic pathways, thereby shortening the lag period, improving the substrate decomposition rate, and increasing the enrichment of functional components.

[0018] Preferably, in step S2, the liquid fermentation stage adopts a stepwise oxygen control method, with dissolved oxygen maintained at 4-5 mg / L from 0 to 12 hours, decreasing to 3-4 mg / L from 12 to 36 hours, and controlled at 2-3 mg / L from 36 hours until the end of fermentation.

[0019] By adopting the above technical solution, an overwhelming bacterial community dominance can be formed at the moment of fermentation initiation, rapidly occupying the substrate surface and pore niches and simultaneously triggering the lactic acid and protease dual pathways.

[0020] Preferably, in step S3, the auxiliary material is composed of wheat bran and soybean meal in a mass ratio of 2:1-3:1, and the amount of the auxiliary material added is 25-40% of the dry weight of the liquid fermentation product. Before adding, it is sterilized at 121°C for 15-20 minutes.

[0021] By adopting the above technical solution, a loose framework, a slow-release nitrogen source, and complete inactivation of miscellaneous bacteria can be achieved simultaneously during the solid-state conversion stage.

[0022] Preferably, in step S4, a nutrient solution is added every 24 hours during the dynamic fermentation period. The solution contains 1-3 g / L glucose and 0.5-1.5 g / L potassium dihydrogen phosphate, and the amount added is 0.5-1% of the material mass.

[0023] By adopting the above technical solution, it can continuously provide fast-acting carbon sources and phosphorus and potassium elements during the rapid growth period of mycelium, and maintain osmotic pressure and pH stability.

[0024] Preferably, in step S5, sterile air is introduced during the heating stage of the post-ripening treatment, wherein the sterile air flow rate is 0.1-0.3 L / kg·min.

[0025] By adopting the above technical solution, it can continuously supply readily available carbon sources and phosphorus and potassium elements during the rapid growth period of mycelium, and maintain osmotic pressure and pH stability.

[0026] Preferably, in step S6, the vacuum drying adopts a two-stage temperature control: the first stage is drying at 45-48℃ for 2-3 hours, and the second stage is drying at 50-55℃ for 1-2 hours.

[0027] By adopting the above technical solution, the water activity can be rapidly reduced under low temperature and low oxygen conditions, the heat-sensitive functional substances can be preserved to the maximum extent, and the Maillard reaction can be prevented.

[0028] Preferably, in step S2, the compound microbial agent is composed of Lactobacillus plantarum and Aspergillus oryzae in a live bacteria ratio of 1:2 to 1:3.

[0029] By adopting the above technical solutions, the synergistic advantage of lactic acid bacteria and fungi can be established in the early stage of fermentation, the metabolic pathways of lactic acid and protease can be started simultaneously, and the substrate niche can be occupied rapidly.

[0030] Preferably, in step S2, after the liquid fermentation begins, a compound enzyme preparation is added at 24 hours. The compound enzyme preparation contains 1000-1500 U / g of neutral protease, 800-1200 U / g of cellulase, and 500-800 U / g of phytase, and the amount added is 0.05-0.1% of the dry weight of the material.

[0031] By adopting the above technical solution, the plant cell wall is synergistically lysed in the early logarithmic phase of mycelium, releasing proteins and bound phosphorus, and reducing phytic acid anti-nutritional factors.

[0032] In summary, this application has the following beneficial effects:

[0033] 1. Because this application dynamically adjusts the dissolved oxygen level during the fermentation process to match the different respiratory requirements of Aspergillus oryzae and Lactobacillus plantarum, it effectively avoids the metabolic inhibition phenomenon of a single species under constant dissolved oxygen mode, thereby significantly accelerating the overall oxygen consumption rate of the system and greatly improving the activity maintenance ability of Lactobacillus plantarum.

[0034] 2. Since this application uses Lactobacillus plantarum and Aspergillus oryzae in a specific live bacteria ratio of 1:2–1:3 to form a compound microbial agent as a fermentation starter, this synergistic relationship allows the lactic acid bacteria and fungi to play a synergistic role in the early stage of fermentation, simultaneously start their respective metabolic pathways and quickly establish growth advantages. Therefore, it effectively shortens the fermentation lag period, significantly improves the substrate decomposition efficiency, and promotes the enrichment of functional metabolites such as GABA.

[0035] 3. In this application, it is preferred to add a compound enzyme preparation with a specific composition at the 24th hour of liquid fermentation. Because the addition time and the combination of specific enzyme species have a synergistic effect during the logarithmic growth phase of mycelium, they can effectively lyse the plant cell wall structure, release bound nutrients, and degrade anti-nutritional factors such as phytic acid, thereby significantly improving the available nutrients in the fermentation system, optimizing the fermentation process, and helping to increase the yield of the target product GABA.

[0036] 4. The method of this application, by adding a compound enzyme preparation during 24 hours of liquid fermentation, avoids the competition between microbial growth and enzymatic substrate in the early stage of fermentation, and on the other hand, it synergizes with the enzyme system secreted by Aspergillus oryzae to form a synergistic effect, ultimately achieving a dual improvement in enzymatic hydrolysis efficiency and target product accumulation. Attached Figure Description

[0037] Figure 1 This is a flowchart of a microbial fermentation process for food processing provided in this application. Detailed Implementation

[0038] The present application will be further described in detail below with reference to the accompanying drawings and embodiments.

[0039] Technical concept:

[0040] In traditional liquid fermentation, aerobic bacteria (Aspergillus oryzae) compete with facultative anaerobic bacteria (Lactobacillus plantarum) for dissolved oxygen, leading to an imbalance in the microbial community. Under constant dissolved oxygen conditions, the survival rate of Lactobacillus plantarum is too low. The root cause of these phenomena lies in the conflict between microbial respiration types and the lack of a dynamic oxygen regulation mechanism. At the same time, single liquid fermentation lacks a solid-state mass transfer interface, preventing mycelia from colonizing and producing enzymes, thus hindering the deep decomposition of structural substrates.

[0041] This application achieves synergistic respiratory regulation of the microbial community through stepwise oxygen control and control of the microbial community ratio. This is achieved by locking the ratio of *Lactobacillus plantarum* to *Aspergillus oryzae* at 1:2 to 1:3 (viable cell ratio).

[0042] During the hyperoxic period (0-12h): Aspergillus oryzae dominates oxygen consumption and rapidly constructs an anaerobic microenvironment;

[0043] Microaerobic period (12-36h): Lactobacillus plantarum uses residual oxygen to synthesize NADH, driving GABA conversion;

[0044] During the hypoxic period (>36h): Aspergillus oryzae hyphae provide attachment sites for Lactobacillus, forming a symbiotic microbial community.

[0045] Preparation Example 1

[0046] The preparation steps of the highly active compound microbial agent are as follows:

[0047] S1, Aspergillus oryzae spore amplification

[0048] Preparation of culture medium: First, prepare a liquid culture medium containing wheat bran extract (20 g / L) and glucose (5 g / L), where wheat bran provides the growth factor manganese ions and glucose is the readily available carbon source; then, inoculate Aspergillus oryzae into the culture medium and incubate at 30℃; maintain a rotation speed of 200 rpm for the first 48 hours to promote mycelial growth, and then reduce the rotation speed to 100 rpm for the next 72 hours to induce spore formation; then collect the spores by centrifugation, add a compound protectant containing trehalose (5%) and glycerol (10%) and mix well; freeze-dry under vacuum to obtain spores with a survival rate >90% and a concentration ≥5×10⁻⁶. 8 Aspergillus oryzae agent per gram.

[0049] S2, High-density fermentation with Lactobacillus plantarum

[0050] Preparation of modified MRS medium: First, add 0.1% cysteine ​​to the MRS basal medium; then...

[0051] After inoculation, NaOH (2M) was automatically added to maintain pH 6.0; 0.5% yeast extract was added as a nitrogen source, and the culture was carried out until OD reached [value missing]. 600 =8.0 at the end of the logarithmic phase; finally, centrifuge to collect the bacterial sludge, obtaining a viable count ≥1×10 9 CFU / g concentrated Lactobacillus plantarum inoculum.

[0052] Preparation Example 2

[0053] The preparation steps of the compound enzyme stabilizer are as follows:

[0054] S1, Enzyme Combination and Vector Loading

[0055] Weigh and mix the neutral protease, cellulase, and phytase at a ratio of 1.2:1:0.6 (enzyme activity ratio); then thoroughly stir the mixed enzyme with a microcrystalline cellulose carrier with a particle size of 50 μm.

[0056] S2, Microcapsule Encapsulation and Fixation

[0057] The enzyme-carrier complex was added to a 2% sodium alginate solution and then cross-linked in a 0.1 M CaCl2 solution. The gel microspheres were then removed and immersed in a 0.5% chitosan (pH 5.5) solution for electrostatic adsorption for 10 minutes. The cumulative release rate was measured at 37°C after 24 hours and found to be 80%, resulting in a sustained-release enzyme preparation with an encapsulation rate >92%.

[0058] This application provides a microbial fermentation process for food processing. The following details the microbial fermentation process for food processing provided in the embodiments of this application.

[0059] Example 1

[0060] This application provides a microbial fermentation process for food processing, including the following steps:

[0061] S1. Raw material pretreatment: The grain raw material is crushed to a particle size of 1.25mm. The proportion of particles passing through a 50-mesh sieve after crushing is ≥85%. It is mixed with water at a mass ratio of 1:2.25 and cooked at 100℃ for 42.5 minutes. The ratio of broken rice to wheat in the grain raw material is 1:1.

[0062] S2, Liquid fermentation: Cool the cooked material to 36.5℃ and inoculate it with a compound microbial agent, which is composed of Lactobacillus plantarum and Aspergillus oryzae in a live count ratio of 1:2.5. The inoculation amount of the compound microbial agent is 2.0% of the total mass of the material. Ferment for 42 hours at pH 5.75 and dissolved oxygen saturation of 40%.

[0063] The oxygen control method is as follows: dissolved oxygen is maintained at 4.5 mg / L for 0-12 hours, reduced to 3.5 mg / L for 12-36 hours, and controlled at 2.5 mg / L after 36 hours;

[0064] Add a compound enzyme preparation at 24 hours, which contains 1250 U / g of neutral protease, 1000 U / g of cellulase, and 650 U / g of phytase, at an addition amount of 0.075%.

[0065] S3, Solid-state conversion: Add auxiliary materials to the liquid fermentation product, wherein the auxiliary materials consist of wheat bran and soybean meal in a mass ratio of 2.5:1, and the amount added is 32% of the dry weight. Before adding, sterilize at 121℃ for 17.5 minutes, adjust the moisture content to 50%, spread the material to a thickness of 6.5cm, and incubate at 31℃.

[0066] S4. Dynamic fermentation: Stir every 7 hours, control the material temperature ≤40℃, and continue for 84 hours; replenish nutrient salt solution every 24 hours, which contains 2g / L glucose and 1g / L potassium dihydrogen phosphate, with a replenishment amount of 0.75%.

[0067] S5. Post-ripening treatment: Heat to 36℃ and maintain for 15 hours. During the heating stage, sterile air is introduced at a flow rate of 0.2 L / kg·min. Then, the temperature is reduced to 12.5℃ at a rate of 0.75℃ / min.

[0068] S6. Vacuum drying: Two-stage temperature control is adopted, with the first stage drying at 46.5℃ for 2.5 hours and the second stage drying at 52.5℃ for 1.5 hours, under a vacuum of -0.0875MPa until the moisture content is ≤8%.

[0069] Example 2

[0070] This application provides a microbial fermentation process for food processing, including the following steps:

[0071] S1. Raw material pretreatment: The grain raw material is crushed to a particle size of 0.5mm. The proportion of particles passing through a 40-mesh sieve after crushing is ≥85%. It is mixed with water at a mass ratio of 1:1.5 and cooked at 98℃ for 35 minutes. The ratio of broken rice to wheat in the grain raw material is 1:1.5.

[0072] S2, Liquid fermentation: Cool the cooked material to 35°C and inoculate it with a compound microbial agent, which is composed of Lactobacillus plantarum and Aspergillus oryzae in a live count ratio of 1:2. The inoculation amount of the compound microbial agent is 1.5% of the total mass of the material. Ferment for 36 hours at pH 5.5 and dissolved oxygen saturation of 30%.

[0073] The oxygen control method is as follows: maintain dissolved oxygen at 4 mg / L for 0-12 hours, reduce it to 3 mg / L for 12-36 hours, and control it at 2 mg / L after 36 hours;

[0074] Add a compound enzyme preparation at 24 hours, which contains 1000 U / g of neutral protease, 800 U / g of cellulase, and 500 U / g of phytase, at an addition amount of 0.05%.

[0075] S3, Solid-state conversion: Add auxiliary materials to the liquid fermentation product. The auxiliary materials consist of wheat bran and soybean meal in a mass ratio of 2:1. The amount added is 25% of the dry weight. Before adding, sterilize at 121℃ for 15 minutes, adjust the moisture content to 45%, spread the material to a thickness of 5cm, and incubate at 30℃.

[0076] S4. Dynamic fermentation: Stir every 6 hours, control the material temperature ≤40℃, continue for 72 hours; replenish nutrient salt solution every 24 hours, the nutrient salt solution contains 1g / L glucose and 0.5g / L potassium dihydrogen phosphate, the replenishment amount is 0.5%;

[0077] S5. Post-ripening treatment: Heat to 35℃ and maintain for 12 hours. During the heating stage, sterile air is introduced at a flow rate of 0.1 L / kg·min. Then, the temperature is reduced to 10℃ at a rate of 0.5℃ / min.

[0078] S6. Vacuum drying: Two-stage temperature control is adopted, with the first stage drying at 45℃ for 2 hours and the second stage drying at 50℃ for 1 hour, with a vacuum degree of -0.08MPa until the moisture content is ≤8%.

[0079] Example 3

[0080] This application provides a microbial fermentation process for food processing, including the following steps:

[0081] S1. Raw material pretreatment: The grain raw material is crushed to a particle size of 2mm. After crushing, the proportion of particles passing through a 60-mesh sieve is ≥85%. It is mixed with water at a mass ratio of 1:3 and cooked at 102℃ for 50 minutes. The ratio of broken rice to wheat in the grain raw material is 1:3.

[0082] S2, Liquid fermentation: Cool the cooked material to 38°C and inoculate it with a compound microbial agent, which is composed of Lactobacillus plantarum and Aspergillus oryzae in a live count ratio of 1:3. The inoculation amount of the compound microbial agent is 2.5% of the total mass of the material. Ferment for 48 hours at pH 6.0 and dissolved oxygen saturation of 50%.

[0083] The oxygen control method is as follows: maintain dissolved oxygen at 5 mg / L for 0-12 hours, reduce it to 4 mg / L for 12-36 hours, and control it at 3 mg / L after 36 hours;

[0084] Add a compound enzyme preparation at 24 hours, which contains 1500 U / g of neutral protease, 1200 U / g of cellulase, and 800 U / g of phytase, at an addition amount of 0.1%.

[0085] S3, Solid-state conversion: Add auxiliary materials to the liquid fermentation product. The auxiliary materials consist of wheat bran and soybean meal in a mass ratio of 3:1. The amount added is 40% of the dry weight. Before adding, sterilize at 121℃ for 20 minutes, adjust the moisture content to 55%, spread the material to a thickness of 8cm, and incubate at 32℃.

[0086] S4. Dynamic fermentation: Stir every 8 hours, control the material temperature ≤40℃, continue for 96 hours; replenish nutrient salt solution every 24 hours, the nutrient salt solution contains 3g / L glucose and 1.5g / L potassium dihydrogen phosphate, the replenishment amount is 1%;

[0087] S5. Post-ripening treatment: Heat to 37℃ and maintain for 18 hours. During the heating stage, sterile air is introduced at a flow rate of 0.3L / kg·min. Then, the temperature is reduced to 15℃ at a rate of 1℃ / min.

[0088] S6. Vacuum drying: Two-stage temperature control is adopted, with the first stage drying at 48℃ for 3 hours and the second stage drying at 55℃ for 2 hours, with a vacuum degree of -0.095MPa until the moisture content is ≤8%.

[0089] Comparative Example 1

[0090] This comparative example provides a microbial fermentation process for food processing. The only difference between this process and Example 1 is that constant dissolved oxygen is used instead of stepwise oxygen control.

[0091] In step S2, liquid fermentation was carried out at pH 5.75 and dissolved oxygen at a constant 3.8 mg / L for 42 hours; the remaining process steps were exactly the same as in Example 1.

[0092] Comparative Example 2

[0093] This comparative example provides a microbial fermentation process for food processing. The only difference between this process and Example 1 is that the proportion of microbial strains is changed to a conventional industry value.

[0094] In step S2, the ratio of bacteria in the liquid fermentation was changed to Lactobacillus plantarum: Aspergillus oryzae = 1:1; the remaining process steps were exactly the same as in Example 1.

[0095] Comparative Example 3

[0096] This comparative example provides a microbial fermentation process for food processing. The only difference between this process and Example 1 is that the proportion of microbial strains is changed to a conventional industry value.

[0097] In step S2, the ratio of bacteria in the liquid fermentation was changed to Lactobacillus plantarum: Aspergillus oryzae = 1:4; the remaining process steps were exactly the same as in Example 1.

[0098] Comparative Example 4

[0099] This comparative example provides a microbial fermentation process for food processing. The only difference between this process and Example 1 is that step S3, solid-state conversion, is omitted; the remaining process steps are exactly the same as in Example 1.

[0100] Comparative Example 5

[0101] This comparative example provides a microbial fermentation process for food processing. The only difference between this process and Example 1 is that step S4, dynamic fermentation, is omitted; the remaining process steps are exactly the same as in Example 1.

[0102] Comparative Example 6

[0103] This comparative example provides a microbial fermentation process for food processing. The only difference between this process and Example 1 is that step S3 (solid-state conversion) and step S4 (dynamic fermentation) are omitted, and only step S2 (liquid fermentation) is performed. The remaining process steps are exactly the same as in Example 1.

[0104] Comparative Example 7

[0105] This comparative example provides a microbial fermentation process for food processing. The only difference between this process and Example 1 is that the enzyme preparation is added at the initial inoculation time instead of at the 24th hour.

[0106] In step S2, during liquid fermentation, the compound enzyme preparation is added at the start of fermentation (0 hours); the remaining process steps are exactly the same as in Example 1.

[0107] The main performance test results of Examples 1-3 and Comparative Examples 1-7 are shown in Table 1.

[0108] Table 1:

[0109] project Dissolved oxygen consumption rate (mg / L·h) <![CDATA[Survival rate of Lactobacillus plantarum (CFU×10 8 / g)]]> Protease utilization rate (%) Substrate decomposition rate (%) Example 1 12.3±0.5 3.82±0.15 89.6±2.1 94.5±1.3 Example 2 13.1±0.4 4.05±0.12 92.3±1.8 96.8±0.9 Example 3 11.8±0.6 3.74±0.18 87.9±2.4 93.6±1.7 Comparative Example 1 8.9±0.7 2.31±0.23 71.5±3.2 82.4±2.8 Comparative Example 2 8.2±0.5 3.18±0.17 62.8±2.9 74.6±3.1 Comparative Example 3 10.7±0.6 1.64±0.12 49.3±4.1 86.9±2.4 Comparative Example 4 9.4±0.4 2.67±0.19 65.2±3.5 52.7±4.2 Comparative Example 5 7.3±0.8 2.89±0.21 68.1±2.7 80.3±3.0 Comparative Example 6 10.8±0.6 2.14±0.16 72.4±3.0 41.5±5.1 Comparative Example 7 13.6±0.7 3.52±0.14 51.7±4.3 83.7±2.6

[0110] 1. Dissolved oxygen consumption rate (mg / L·h): The test standard is: ISO5815-1:2019 "Water quality - Determination of dissolved oxygen - Electrochemical probe method";

[0111] 2. Survival rate of Lactobacillus plantarum (CFU×10⁻⁶) 8 / g): The test standard is GB4789.35-2016 "National Food Safety Standard for the Examination of Lactic Acid Bacteria";

[0112] 3. Protease utilization rate (%): The test standard is GB / T28715-2012 "Determination of protease activity in feed additives";

[0113] 4. Substrate degradation rate (%): The test standard is: AACC76-13.01 (starch) + AOAC994.12 (protein).

[0114] Based on Example 1 and Comparative Example 1, and referring to Table 1, it can be seen that compared with the constant dissolved oxygen mode, the stepwise oxygen control strategy increases the dissolved oxygen consumption rate by 40.4% and the survival rate of *Lactobacillus plantarum* by 65.4%. The fundamental mechanism is that stepwise oxygen control precisely matches the respiratory needs of *Aspergillus oryzae* and *Lactobacillus plantarum*, avoiding metabolic inhibition under constant oxygen conditions.

[0115] Based on Example 1 and Comparative Examples 2-3, and referring to Table 1, it can be seen that in Comparative Example 2, after changing the bacterial culture ratio of *Lactobacillus plantarum* to *Aspergillus oryzae* in step S2 of liquid fermentation to 1:1, the excessive proliferation of *Lactobacillus plantarum* inhibited protease secretion, resulting in a decrease in substrate degradation rate. In Comparative Example 3, after changing the bacterial culture ratio of *Lactobacillus plantarum* to *Aspergillus oryzae* in step S2 of liquid fermentation to 1:4, the respiration and oxygen consumption of *Aspergillus oryzae* increased, competitively suppressing *Lactobacillus plantarum*, thus reducing its survival rate.

[0116] Combining Example 1 and Comparative Example 4 with Table 1, it can be seen that: deleting step S3 solid-state conversion resulted in: reduced porosity, which led to a sharp drop in substrate decomposition rate of 44.2%; the penetration depth of Aspergillus oryzae hyphae was limited to 320 μm; the porous structure of the solid matrix provided oxygen diffusion channels and hyphal anchoring points for Aspergillus oryzae, promoting extracellular enzyme secretion.

[0117] Combining Example 1 and Comparative Example 5 with Table 1, it can be seen that: Deleting step S4, which initiates dynamic fermentation, greatly improves the fermentation uniformity CV value; dynamic stirring breaks the concentration / temperature gradient, thereby improving the substrate-cell-oxygen mass transfer efficiency.

[0118] Combining Example 1 and Comparative Example 6 with Table 1, it can be seen that pure liquid fermentation results in: a decrease in substrate decomposition rate and an increase in drying energy consumption; the liquid-solid two-phase process achieves the following through spatial partitioning metabolism: rapid consumption of soluble sugars in the liquid stage (dominated by Aspergillus oryzae); and deep decomposition of structural substrates in the solid stage (mycelial colonization + enzyme secretion).

[0119] Combining Example 1 and Comparative Example 7 with Table 1, it can be seen that adding enzyme at 0 hours leads to: decreased protease utilization; an artificially high substrate decomposition rate in the early stage, which collapses in the final stage; adding enzyme at 24 hours avoids the microbial-enzyme-substrate competition in the early stage of fermentation and matches the peak enzyme production period of Aspergillus oryzae.

[0120] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A microbial fermentation process for food processing, characterized in that: Includes the following steps: S1. Raw material pretreatment: Crush the grain raw material to a particle size of 0.5-2mm, mix it with water at a mass ratio of 1:1.5-1:3, and cook it at a temperature of 98-102℃ for 35-50 minutes. S2, Liquid fermentation: Cool the cooked material to 35-38℃, then inoculate with compound microbial agent, and ferment for 36-48 hours at pH 5.5-6.0 and dissolved oxygen saturation of 30-50%. S3, Solid-state conversion: Add auxiliary materials to the liquid fermentation product, adjust the moisture content to 45-55%, and spread the material to a thickness of 5-8cm. Incubate at 30-32℃. S4. Dynamic fermentation: Stir every 6-8 hours, control the material temperature to not exceed 40℃, and continue for 72-96 hours; S5. Post-ripening treatment: Heat to 35-37℃ and maintain for 12-18 hours, then cool down to 10-15℃ at a rate of 0.5-1℃ / minute. S6. Vacuum drying: Dry at a vacuum of -0.08 to -0.095 MPa and a temperature of 45-50℃ until the moisture content is ≤8%.

2. The microbial fermentation process for food processing according to claim 1, characterized in that: In step S1, the grain raw material includes at least two of broken rice, wheat, and oats, and the proportion of particles passing through a 40-60 mesh sieve after crushing is ≥85%.

3. The microbial fermentation process for food processing according to claim 1, characterized in that: In step S2, the inoculation amount of the compound microbial agent is 1.5-2.5% of the total mass of the material, wherein the viable count of *Lactobacillus plantarum* is ≥1×10⁻⁶. 9 CFU / g, Aspergillus oryzae spore count ≥5×10 8 per g.

4. The microbial fermentation process for food processing according to claim 1, characterized in that: In step S2, the liquid fermentation stage adopts a stepwise oxygen control method: dissolved oxygen is maintained at 4-5 mg / L from 0 to 12 hours, reduced to 3-4 mg / L from 12 to 36 hours, and controlled at 2-3 mg / L from 36 hours until the end of fermentation.

5. The microbial fermentation process for food processing according to claim 1, characterized in that: In step S3, the auxiliary material is composed of wheat bran and soybean meal in a mass ratio of 2:1-3:

1. The amount of the auxiliary material added is 25-40% of the dry weight of the liquid fermentation product. Before adding, it is sterilized at 121℃ for 15-20 minutes.

6. The microbial fermentation process for food processing according to claim 1, characterized in that: In step S4, a nutrient solution is added every 24 hours during the dynamic fermentation period. The solution contains 1-3 g / L glucose and 0.5-1.5 g / L potassium dihydrogen phosphate, and the amount added is 0.5-1% of the material mass.

7. The microbial fermentation process for food processing according to claim 1, characterized in that: In step S5, sterile air is introduced during the heating stage of the post-ripening treatment, wherein the sterile air flow rate is 0.1-0.3 L / kg·min.

8. The microbial fermentation process for food processing according to claim 1, characterized in that: In step S6, the vacuum drying adopts a two-stage temperature control: the first stage is drying at 45-48℃ for 2-3 hours, and the second stage is drying at 50-55℃ for 1-2 hours.

9. The microbial fermentation process for food processing according to claim 1, characterized in that: In step S2, the compound microbial agent is composed of Lactobacillus plantarum and Aspergillus oryzae in a live bacteria ratio of 1:2 to 1:

3.

10. The microbial fermentation process for food processing according to claim 1, characterized in that: In step S2, after the liquid fermentation begins, a compound enzyme preparation is added at 24 hours. The compound enzyme preparation contains 1000-1500 U / g of neutral protease, 800-1200 U / g of cellulase, and 500-800 U / g of phytase. The amount added is 0.05-0.1% of the dry weight of the material.

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