A method for preparing a microbial inoculant by mixed fermentation of bacillus subtilis and bacillus cereus

Abstract

This invention belongs to the field of microbial fermentation technology, specifically relating to a method for preparing microbial inoculants through mixed fermentation of Bacillus subtilis and Bacillus cereus. The method includes: separately activating Bacillus subtilis and Bacillus cereus to obtain a seed culture; adding a basic fermentation medium to a fermenter, first inoculating Bacillus subtilis, and then inoculating Bacillus cereus after it enters the mid-logarithmic growth phase, achieving sequential mixed inoculation; employing segmented temperature control, two-stage pH control, and dynamic sugar feeding during fermentation, while simultaneously adding malic acid in a coupled manner between 20 and 35 hours to alleviate oxidative stress; collecting the cells after fermentation, adding a protective agent, and drying to obtain the inoculant. This invention, through the synergistic effect of sequential inoculation and malic acid coupled feeding, achieves an effective viable cell count of up to 1.0 × 10⁻⁶. 13 With a CFU / mL or higher, the spore formation rate is ≥89%, which is significantly better than the existing mixed-strain fermentation process.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, specifically relating to a method for preparing microbial agents by mixed fermentation of Bacillus subtilis and Bacillus cereus. Background Technology

[0002] Bacillus subtilis is a probiotic widely used in agriculture, environmental protection, and feed additives. High-density fermentation is a key method to increase the number of viable bacteria per unit volume. In existing technologies, mixed fermentation of Bacillus subtilis and Bacillus licheniformis has achieved relatively high viable bacterial counts, such as 2.8 × 10⁻⁶. 14 The concentration of CFU / mL is high, but this combination suffers from problems such as strain competition imbalance and significant inhibition of byproduct accumulation in long-term industrial applications. *Bacillus cereus* has strong protease and amylase secretion capabilities, exhibiting metabolic complementarity with *Bacillus subtilis* without antagonistic effects. However, no systemic application of both has yet been shown to achieve a concentration of 10 CFU / mL in high-density fed-batch fermentation. 12 Reports of viable cell counts above CFU / mL have been published. Furthermore, existing processes lack integrated solutions for sequential inoculation of mixed strains, oxidative stress regulation, and dynamic feeding. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention provides a method for preparing microbial inoculants through mixed fermentation of Bacillus subtilis and Bacillus cereus. By employing a strategy of sequential inoculation, segmented temperature control, dynamic glucose control, and malic acid-coupled feeding, an effective viable cell count ≥1.0 × 10⁻⁶ is achieved. 12 CFU / mL.

[0004] A method for preparing microbial inoculants through high-density fermentation of a mixture of Bacillus subtilis and Bacillus cereus includes the following steps: (1) Strain activation and seed culture: Activate the freeze-dried bacterial powder or slant culture of Bacillus subtilis and Bacillus cereus, respectively; Activation of Bacillus subtilis: LB slant medium, cultured at 36-38℃ for 23-25h; pick a single colony and inoculate it into LB liquid medium, cultured at 36-38℃ and 170-190 rpm for 11-13h with shaking to obtain primary seed culture of Bacillus subtilis; Activation of Bacillus cereus: LB slant medium, cultured at 34-36℃ for 23-25 ​​h; single colonies were picked and inoculated into LB liquid medium, cultured at 34-36℃ and 170-190 rpm for 13-15 h with shaking to obtain primary seed culture of Bacillus cereus; The above-mentioned primary seed culture was transferred to the seed tank culture medium at an inoculation rate of 5%-10% by volume for expansion culture. Seed culture conditions for Bacillus subtilis: temperature 36-38℃, pH 7.0, aeration rate 0.9-1.1 vvm, stirring speed 190-210 rpm, culture time 8-10 h, OD600 reaches 4.0-5.0; Seed culture conditions for Bacillus cereus: temperature 34-36℃, pH 7.0, aeration rate 1.0 vvm, stirring speed 190-210 rpm, culture time 10-12 h, OD600 reaches 3.5-4.5; (2) Sequential mixed inoculation: Add basic fermentation medium to the fermenter, with the initial volume being 60%-70% of the fermenter's volume. First, inoculate with Bacillus subtilis primary seed culture at a volume of 5% of the basic fermentation medium. Incubate at 35-37℃ and pH 6.8 for 7-9 hours. At this point, Bacillus subtilis enters the mid-logarithmic growth phase, with an OD600 of 3.0-4.0. Then, inoculate with Bacillus cereus primary seed culture at a Bacillus subtilis:Bacillus cereus primary seed culture volume ratio of 2:1, so that the total inoculation volume reaches 7.5%-8% of the fermentation liquid volume, and continue fermentation. (3) High-density fed-batch fermentation: Fed-batch fermentation involves a combination of two feeding methods: A. Dynamic glucose control and feeding: When the residual sugar concentration in the fermentation broth drops to 2.9-3.1 g / L, fed culture medium is started. The feeding rate is controlled by feedback from an online sugar analyzer to maintain the glucose concentration in the fermentation broth at 3.0-5.0 g / L.

[0005] Feeding rate segmented setting: 10-18 h: constant feed rate 2.4-2.6 mL / (L·min); 18-30 h: Accelerate feeding to 3.9-4.1 mL / (L·min); 30-40 h: Reduce feed rate to 1.4-1.6 mL / (L·min).

[0006] B. Malic acid coupled feeding: During the 20-35 h of fermentation, an additional malic acid solution with a concentration of 99-101 g / L was added at a flow rate of 0.5-1.0 g / (L·h), alternating with glucose feeding, with the total malic acid addition controlled at 8-12 g / L of fermentation broth; Control parameters for high-density fed-batch fermentation process: Temperature control in stages: 0-16 h constant temperature 36℃, 16-30 h constant temperature 38℃, 30 h until the end of fermentation constant temperature 34℃; pH is controlled in two stages: pH is 6.7-6.9 from 0 to 12 h, and pH is 7.4-7.6 after 12 h, adjusted with ammonia. Dissolved oxygen control: Dissolved oxygen is controlled to be ≥30% by linking stirring speed and aeration rate. The stirring speed range is 300-800 rpm, and the aeration rate is 1.2-1.8 vvm. (4) Determination of fermentation endpoint and preparation of inoculum: When the total fermentation time reaches 40-48 hours, samples are taken and the total viable count and spore formation rate are determined using the plate count method. When the total viable count is ≥1.0 × 10⁻⁶, the result is considered satisfactory. 12 Fermentation was terminated when CFU / mL and spore formation rate ≥85%; The fermentation broth was concentrated by centrifugation at 8000-10000 rpm for 9-10 min, and the bacterial sludge was collected. A protectant was added at a mass ratio of 1:1, and the mixture was freeze-dried after thorough mixing to obtain a microbial inoculum powder. The viable count of the finished product was ≥1.0×10⁻⁶. 12 CFU / g.

[0007] The seed tank culture medium in step (1) consists of: glucose 9-10 g / L, yeast extract 4-5 g / L, peptone 9-10 g / L, NaCl 4-5 g / L, pH 7.0.

[0008] The basic fermentation medium formula in step (2) is as follows: glucose 14-16 g / L, molasses 4-5 g / L, soybean meal powder 34-36 g / L, yeast powder 24-26 g / L, urea 2-3 g / L, KH2PO4 2.4-2.6 g / L, MgSO4·7H2O 0.5-0.7 g / L, CaCl2 0.14-0.16 g / L, MnSO4 0.04-0.06 g / L, initial pH 7.0.

[0009] The formula of the feeding medium in step (3) is: glucose 490-500 g / L, yeast powder 9-10 g / L, MgSO4·7H2O 0.4-0.6 g / L.

[0010] The formulation of the protective agent in step (4) is: trehalose 1-2% (w / v), skim milk powder 4-5% (w / v), and glycerin 0.9-1% (v / v).

[0011] This invention employs high-density fermentation using Bacillus subtilis and Bacillus cereus in synergy. Firstly, Bacillus cereus is a Gram-positive aerobic bacterium with an optimal growth temperature of 28-35℃, an optimal pH of 6.5-7.5, and can tolerate glucose concentrations up to 15-20 g / L. It exhibits strong spore-forming ability and good compatibility with Bacillus subtilis in fermentation conditions.

[0012] Secondly, there is a significant synergistic growth effect between Bacillus subtilis and Bacillus cereus. This synergistic mechanism mainly stems from the following: Bacillus cereus has a strong protease and amylase secretion capacity, which can effectively degrade macromolecular nutrients and release small molecule amino acids and reducing sugars, providing Bacillus subtilis with more readily available nutrient substrates; the two bacteria have complementary metabolic pathways, with Bacillus subtilis preferentially utilizing glucose, while Bacillus cereus has a better ability to utilize non-glucose carbon sources, which can reduce carbon source competition; both bacteria have aerobic dissolved oxygen requirements, but in the later stages of fermentation, they can form spatial differentiation between microaerobic and aerobic zones to avoid excessive competition. Compared with the Bacillus subtilis plus Bacillus licheniformis combination, this combination is novel and unpredictable in terms of strain combination. The synergistic mechanism of Bacillus cereus and Bacillus subtilis has not been systematically applied to high-density fed-batch fermentation processes, and there are no high-density co-fermentation products based on this combination on the market.

[0013] Advantages of sequential inoculation: It effectively avoids early metabolic competition that may occur when two bacteria are inoculated simultaneously, while utilizing the "first-mover advantage" of Bacillus subtilis and the "second-mover complementarity" of Bacillus cereus to form metabolic synergy. Unlike the isothermal fermentation of conventional processes, this scheme takes advantage of the fact that Bacillus cereus is more sensitive to temperature than Bacillus subtilis. It promotes the co-colonization of the two bacteria at 36℃ in the early stage, accelerates the metabolism and spore transformation of Bacillus subtilis at 38℃ in the middle stage, and reduces the accumulation of by-products and maintains a high viable cell count at 34℃ in the later stage.

[0014] Sugar thresholds were designed for each of the two bacteria in the mixed strain study. The optimal sugar control range for *Bacillus subtilis* was 2-5 g / L, while *Bacillus cereus* showed higher sugar tolerance at 5-10 g / L. A combined range of 3-5 g / L was chosen to meet the growth requirements of *Bacillus subtilis* while avoiding excessively low sugar concentrations that could cause *Bacillus cereus* to enter the lag phase. Furthermore, *Bacillus cereus* generates high levels of reactive oxygen species (ROS) during the late-stage sporulation process. The addition of malic acid can mitigate this oxidative damage, forming an antioxidant synergy with *Bacillus subtilis*, a novel feature not found in existing *Bacillus licheniformis* strains.

[0015] Beneficial effects of this invention: (1) Novel strain combination: For the first time, Bacillus subtilis and Bacillus cereus were applied to high-density fed-batch fermentation. By utilizing the extracellular enzyme secretion capacity and metabolic complementarity of Bacillus cereus, the synergistic growth of the two strains was achieved, with an effective viable count of 1.0 × 10⁻⁶. 12 -1.0×10 14 CFU / mL.

[0016] (2) Sequential inoculation avoids competitive imbalance: The strategy of inoculating Bacillus subtilis first and then Bacillus cereus allows Bacillus subtilis to occupy the ecological niche first, reduces early carbon source competition, and improves the stability of mixed culture.

[0017] (3) Malic acid coupled feeding alleviates oxidative damage: Bacillus cereus is prone to producing reactive oxygen species during spore formation. Malic acid feeding can upregulate the expression of antioxidant enzyme genes and reduce ROS levels, thereby increasing the final viable count and spore rate.

[0018] (4) Segmented temperature control and segmented pH optimization: Based on the temperature sensitivity difference between the two bacteria, three-stage temperature control was designed, combined with two-stage pH control, to promote bacterial proliferation and spore transformation respectively. Detailed Implementation

[0019] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; and the materials and reagents used are commercially available unless otherwise specified.

[0020] Bacillus subtilis, accession number: CCTCC AB 130031, purchased from China Center for Type Culture Collection.

[0021] Bacillus cereus, accession number: CMCC63301, purchased from the China Center for Medical Bacteriological Culture Collection.

[0022] Example 1

[0023] A method for preparing microbial inoculants through high-density fermentation of a mixture of Bacillus subtilis and Bacillus cereus includes the following steps: (1) Activation and seed culture of strains: freeze-dried bacterial powder or slant culture of Bacillus subtilis and Bacillus cereus were taken and activated respectively; Activation of Bacillus subtilis: LB slant medium, cultured at 37℃ for 24 h; single colonies were picked and inoculated into LB liquid medium, cultured at 37℃ and 180 rpm for 12 h with shaking, to obtain Bacillus subtilis primary seed culture with OD600=4.2; Activation of Bacillus cereus: LB slant medium, cultured at 35℃ for 24 h; single colonies were picked and inoculated into LB liquid medium, cultured at 35℃ and 180 rpm for 14 h with shaking, to obtain primary seed culture of Bacillus cereus with OD600=3.8; The above-mentioned primary seed culture was transferred to a 5 L seed tank culture medium at an inoculation rate of 8% by volume for expansion culture. Bacillus subtilis seed culture conditions: temperature 37℃, pH 7.0, aeration rate 1.0 vvm, stirring speed 200 rpm, culture time 9 h, OD600 reached 4.5; Cultivation conditions for Bacillus cereus in seed tanks: temperature 35℃, pH 7.0, aeration rate 1.0 vvm, stirring speed 200 rpm, cultivation for 11 h, OD600 reached 4.0; (2) Sequential inoculation: Add 35 L of basic fermentation medium to a 50 L fermenter, sterilize it, and then inoculate it with 1.75 L of Bacillus subtilis primary seed culture. Incubate at 36℃ and pH=6.8 for 8 h, at which point OD600=3.5; then add 0.875 L of Bacillus cereus primary seed culture. (3) Fermentation control: The temperature was maintained at 36℃ for 0-16h, 38℃ for 16-30h, and 34℃ for 30-44h; the pH was maintained at 6.8 for 0-12h and adjusted to 7.5 after 12h; the dissolved oxygen was maintained at 35% by stirring at a rate of 500 rpm and aeration at a rate of 1.5 vvm. (4) Feeding: When the residual sugar drops to 3.0 g / L, feed culture medium is started, and the feeding rate is set in stages: Feeding rate constant: 2.5 mL / (L·min) from 10 to 18 h; 18-30 h: Increase feed rate to 4.0 mL / (L·min); 30-40 h: Reduce feed rate to 1.5 mL / (L·min); A 100 g / L malic acid solution was simultaneously added from 20 to 35 h at a rate of 0.6 g / (L·h). (5) After 44 hours of fermentation, plate samples were taken for counting: total viable count = 1.2 × 10⁻⁶ 13 CFU / mL, of which Bacillus subtilis accounted for 75% and Bacillus cereus accounted for 25%, with a spore formation rate of 91%; (6) Post-treatment: Centrifuge the fermentation broth at 8000 rpm for 10 min, collect the bacterial sludge, add the protectant, mix the bacterial sludge and protectant at a ratio of 1:1, freeze dry, and obtain bacterial powder.

[0024] The seed tank culture medium in step (1) consists of: 10 g / L glucose, 5 g / L yeast extract, 10 g / L peptone, 5 g / L NaCl, and pH 7.0.

[0025] The basic fermentation medium formula in step (2) is as follows: glucose 15 g / L, molasses 5 g / L, soybean meal powder 35 g / L, yeast powder 25 g / L, urea 3 g / L, KH2PO4 2.5 g / L, MgSO4·7H2O 0.6 g / L, CaCl2 0.15 g / L, MnSO4 0.05 g / L, initial pH 7.0.

[0026] The formula for the feeding medium in step (4) is: 500 g / L glucose, 10 g / L yeast powder, and 0.5 g / L MgSO4·7H2O.

[0027] The formula for the protective agent in step (6) is: trehalose 2% (w / v), skim milk powder 5% (w / v), and glycerin 1% (v / v).

[0028] Example 2

[0029] A method for preparing microbial inoculants through high-density fermentation of a mixture of Bacillus subtilis and Bacillus cereus includes the following steps: (1) Activation and seed culture of strains: freeze-dried bacterial powder or slant culture of Bacillus subtilis and Bacillus cereus were taken and activated respectively; Activation of Bacillus subtilis: LB slant medium, cultured at 37℃ for 24 h; single colonies were picked and inoculated into LB liquid medium, cultured at 37℃ and 180 rpm for 12 h with shaking, to obtain Bacillus subtilis primary seed culture with OD600=4.2; Activation of Bacillus cereus: LB slant medium, cultured at 35℃ for 24 h; single colonies were picked and inoculated into LB liquid medium, cultured at 35℃ and 180 rpm for 14 h with shaking, to obtain primary seed culture of Bacillus cereus with OD600=3.8; The above-mentioned primary seed culture was transferred to a 5 L seed tank culture medium at an inoculation rate of 8% by volume for expansion culture. Bacillus subtilis seed culture conditions: temperature 37℃, pH 7.0, aeration rate 1.0 vvm, stirring speed 200 rpm, culture time 9 h, OD600 reached 4.5; Cultivation conditions for Bacillus cereus in seed tanks: temperature 35℃, pH 7.0, aeration rate 1.0 vvm, stirring speed 200 rpm, cultivation for 11 h, OD600 reached 4.0; (2) Sequential inoculation: Add 35 L of basic fermentation medium to a 50 L fermenter, sterilize it, and then inoculate it with 1.75 L of Bacillus subtilis primary seed culture. Incubate at 36℃ and pH=6.8 for 8 h, at which point OD600=3.5; then add 0.875 L of Bacillus cereus primary seed culture. (3) Fermentation control: The temperature was maintained at 36℃ for 0-16h, 38℃ for 16-30h, and 34℃ for 30-44h; the pH was maintained at 6.8 for 0-12h and adjusted to 7.5 after 12h; the dissolved oxygen was maintained at 35% by stirring at a rate of 500 rpm and aeration at a rate of 1.5 vvm. (4) Feeding: When the residual sugar drops to 3.0 g / L, feed culture medium is started, and the feeding rate is set in stages: Feeding rate constant: 2.5 mL / (L·min) from 10 to 18 h; 18-30 h: Increase feed rate to 4.0 mL / (L·min); 30-40 h: Reduce feed rate to 1.5 mL / (L·min); (5) Fermentation ends after 44 hours; (6) Post-treatment: Centrifuge the fermentation broth at 8000 rpm for 10 min, collect the bacterial sludge, add the protectant, mix the bacterial sludge and protectant at a ratio of 1:1, freeze dry, and obtain bacterial powder.

[0030] The seed tank culture medium in step (1) consists of: 10 g / L glucose, 5 g / L yeast extract, 10 g / L peptone, 5 g / L NaCl, and pH 7.0.

[0031] The basic fermentation medium formula in step (2) is as follows: glucose 15 g / L, molasses 5 g / L, soybean meal powder 35 g / L, yeast powder 25 g / L, urea 3 g / L, KH2PO4 2.5 g / L, MgSO4·7H2O 0.6 g / L, CaCl2 0.15 g / L, MnSO4 0.05 g / L, initial pH 7.0.

[0032] The formula for the feeding medium in step (4) is: 500 g / L glucose, 10 g / L yeast powder, and 0.5 g / L MgSO4·7H2O.

[0033] The formula for the protective agent in step (6) is: trehalose 2% (w / v), skim milk powder 5% (w / v), and glycerin 1% (v / v).

[0034] Comparative Example 1 A method for preparing microbial inoculants through high-density fermentation of a mixture of Bacillus subtilis and Bacillus cereus includes the following steps: (1) Activation and seed culture of strains: freeze-dried bacterial powder or slant culture of Bacillus subtilis and Bacillus cereus were taken and activated respectively; Activation of Bacillus subtilis: LB slant medium, cultured at 37℃ for 24 h; single colonies were picked and inoculated into LB liquid medium, cultured at 37℃ and 180 rpm for 12 h with shaking, to obtain Bacillus subtilis primary seed culture with OD600=4.2; The above-mentioned Bacillus subtilis primary seed culture was transferred to a 5 L seed tank culture medium at an inoculation rate of 8% by volume for scale-up culture. Bacillus subtilis seed culture conditions: temperature 37℃, pH 7.0, aeration rate 1.0 vvm, stirring speed 200 rpm, culture time 9 h, OD600 reached 4.5; (2) Sequential inoculation: Add 35 L of basic fermentation medium to a 50 L fermenter, sterilize it, and then inoculate it with 1.75 L of Bacillus subtilis primary seed culture. Incubate at 36℃ and pH=6.8 for 8 h, at which point OD600=3.5; then add 0.875 L of Bacillus subtilis primary seed culture. (3) Fermentation control: The temperature was maintained at 36℃ for 0-16h, 38℃ for 16-30h, and 34℃ for 30-44h; the pH was maintained at 6.8 for 0-12h and adjusted to 7.5 after 12h; the dissolved oxygen was maintained at 35% by stirring at a rate of 500 rpm and aeration at a rate of 1.5 vvm. (4) Feeding: When the residual sugar drops to 3.0 g / L, feed culture medium is started, and the feeding rate is set in stages: Feeding rate constant: 2.5 mL / (L·min) from 10 to 18 h; 18-30 h: Increase feed rate to 4.0 mL / (L·min); 30-40 h: Reduce feed rate to 1.5 mL / (L·min); (5) Fermentation ends after 44 hours; (6) Post-treatment: Centrifuge the fermentation broth at 8000 rpm for 10 min, collect the bacterial sludge, add the protectant, mix the bacterial sludge and protectant at a ratio of 1:1, freeze dry, and obtain bacterial powder.

[0035] The seed tank culture medium in step (1) consists of: 10 g / L glucose, 5 g / L yeast extract, 10 g / L peptone, 5 g / L NaCl, and pH 7.0.

[0036] The basic fermentation medium formula in step (2) is as follows: glucose 15 g / L, molasses 5 g / L, soybean meal powder 35 g / L, yeast powder 25 g / L, urea 3 g / L, KH2PO4 2.5 g / L, MgSO4·7H2O 0.6 g / L, CaCl2 0.15 g / L, MnSO4 0.05 g / L, initial pH 7.0.

[0037] The formula for the feeding medium in step (4) is: 500 g / L glucose, 10 g / L yeast powder, and 0.5 g / L MgSO4·7H2O.

[0038] The formula for the protective agent in step (6) is: trehalose 2% (w / v), skim milk powder 5% (w / v), and glycerin 1% (v / v).

[0039] Comparative Example 2 A method for preparing microbial inoculants through high-density fermentation of a mixture of Bacillus subtilis and Bacillus licheniformis includes the following steps: (1) Activation and seed culture of strains: freeze-dried bacterial powder or slant culture of Bacillus subtilis and Bacillus cereus were taken and activated respectively; Activation of Bacillus subtilis: LB slant medium, cultured at 37℃ for 24 h; single colonies were picked and inoculated into LB liquid medium, cultured at 37℃ and 180 rpm for 12 h to obtain primary seed culture of Bacillus subtilis; Activation of Bacillus licheniformis: LB slant medium, cultured at 35℃ for 24 h; single colonies were picked and inoculated into LB liquid medium, cultured at 35℃ and 180 rpm for 14 h with shaking to obtain primary seed culture of Bacillus licheniformis; The above-mentioned primary seed culture was transferred to a 5 L seed tank culture medium at an inoculation rate of 8% by volume for expansion culture. Bacillus subtilis seed culture conditions: temperature 37℃, pH 7.0, aeration rate 1.0 vvm, stirring speed 200 rpm, culture time 9 h; Seed culture conditions for Bacillus licheniformis: temperature 35℃, pH 7.0, aeration rate 1.0 vvm, stirring speed 200 rpm, culture time 11 h; (2) Simultaneous inoculation: Add 35 L of basic fermentation medium to a 50 L fermenter, sterilize it, and then inoculate it with 1.75 L of Bacillus subtilis primary seed culture and 1.75 L of Bacillus licheniformis primary seed culture. Incubate at 36℃ and pH=6.8 for 8 h. (3) Fermentation control: The temperature was maintained at 36℃ for 0-16h, 38℃ for 16-30h, and 34℃ for 30-44h; the pH was maintained at 6.8 for 0-12h and adjusted to 7.5 after 12h; the dissolved oxygen was maintained at 35% by stirring at a rate of 500 rpm and aeration at a rate of 1.5 vvm. (4) Feeding: When the residual sugar drops to 3.0 g / L, feed culture medium is started, and the feeding rate is set in stages: Feeding rate constant: 2.5 mL / (L·min) from 10 to 18 h; 18-30 h: Increase feed rate to 4.0 mL / (L·min); 30-40 h: Reduce feed rate to 1.5 mL / (L·min); (5) Fermentation ends after 44 hours; (6) Post-treatment: Centrifuge the fermentation broth at 8000 rpm for 10 min, collect the bacterial sludge, add the protectant, mix the bacterial sludge and protectant at a ratio of 1:1, freeze dry, and obtain bacterial powder.

[0040] The seed tank culture medium in step (1) consists of: 10 g / L glucose, 5 g / L yeast extract, 10 g / L peptone, 5 g / L NaCl, and pH 7.0.

[0041] The basic fermentation medium formula in step (2) is as follows: glucose 15 g / L, molasses 5 g / L, soybean meal powder 35 g / L, yeast powder 25 g / L, urea 3 g / L, KH2PO4 2.5 g / L, MgSO4·7H2O 0.6 g / L, CaCl2 0.15 g / L, MnSO4 0.05 g / L, initial pH 7.0.

[0042] The formula for the feeding medium in step (4) is: 500 g / L glucose, 10 g / L yeast powder, and 0.5 g / L MgSO4·7H2O.

[0043] The formula for the protective agent in step (6) is: trehalose 2% (w / v), skim milk powder 5% (w / v), and glycerin 1% (v / v).

[0044] Comparative Example 3 A method for preparing microbial inoculants through high-density fermentation of a mixture of Bacillus subtilis and Bacillus cereus includes the following steps: (1) Activation and seed culture of strains: freeze-dried bacterial powder or slant culture of Bacillus subtilis and Bacillus cereus were taken and activated respectively; Activation of Bacillus subtilis: LB slant medium, cultured at 37℃ for 24 h; single colonies were picked and inoculated into LB liquid medium, cultured at 37℃ and 180 rpm for 12 h with shaking, to obtain Bacillus subtilis primary seed culture with OD600=4.2; Activation of Bacillus cereus: LB slant medium, cultured at 35℃ for 24 h; single colonies were picked and inoculated into LB liquid medium, cultured at 35℃ and 180 rpm for 14 h with shaking, to obtain primary seed culture of Bacillus cereus with OD600=3.8; The above-mentioned primary seed culture was transferred to a 5 L seed tank culture medium at an inoculation rate of 8% by volume for expansion culture. Bacillus subtilis seed culture conditions: temperature 37℃, pH 7.0, aeration rate 1.0 vvm, stirring speed 200 rpm, culture time 9 h, OD600 reached 4.5; Cultivation conditions for Bacillus cereus in seed tanks: temperature 35℃, pH 7.0, aeration rate 1.0 vvm, stirring speed 200 rpm, cultivation for 11 h, OD600 reached 4.0; (2) Simultaneous mixed inoculation: Add 35 L of basic fermentation medium to a 50 L fermenter, sterilize it, and then inoculate it with 1.75 L of Bacillus subtilis primary seed culture and 0.875 L of Bacillus cereus primary seed culture. Incubate at 36℃ and pH=6.8 for 8 h. (3) Fermentation control: The temperature was maintained at 36℃ for 0-16h, 38℃ for 16-30h, and 34℃ for 30-44h; the pH was maintained at 6.8 for 0-12h and adjusted to 7.5 after 12h; the dissolved oxygen was maintained at 35% by stirring at a rate of 500 rpm and aeration at a rate of 1.5 vvm. (4) Feeding: When the residual sugar drops to 3.0 g / L, feed culture medium is started, and the feeding rate is set in stages: Feeding rate constant: 2.5 mL / (L·min) from 10 to 18 h; 18-30 h: Increase feed rate to 4.0 mL / (L·min); 30-40 h: Reduce feed rate to 1.5 mL / (L·min); A 100 g / L malic acid solution was simultaneously added from 20 to 35 h at a rate of 0.6 g / (L·h). (5) Fermentation ends after 44 hours; (6) Post-treatment: Centrifuge the fermentation broth at 8000 rpm for 10 min, collect the bacterial sludge, add the protectant, mix the bacterial sludge and protectant at a ratio of 1:1, freeze dry, and obtain bacterial powder.

[0045] The seed tank culture medium in step (1) consists of: 10 g / L glucose, 5 g / L yeast extract, 10 g / L peptone, 5 g / L NaCl, and pH 7.0.

[0046] The basic fermentation medium formula in step (2) is as follows: glucose 15 g / L, molasses 5 g / L, soybean meal powder 35 g / L, yeast powder 25 g / L, urea 3 g / L, KH2PO4 2.5 g / L, MgSO4·7H2O 0.6 g / L, CaCl2 0.15 g / L, MnSO4 0.05 g / L, initial pH 7.0.

[0047] The formula for the feeding medium in step (4) is: 500 g / L glucose, 10 g / L yeast powder, and 0.5 g / L MgSO4·7H2O.

[0048] The formula for the protective agent in step (6) is: trehalose 2% (w / v), skim milk powder 5% (w / v), and glycerin 1% (v / v).

[0049] Test Example 1 Total viable cell count and spore formation rate of Examples 1, 2 and Comparative Examples 1-3 after 44 h of fermentation.

[0050] Table 1: Total viable count and spore formation rate

[0051] As shown in Table 1, the method for preparing microbial inoculants using high-density fermentation of mixed Bacillus subtilis and Bacillus cereus in Example 1 of this invention yielded the best total viable cell count and spore formation rate after 44 hours of fermentation. A comparison between Example 2 and Example 1 shows that malic acid feeding can further increase the viable cell count and spore formation rate. This invention suggests that this is because Bacillus cereus readily generates reactive oxygen species (ROS) during spore formation, and malic acid feeding can upregulate the expression of antioxidant enzyme genes and reduce ROS levels, thereby increasing the final viable cell count and spore formation rate.

[0052] Comparative Example 1 used single-strain fermentation of Bacillus subtilis, and the viable cell count was only 4.2 × 10⁻⁶. 11 CFU / mL, spore count 48%; after introducing Bacillus cereus, even with simultaneous inoculation in control ratio 3, the viable count increased to 6.2 × 10⁻⁶. 12 The CFU / mL concentration increased by approximately 15 times. This invention suggests that this is due to *Bacillus cereus*'s strong protease and amylase secretion capabilities, which can degrade macromolecular nutrients such as soybean meal and molasses in the culture medium into smaller amino acids and reducing sugars, providing *Bacillus subtilis* with a more readily available carbon and nitrogen source. Simultaneously, the two bacteria exhibit complementary metabolic pathways, reducing carbon source competition. This metabolic interaction is the fundamental reason why mixed-culture fermentation is superior to single-culture fermentation.

[0053] A comparison of Example 1 and Comparative Example 2 shows that the combination of Bacillus subtilis and Bacillus cereus used in Example 1 of this invention is superior to the combination of Bacillus subtilis and Bacillus licheniformis in the prior art. This invention argues that Bacillus licheniformis and Bacillus subtilis are closely related species with highly overlapping metabolic needs, easily leading to resource competition under limited nutrient conditions, resulting in the suppression of one in the later stages of fermentation. However, Bacillus cereus exhibits different tolerance ranges for temperature and pH compared to Bacillus subtilis, especially in the mid-to-late spore transformation stage. The segmented temperature control designed in this invention can optimize this process by: 36℃ in the early stage facilitates co-colonization of both bacteria; 38℃ in the mid-stage promotes spore transformation of Bacillus subtilis, while Bacillus cereus' metabolism slows down but it still survives at this temperature; and 34℃ in the late stage reduces apoptosis caused by heat stress. The segmented temperature control strategy is not significantly effective in the Bacillus subtilis and Bacillus cereus combination because their temperature preferences are more similar.

[0054] A comparison of Example 1 and Comparative Example 3 shows that the strategy of inoculating Bacillus subtilis first and then Bacillus cereus allows Bacillus subtilis to preferentially occupy the ecological niche, reduces early carbon source competition, and improves the stability of the mixed culture. In Comparative Example 3, the two bacteria competed for resources such as glucose and dissolved oxygen simultaneously at the beginning of fermentation. Although Bacillus subtilis had a slightly faster growth rate, the early intervention of Bacillus cereus led to a continuous competitive state between the two, disrupting the expected microbial community structure and accumulating more organic acids, which inhibited spore formation. In contrast, in Example 1, Bacillus subtilis was inoculated first and cultured for 8 hours to allow it to enter the mid-logarithmic growth phase (OD600≈3.5). At this point, Bacillus subtilis had consumed most of the initial glucose and secreted small-molecule amino acids, organic acids, and other metabolites. When Bacillus cereus was introduced at this time, the cereus bacteria could utilize these pre-digested nutrients to quickly adapt to the environment without fiercely competing with Bacillus subtilis for glucose. The two bacteria changed from a competitive relationship to a synergistic relationship, ultimately achieving a higher total bacterial count and spore rate.

[0055] In summary, the core technical feature of this invention lies in the synergistic effect between the strain combination of Bacillus subtilis and Bacillus cereus, the sequential inoculation strategy, and the malic acid-coupled feeding; none of these can be omitted. Specifically, sequential inoculation solves the problem of competitive imbalance, and malic acid feeding overcomes the bottleneck of oxidative damage. The combination of these two factors further improves the viable cell count and spore rate.

Claims

1. A method for preparing microbial inoculants through high-density fermentation of a mixture of Bacillus subtilis and Bacillus cereus, characterized in that, Includes the following steps: (1) Activate and culture Bacillus subtilis and Bacillus cereus respectively to obtain seed liquid; (2) Add basic fermentation culture medium to the fermenter, first inoculate with Bacillus subtilis seed liquid for culture, and after Bacillus subtilis enters the mid-log growth phase, inoculate with Bacillus cereus seed liquid for sequential mixed fermentation. (3) During the fermentation process, a combination of strategies including segmented temperature control, two-stage pH control, dynamic glucose control feeding, and malic acid coupled feeding was adopted to carry out high-density feeding batch fermentation. (4) After fermentation, collect the cells, add a protectant, and dry to obtain the microbial inoculant; The total viable count obtained by the method is ≥1.0×10⁻⁶. 12 CFU / mL.

2. The method for preparing microbial inoculants by high-density fermentation of Bacillus subtilis and Bacillus cereus as described in claim 1, characterized in that, In step (2), Bacillus subtilis is first inoculated and cultured for 6-10 h. After the OD600 reaches 2.5-5.0, Bacillus cereus is inoculated. The inoculation volume ratio of Bacillus subtilis to Bacillus cereus seed liquid is 2:

1.

3. The method for preparing microbial inoculants by high-density fermentation of Bacillus subtilis and Bacillus cereus as described in claim 1, characterized in that, In step (3), the temperature is controlled in stages: 36℃ for 0-16 h, 38℃ for 16-30 h, and 34℃ from 30 h to the end of fermentation; the pH is controlled in two stages: pH 6.7-6.9 for 0-12 h, and pH 7.4-7.6 after 12 h.

4. The method for preparing microbial inoculants by high-density fermentation of Bacillus subtilis and Bacillus cereus as described in claim 1, characterized in that, In step (3), the glucose dynamic control feeding is as follows: maintain the glucose concentration in the fermentation broth at 3.0-5.0 g / L, and set the feeding rate in segments: constant rate of 2.4-2.6 mL / (L·min) from the 10th to the 18th h, constant rate of 3.9-4.1 mL / (L·min) from the 18th to the 30th h, and constant rate of 1.4-1.6 mL / (L·min) from the 30th to the 40th h.

5. The method for preparing microbial inoculants by high-density fermentation of Bacillus subtilis and Bacillus cereus as described in claim 1, characterized in that, In step (3), the malic acid coupled feeding is as follows: during the 20-35 h of fermentation, malic acid solution is added to make the total malic acid addition 8-12 g / L of fermentation broth, and the flow rate is 0.5-1.0 g / (L·h).

6. The method for preparing microbial inoculants by high-density fermentation of Bacillus subtilis and Bacillus cereus as described in claim 1, characterized in that, The basic fermentation medium consists of: glucose 14-16 g / L, molasses 4-5 g / L, soybean meal powder 34-36 g / L, yeast powder 24-26 g / L, urea 2-3 g / L, KH2PO4 2.4-2.6 g / L, MgSO4·7H2O 0.5-0.7 g / L, CaCl2 0.14-0.16 g / L, MnSO4 0.04-0.06 g / L, with an initial pH of 7.

0.

7. The method for preparing microbial inoculants by high-density fermentation of Bacillus subtilis and Bacillus cereus as described in claim 1, characterized in that, The protective agent mentioned in step (4) consists of 1-2% (w / v) trehalose, 4-5% (w / v) skim milk powder, and 0.9-1% (v / v) glycerin.

8. The microbial inoculant prepared by the method for preparing microbial inoculants by high-density fermentation of Bacillus subtilis and Bacillus cereus as described in any one of claims 1-7, characterized in that, The viable count of the bacterial agent is ≥1.0×10⁻⁶. 12 CFU / g.

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