High-density fermentation method of fermented lactobacillus mucilaginosus, fermented lactobacillus mucilaginosus freeze-dried powder and preparation method

Abstract

The invention relates to a high-density fermentation method of lactobacillus mucus fermentation, lactobacillus mucus fermentation freeze-dried powder and a preparation method, and belongs to the technical field of microbial fermentation, and the method comprises the following steps: inoculating a bouillon culture medium with a lactobacillus mucus fermentation MSJK0025 strain for primary activation; inoculating the primary activation solution into an optimized culture medium for secondary propagation, wherein the optimized culture medium contains yeast peptone FP103, a beef extract, yeast extract powder FM902, glucose, dipotassium phosphate, citric acid monohydrate, sodium acetate, magnesium sulfate, manganese sulfate and Tween 80; and inoculating the second-stage expanding culture solution into the optimized culture medium, and introducing nitrogen into a fermentation tank with the pressure of 0.01-0.04 MPa to carry out third-stage fermentation, so as to obtain the fermented lactobacillus mucus MSJK0025 culture solution. By adopting the fermentation method disclosed by the invention, the thallus yield of the fermented lactobacillus mucus MSJK0025 can be improved, and the fermentation period is shortened.

Description

Technical Field

[0001] This application relates to the field of microbial fermentation technology, and in particular to a high-density fermentation method for fermenting *Lactobacillus myxoidis*, a freeze-dried powder of *Lactobacillus myxoidis* and its preparation method, especially to a high-density fermentation method for *Lactobacillus myxoidis* MSJK0025 based on nitrogen-pressure culture, a freeze-dried powder of *Lactobacillus myxoidis* MSJK0025 and its preparation method. Background Technology

[0002] Limosilactobacillus mucosae MSJK0025, a natural probiotic with excellent probiotic functions, has broad application prospects in functional foods and microecological preparations. However, its industrial application faces many technical bottlenecks. Traditional MRS culture media and conventional fermentation processes are insufficient to meet its high-density growth requirements. An unreasonable ratio of carbon and nitrogen sources leads to low viable cell counts in the fermentation broth, restricting the efficiency of subsequent powder preparation. Simultaneously, insufficient dynamic control of parameters such as temperature and pH further restricts proliferation efficiency, making it difficult to meet the high-yield demands of industrial production. Furthermore, the long fermentation time results in energy waste. High-density fermentation is a crucial step in preparing highly active Limosilactobacillus mucosae powder. The initial viable cell count of Limosilactobacillus mucosae MSJK0025 powder prepared using existing technologies is low, and the cell stability deteriorates after a period of storage at room temperature, making it prone to inactivation during storage. Summary of the Invention

[0003] The purpose of this application is to address the shortcomings of existing technologies in the conventional fermentation of *Lactobacillus myxoidis* MSJK0025, which suffers from low yield and long fermentation time. Therefore, this application proposes a high-density fermentation method for *Lactobacillus myxoidis*, a freeze-dried powder of *Lactobacillus myxoidis*, and a preparation method thereof. The fermentation method described in this application uses a specific optimized culture medium to cultivate *Lactobacillus myxoidis* MSJK0025 at high density, and nitrogen gas is introduced during the fermentation process while maintaining a constant pressure to increase the yield of *Lactobacillus myxoidis* MSJK0025 cells and shorten the fermentation cycle.

[0004] The *Lactobacillus fermentans* MSJK0025 used in this application has been disclosed in patent application number CN 202311070808.2 and was deposited on January 9, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 26426, located at the Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.

[0005] Firstly, the high-density fermentation method for *Lactobacillus mucinus* provided in this application adopts the following technical solution: the fermentation method includes the following steps: S1. Inoculate Lactobacillus fermentum strain MSJK0025 into broth medium for primary activation to obtain primary activation solution; S2. The primary activation solution is inoculated into an optimized culture medium for secondary expansion to obtain a secondary expansion solution, wherein the optimized culture medium contains yeast peptone FP103, beef extract, yeast extract FM902, glucose, dipotassium hydrogen phosphate, citric acid monohydrate, sodium acetate, magnesium sulfate, manganese sulfate and Tween 80. S3. The secondary expansion broth is inoculated into the optimized culture medium, and nitrogen gas is introduced into a fermenter at a pressure of 0.01~0.04 MPa for tertiary fermentation. Specifically, for example, the pressure can be 0.01 MPa, 0.02 MPa, 0.03 MPa or 0.04 MPa to obtain fermented Lactobacillus mucinus MSJK0025 culture medium.

[0006] In the early stages of fermentation, air (containing 21% oxygen) is present in the fermenter. Oxygen induces the production of reactive oxygen species (ROS, such as superoxide anion and hydrogen peroxide), which damage cell membrane phospholipids, DNA, and key metabolic enzymes (such as lactate dehydrogenase (LDH) and phosphofructokinase (PFK), leading to apoptosis. Introducing nitrogen gas can prevent cells from prematurely entering the apoptosis phase. Simultaneously, the introduction of nitrogen gas generates microbubbles, creating a gentle stirring effect that ensures even distribution of nutrients such as carbon, nitrogen, and minerals in the culture medium, preventing "growth arrest" caused by localized nutrient deficiencies. Furthermore, it reduces the speed of the agitator, preventing cell shearing and loss of activity, and allows for rapid diffusion of fermentation products (lactic acid), avoiding excessively low local pH (<4.0) that inhibits LDH production. The active enzyme (a key rate-limiting enzyme in lactic acid fermentation) maintains the continuous operation of the glycolysis pathway, thereby increasing the yield of fermented Lactobacillus mucilaginosus MSJK0025 cells and shortening the fermentation cycle. By regulating the gas pressure inside the fermenter, cell metabolism and environmental stability are further optimized. Moderate pressurization can create a gradient of "high pressure outside the membrane → low pressure inside the membrane," which helps nutrients cross the cell membrane and enter the cell, reducing the energy consumption of active transport, while improving substrate utilization and accelerating cell proliferation. Precise pressure control can balance the pressure difference between CO2 release and nitrogen introduction, maintain stable system pressure, and avoid "stress responses" of cells due to environmental fluctuations (such as stopping proliferation and initiating apoptosis pathways). At the same time, it reduces the production of foam in the fermentation broth (foam can cause oxygen to re-enter the system, disrupting the anaerobic environment). When the pressure of the fermenter is less than 0.01 MPa, it is difficult to achieve an anaerobic environment, and the nutrient transfer efficiency is reduced. When the pressure of the fermenter is greater than 0.04 MPa, excessive pressure can easily cause cell aggregation, cell membrane damage, and other situations that lead to bacterial inactivation, while also affecting the stability of the bacterial powder in the later stages.

[0007] "Nitrogen introduction + pressure control" does not work independently, but forms a closed loop of "environmental optimization → metabolic enhancement → accelerated proliferation," producing a synergistic effect of 1+1>2. 1. First, eliminate inhibition, then enhance metabolism: Nitrogen introduction first creates a "safe fermentation environment" free of oxygen and bacteria, preventing cells from entering growth stagnation due to oxidative damage or nutrient competition; on this basis, pressure control improves nutrient absorption efficiency and metabolic rate, allowing cells to enter a virtuous cycle of "rapid proliferation + efficient metabolism"; the inert nature of nitrogen avoids changes in gas composition during pressure regulation, ensuring a stable anaerobic environment, so that the nutrient absorption enhancement brought about by the pressure gradient synergizes with the oxidative damage-free environment; 2. Reduce metabolic burden and byproducts: Oxygen stress causes cells to consume a lot of energy for ROS removal (such as SOD synthesis), while the anaerobic environment + stable pressure can reduce this "ineffective energy consumption," allowing more carbon sources and energy to be used for cell proliferation and target product (lactic acid, EPS) synthesis, reducing the proportion of byproducts (such as acetic acid, ethanol).

[0008] In a specific embodiment of the fermentation method described in this application, the frozen glycerol storage tube containing fermenting Lactobacillus mucinus MSJK0025 is placed in a water bath at 37±1℃ for rapid thawing, and then the fermenting Lactobacillus mucinus MSJK0025 is subjected to primary activation.

[0009] In a specific embodiment of the fermentation method described in this application, the broth culture medium is a conventional choice in the art. Specifically, the composition of the broth culture medium is: 10.0 g / L peptone, 8.0 g / L beef extract, 4.0 g / L yeast extract, 20.0 g / L glucose, 2.0 g / L dipotassium hydrogen phosphate, 2.0 g / L diammonium hydrogen citrate, 5.0 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.04 g / L manganese sulfate, and 1.0 g / L Tween 80, with a pH of 5.7±0.2. Peptone, beef extract, and yeast extract provide nitrogen, vitamins, and growth factors; glucose is a fermentable sugar; dipotassium hydrogen phosphate is an acid-base buffer; diammonium hydrogen citrate, magnesium sulfate, manganese sulfate, Tween 80, and sodium acetate provide growth factors for cultivating various lactic acid bacteria. Its components can also inhibit certain miscellaneous bacteria and neutralize cytotoxic substances, providing a good growth environment for lactic acid bacteria.

[0010] Optionally, in the optimized culture medium, the content of yeast peptone FP103 is 18-25 g / L, specifically, for example, 18 g / L, 22 g / L, or 25 g / L; the content of beef extract is 2-6 g / L, specifically, for example, 2 g / L, 3 g / L, or 6 g / L; the content of yeast extract FM902 is 4-8 g / L, specifically, for example, 4 g / L, 6 g / L, or 8 g / L; the content of glucose is 28-32 g / L, specifically, for example, 28 g / L, 30 g / L, or 32 g / L; the content of dipotassium hydrogen phosphate is 1-4 g / L, specifically, for example, 1 g / L, 2 g / L, or 4 g / L; the content of citric acid monohydrate is 1-4 g / L, specifically, for example, 1 g / L, 2 g / L, or 4 g / L; and the content of sodium acetate is 3-8 g / L. The concentration of manganese sulfate is 0.01-0.05 g / L, specifically, for example, 0.01 g / L, 0.02 g / L, or 0.05 g / L; the concentration of Tween 80 is 0.1-0.4 g / L, specifically, for example, 0.1 g / L, 0.2 g / L, or 0.4 g / L. Specifically, the pH of the optimized culture medium is adjusted to 7.0 and sterilized at 115 °C for 20 min. In actual operation, in order to reduce the generation of foam and prevent foam from affecting the quality of the optimized culture medium, polydimethylsiloxane emulsion can be added. The content of the polydimethylsiloxane emulsion is 0.01~0.08 g / L, specifically, for example, it can be 0.01 g / L, 0.05 g / L or 0.08 g / L, preferably 0.05 g / L.

[0011] In a preferred embodiment, the optimized culture medium contains 22 g / L of yeast peptone FP103, 3 g / L of beef extract, 6 g / L of yeast extract FM902, 30 g / L of glucose, 2 g / L of dipotassium hydrogen phosphate, 2 g / L of citric acid monohydrate, 5 g / L of sodium acetate, 0.5 g / L of magnesium sulfate, 0.02 g / L of manganese sulfate, and 0.2 g / L of Tween 80.

[0012] The inventors of this application used the number of viable bacteria in the bacterial solution, the yield of the bacterial sludge, and the OD (octane number) as the basis for their claims. 600The primary evaluation indicator was the viable cell count. Forty-five formulations were screened, with different carbon sources (anhydrous glucose, white sugar, brown sugar, fructooligosaccharides, isomaltooligosaccharides, inulin), nitrogen sources (yeast extract FM502, yeast extract FM503, yeast extract 985, yeast extract 986, yeast extract 786, yeast peptone 558, soybean peptone, wheat peptone, pea protein, beef extract, etc.), and the carbon-nitrogen source ratio being used as the main evaluation indicator. Through small-scale validation, the optimal culture medium for fermenting *Lactobacillus mucilaginosus* MSJK0025 was finally determined. Under this optimized medium, the viable cell count in a 5L fermenter was ≥6.0 × 10⁻⁶. 9 CFU / mL, mycelium sludge yield ≥1.70%, OD 600 Value ≥ 12.000.

[0013] Optionally, in step S3, the flow rate of nitrogen gas introduced into the fermenter is 0.001~0.004 VVM, specifically, for example, it can be 0.001 VVM, 0.002 VVM, 0.003 VVM or 0.004 VVM.

[0014] By introducing a certain amount of nitrogen gas into the fermenter through the above technical solution, the yield of fermented Lactobacillus mucinus MSJK0025 cells can be further increased, and the fermentation cycle can be further shortened.

[0015] Optionally, in step S1, the inoculation amount of the fermenting *Lactobacillus mucinus* MSJK0025 strain is 3% of the volume of the broth culture medium; in step S2, the inoculation amount of the primary activation solution is 3% of the volume of the optimized culture medium; and in step S3, the inoculation amount of the secondary expansion solution is 3% of the volume of the optimized culture medium.

[0016] Through the above technical solutions, such as Figure 1 As shown, when the inoculum size is 3%, the number of viable bacteria further increases.

[0017] Optionally, the culture conditions for the primary activation, secondary expansion and tertiary fermentation include: a culture temperature of 37 ℃ and a culture time of 10~16 h; in step S3, the initial pH value of the tertiary fermentation is 7.0±0.2, preferably 7.0.

[0018] Through the above technical solutions, such as Figures 2-4 The cell OD values ​​shown were obtained when the culture temperature was 37 ℃, the initial pH was 7.0, and the culture time was 16 h. 600 Both the value and yield are high.

[0019] In a specific embodiment of the fermentation method described in this application, in step S3, the culture conditions for the three-stage fermentation further include: fermentation culture at a rotation speed of 150 rpm.

[0020] Secondly, this application provides a method for preparing lyophilized powder of *Lactobacillus fermentans*, the preparation method comprising the following steps: (1) Centrifuge the fermented Lactobacillus mucinus MSJK0025 culture medium obtained by any one of the fermentation methods described in claims 1 to 5 to obtain bacterial sludge; (2) The bacterial mud is mixed with a freeze-drying protectant and then freeze-dried under vacuum to obtain a freeze-dried powder of fermented Lactobacillus mucilaginosus, wherein the freeze-drying protectant includes trehalose, sodium ascorbate, monosodium glutamate and glycerol.

[0021] In order to obtain a highly active and stable freeze-dried powder, the inventors of this application screened the raw materials for freeze-drying protectants, and compounded trehalose, maltodextrin, white sugar, xylooligosaccharides, lactose, sodium ascorbate, monosodium glutamate, glycerol, and potassium dihydrogen phosphate. It was found that the compound of trehalose, sodium ascorbate, monosodium glutamate, and glycerol had the best protective effect on Lactobacillus fermentum MSJK0025.

[0022] In a specific embodiment of the preparation method described in this application, in step (1), the centrifugation conditions include: a temperature of 4 ℃, a rotation speed of 5000 rpm, and a time of 15 min.

[0023] Optionally, based on 100% of the total mass of the freeze-drying protectant, the freeze-drying protectant comprises: 10-14% trehalose, specifically, for example, 10%, 11%, 12%, 13%, or 14%; 0.1-0.5% sodium ascorbate, specifically, for example, 0.1%, 0.2%, 0.3%, 0.4%, or 0.5%; 1-5% monosodium glutamate, specifically, for example, 1%, 2%, 3%, 4%, or 5%; 1-5% glycerol, specifically, for example, 1%, 2%, 3%, 4%, or 5%, with the balance being water; in a preferred embodiment, based on 100% of the total mass of the freeze-drying protectant, the freeze-drying protectant comprises: 12-13% trehalose, 0.1-0.2% sodium ascorbate, 1-2% monosodium glutamate, 1-2% glycerol, with the balance being water.

[0024] By limiting the content of trehalose, sodium ascorbate, monosodium glutamate, and glycerol in the freeze-drying protectant to a certain range through the above technical solution, the initial viable count of the prepared freeze-dried powder can be further increased, and the stability of the freeze-dried powder can be significantly improved.

[0025] Optionally, in step (2), the mass ratio of the fungal sludge to the freeze-drying protectant is 1:12 to 18, specifically, for example, it can be 1:12, 1:13, 1:14, 1:15, 1:16, 1:17 or 1:18; in the preferred case, the mass ratio of the fungal sludge to the freeze-drying protectant is 1:14 to 16.

[0026] By limiting the mass ratio of the bacterial sludge to the freeze-drying protectant within a certain range through the above technical solution, the initial viable count of the prepared freeze-dried powder can be further increased, and the stability of the freeze-dried powder can be effectively improved.

[0027] In a specific embodiment of the preparation method described in this application, the vacuum freeze-drying procedure in step (2) is shown in Table 1: Table 1 Vacuum freeze-drying process

[0028] Thirdly, this application provides a freeze-dried Lactobacillus fermentans powder obtained according to the above-described method for preparing freeze-dried Lactobacillus fermentans powder.

[0029] In summary, this application includes at least one of the following beneficial technical effects: 1. The high-density fermentation method described in this application uses a specific optimized culture medium to cultivate *Lactobacillus mucinus* MSJK0025 at high density, and introduces nitrogen gas and applies a specific pressure during the fermentation process, which enables the viable cell count in the fermentation broth to reach 4.65 × 10⁻⁶ after 10 hours of fermentation. 10 The cfu / mL level was significantly higher than the original fermentation level (Comparative Example 1, 4.2 × 10⁻⁶). 9 (cfu / mL, no nitrogen gas introduced, and pressure 0), and the fermentation time was shortened by 6 h; 2. The Lactobacillus fermentum MSJK0025 cells obtained by the high-density fermentation method in this application were used to prepare lyophilized powder with a lyophilization protectant composed of trehalose, sodium ascorbate, monosodium glutamate and glycerol. The initial viable count of the lyophilized powder was effectively increased, and the stability of the lyophilized powder (stored at 25°C for 1 year) was also significantly improved. Attached Figure Description

[0030] Figure 1 Figure showing the effect of different inoculum sizes on the growth of fermenting Lactobacillus mucilaginosus MSJK0025; Figure 2 Figure 1 shows the effect of different temperatures on the growth of fermented Lactobacillus mucilaginosus MSJK0025. Figure 3 Figure showing the effect of different initial pH values ​​on the growth of fermented Lactobacillus mucilaginosus MSJK0025; Figure 4Figure showing the effect of different culture durations on the growth of fermented Lactobacillus mucinus MSJK0025; Figure 5 The graph shows the stability test results of the lyophilized Lactobacillus fermentation powder prepared in Example 6. Figure 6 The figure shows the stability test results of the lyophilized Lactobacillus fermentation powder prepared in Comparative Example 3. Detailed Implementation

[0031] The following combination Figures 1-6 The present application will be further described in detail with reference to specific embodiments.

[0032] The following examples further illustrate the high-density fermentation method for *Lactobacillus mucinus*, the freeze-dried *Lactobacillus mucinus* powder, and the preparation method described in this application. These examples are implemented based on the technical solution of this application, providing detailed implementation methods and specific operating procedures. However, the scope of protection of this application is not limited to the following examples.

[0033] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods in the art. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available.

[0034] Broth culture medium: MRS broth (product number: HB0384-1), with the following components: 10.0 g / L peptone, 8.0 g / L beef extract, 4.0 g / L yeast extract, 20.0 g / L glucose, 2.0 g / L dipotassium hydrogen phosphate, 2.0 g / L diammonium hydrogen citrate, 5.0 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.04 g / L manganese sulfate, 1.0 g / L Tween 80, pH 5.7, Qingdao Haibo Biotechnology Co., Ltd.; Yeast peptone FP103: Purchased from Angel Yeast Co., Ltd.; Beef extract: purchased from Qingdao Haibo Biotechnology Co., Ltd.; Yeast extract powder FM902: Purchased from Angel Yeast Co., Ltd.; Tween 80: Purchased from Sinopharm Chemical Reagent Co., Ltd.; Polydimethylsiloxane emulsion: Purchased from Chongqing Yinglong Chemical Plant; Trehalose: Purchased from Shandong Tianli Pharmaceutical Co., Ltd.; Sodium ascorbate: Purchased from Sinopharm Chemical Reagent Co., Ltd. Monosodium glutamate (MSG): Purchased from Hangzhou West Lake MSG Co., Ltd. Glycerin: Purchased from Sinopharm Chemical Reagent Co., Ltd.; Soluble starch: Purchased from Sinopharm Chemical Reagent Co., Ltd.; Skim milk powder: Purchased from Fonterra Ltd., New Zealand; Xylooligosaccharides: Purchased from Shandong Longli Biotechnology Co., Ltd.

[0035] The fermenting mucoid lactobacillus used in the embodiments of this application is (Limosilactobacillus mucosae) MSJK0025.

[0036] The vacuum freeze-drying procedures in each embodiment are shown in Table 1. Example 1

[0037] A high-density fermentation method for Lactobacillus mucinus, the fermentation method comprising the following steps: S1. The fermented Lactobacillus mucinus MSJK0025, which was frozen in glycerol tubes, was rapidly thawed in a water bath at 37°C. The thawed fermented Lactobacillus mucinus MSJK0025 strain was inoculated into broth medium at a 3% inoculation rate and subjected to primary activation at 37°C for 16 h to obtain the primary activation solution. S2. The primary activation solution is inoculated into the optimized culture medium at an inoculation rate of 3%, and secondary expansion is carried out at 37°C for 16 hours to obtain the secondary expansion culture medium. The optimized culture medium includes: 22 g / L yeast peptone FP103, 3 g / L beef extract, 6 g / L yeast extract FM902, 30 g / L glucose, 2 g / L dipotassium hydrogen phosphate, 2 g / L citric acid monohydrate, 5 g / L sodium acetate, 0.5 g / L magnesium sulfate, 0.02 g / L manganese sulfate, 0.2 g / L Tween 80 and 0.05 g / L polydimethylsiloxane emulsion, with the remainder being water. The pH of the optimized culture medium is adjusted to 7.0 and sterilized at 115°C for 20 min. S3. Start the fermenter in advance, adjust the pH of the optimized culture medium to 7.0 using 20% ​​sodium hydroxide solution, and introduce nitrogen into the fermenter to reduce the oxygen content in the culture medium to 0. Inoculate the secondary expansion broth into the optimized culture medium at an inoculation rate of 3%, and continuously introduce nitrogen at a flow rate of 0.002 VVM. Maintain the fermenter pressure at 0.02 MPa, and ferment at 37 ℃ and 150 rpm for 10 h to obtain fermented Lactobacillus mucilaginosus MSJK0025 culture medium. Example 2

[0038] A high-density fermentation method for Lactobacillus mucinus is implemented according to Example 1, except that the nitrogen flow rate is 0.001 VVM and the fermenter pressure is maintained at 0.01 MPa. Example 3

[0039] A high-density fermentation method for Lactobacillus mucinus is implemented according to Example 1, except that the nitrogen flow rate is 0.003 VVM and the fermenter pressure is maintained at 0.04 MPa. Example 4

[0040] A high-density fermentation method for *Lactobacillus mucinus* is implemented according to Example 1, except that the optimized culture medium comprises: 18 g / L yeast peptone FP103, 2 g / L beef extract, 4 g / L yeast extract FM902, 28 g / L glucose, 1 g / L dipotassium hydrogen phosphate, 1 g / L citric acid monohydrate, 3 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.01 g / L manganese sulfate, 0.1 g / L Tween 80, and 0.03 g / L polydimethylsiloxane emulsion, with the balance being water. Example 5

[0041] A high-density fermentation method for *Lactobacillus mucinus* is implemented according to Example 1, except that the optimized culture medium comprises: 25 g / L yeast peptone FP103, 6 g / L beef extract, 8 g / L yeast extract FM902, 32 g / L glucose, 4 g / L dipotassium hydrogen phosphate, 4 g / L citric acid monohydrate, 8 g / L sodium acetate, 0.8 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 0.4 g / L Tween 80, and 0.05 g / L polydimethylsiloxane emulsion, with the balance being water. Example 6

[0042] A method for preparing lyophilized Lactobacillus fermentum powder, the method comprising the following steps: (1) The culture medium of fermented Lactobacillus mucinus MSJK0025 obtained in Example 1 was centrifuged at 4 ℃ and 5000 rpm for 15 min to obtain bacterial sludge; (2) The bacterial mud and the freeze-drying protectant are mixed at a mass ratio of 1:15, and then the mixture is freeze-dried under vacuum according to Table 1 to obtain freeze-dried Lactobacillus fermentum powder. The freeze-drying protectant includes: 12% trehalose, 0.1% sodium ascorbate, 1% sodium glutamate, 1% glycerol, and the remainder is water. Example 7

[0043] A method for preparing freeze-dried Lactobacillus fermentum powder is carried out according to Example 6, except that the bacterial sludge and freeze-drying protectant are mixed at a mass ratio of 1:12. Example 8

[0044] A method for preparing freeze-dried Lactobacillus fermentum powder is carried out according to Example 6, except that the bacterial sludge and freeze-drying protectant are mixed at a mass ratio of 1:18. Example 9

[0045] A method for preparing lyophilized Lactobacillus fermentum powder is carried out according to Example 6, except that the lyophilization protectant includes: 10% trehalose, 0.5% sodium ascorbate, 5% monosodium glutamate, 5% glycerol, and the balance being water. Example 10

[0046] A method for preparing lyophilized Lactobacillus fermentum powder is carried out according to Example 6, except that the lyophilization protectant includes: 14% trehalose, 0.3% sodium ascorbate, 3% monosodium glutamate, 3% glycerol, and the balance being water.

[0047] Comparative Example 1 A high-density fermentation method for *Lactobacillus mucinus* is implemented according to Example 1, except that nitrogen gas is not introduced in step S3. Specifically, the fermenter is started in advance, the pH of the optimized culture medium is adjusted to 7.0 using 20% ​​sodium hydroxide solution, the secondary expansion broth is inoculated into the optimized culture medium at a 3% inoculation rate, the fermenter pressure is 0 MPa, and fermentation is carried out at 37 ℃ and 150 rpm for 16 h to obtain *Lactobacillus mucinus* MSJK0025 culture medium.

[0048] Comparative Example 2 A high-density fermentation method for *Lactobacillus mucinus* is implemented according to Example 1, except that nitrogen gas is not introduced in step S3. Specifically, the fermenter is started in advance, the pH of the optimized culture medium is adjusted to 7.0 using 20% ​​sodium hydroxide solution, the secondary expansion broth is inoculated into the optimized culture medium at a 3% inoculation rate, the pressure of the fermenter is maintained at 0.06 MPa, and fermentation is carried out at 37 ℃ and 150 rpm for 16 h to obtain fermented *Lactobacillus mucinus* MSJK0025 culture medium.

[0049] Comparative Example 3 A method for preparing freeze-dried Lactobacillus fermentum powder was carried out according to Example 6, except that all of the Lactobacillus fermentum MSJK0025 obtained in Example 1 were replaced with Lactobacillus fermentum MSJK0025 obtained in Comparative Example 1.

[0050] Comparative Example 4 A method for preparing freeze-dried Lactobacillus fermentum powder was carried out according to Example 6, except that all of the Lactobacillus fermentum MSJK0025 obtained in Example 1 were replaced with Lactobacillus fermentum MSJK0025 obtained in Comparative Example 2.

[0051] Comparative Example 5 A method for preparing lyophilized Lactobacillus fermentum powder is carried out according to Example 6, except that the lyophilization protectant includes: 10% soluble starch, 3% sodium VC salt, 12% skim milk powder, 14% xylooligosaccharide, and the balance being water.

[0052] Test case Absorbance test: The spectrophotometer was set to 600 nm wavelength. The absorbance of the bacterial suspension was zeroed to optimize the culture medium. If the absorbance value exceeded 0.600, the suspension was diluted before measurement. Viable cell count test: The number of viable cells per unit is calculated using the plate count method; Stability test of freeze-dried powder: The test was conducted in accordance with the "National Food Safety Standard for Microbiological Examination of Food - Lactic Acid Bacteria Examination" GB4789.35, and the stability test temperature was 25℃.

[0053] In step S3 of Example 1 and Comparative Example 1, samples were taken every 2 hours to measure absorbance and viable count, and the results are shown in Table 2 and Table 3, respectively. Table 2

[0054] As can be seen from Table 2, Example 1 used a specific optimized culture medium to culture Lactobacillus mucinus MSJK0025 at high density, and nitrogen gas was introduced during the fermentation process while maintaining a constant pressure. As a result, the absorbance value of the fermented Lactobacillus mucinus MSJK0025 culture medium was high, indicating that the concentration of fermented Lactobacillus mucinus MSJK0025 was high. In contrast, Comparative Example 1 did not introduce nitrogen gas during the culture, and the pressure was not within the specified range. As a result, the absorbance value of the fermented Lactobacillus mucinus MSJK0025 culture medium was low, indicating that the concentration of Lactobacillus mucinus MSJK0025 was low.

[0055] Table 3

[0056] As shown in Table 3, the viable cell count of the bacterial solution in Example 1 reached 4.65 × 10⁻⁶ after 10 hours of fermentation. 10 The cfu / mL concentration was significantly higher than that of Comparative Example 1 (4.2 × 10⁻⁶). 9 The high-density fermentation method described in this application uses a specific optimized culture medium to culture *Lactobacillus mucinus* MSJK0025 at high density, and introduces nitrogen gas and applies specific pressure during the fermentation process, which can effectively increase the yield of *Lactobacillus mucinus* MSJK0025 cells and shorten the fermentation cycle.

[0057] The viable cell counts of *Lactobacillus fermentans* MSJK0025 culture media obtained in Examples 1-5 and Comparative Examples 1-2 were tested, and the results are shown in Table 4. Table 4

[0058] As shown in Table 4, the viable cell counts of *Lactobacillus fermentatus* MSJK0025 culture media obtained in Examples 1-5 were significantly higher than those obtained in Comparative Examples 1-2. This indicates that the fermentation method described in this application uses a specific optimized culture medium to culture *Lactobacillus fermentatus* MSJK0025 at high density, and introduces nitrogen gas and maintains a constant pressure during the fermentation process, which is more conducive to the growth of *Lactobacillus fermentatus* and results in higher culture efficiency.

[0059] The initial viable counts of the lyophilized Lactobacillus fermentans prepared in Examples 6-10 and Comparative Examples 3-5 were tested, and the results are shown in Table 5. Table 5

[0060] The freeze-dried powders of *Lactobacillus fermentatus* prepared in Example 6 and Comparative Example 3 were subjected to freeze-dried powder stability tests, with viable cell count as the test data. The test periods were 1 month, 2 months, 3 months, 6 months, 9 months, and 12 months, and the test results are as follows: Figure 5 and Figure 6 As shown, according to Table 5 and Figures 5-6 The data shows that when the Lactobacillus fermentum MSJK0025 cells obtained by the high-density fermentation method in this application are combined with a freeze-drying protectant composed of trehalose, sodium ascorbate, monosodium glutamate and glycerol to prepare freeze-dried powder, the initial viable count of the freeze-dried powder is effectively increased, and the stability of the freeze-dried powder is also significantly improved.

[0061] The freeze-dried powders of *Lactobacillus fermentum* prepared in Examples 6-10 and Comparative Examples 3-5 were subjected to freeze-dried powder stability tests. The viable cell count was used as the test data, with the unit of viable cell count being cfu / g. The test periods were 1 month, 2 months, 3 months, 6 months, 9 months, and 12 months. The test results are shown in Table 6. Table 6

[0062] As can be seen from the data in Table 6, compared to the fermentation culture of Lactobacillus mucinus MSJK0025 without nitrogen gas and with pressure outside the specified range, the freeze-dried powder prepared by using the specific optimized culture medium in this application to culture Lactobacillus mucinus MSJK0025 at high density, purging nitrogen gas and maintaining constant pressure during the fermentation culture, and using a specific cryoprotectant, has significantly improved stability at room temperature (25℃).

[0063] The embodiments described in this specific implementation are preferred embodiments of this application and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made to the structure, shape, and principle of this application should be covered within the scope of protection of this application.

Claims

1. A method for high-density fermentation of *Lactobacillus mucilaginosus*, characterized in that, The fermentation method includes the following steps: S1. Inoculate Lactobacillus fermentum strain MSJK0025 into broth medium for primary activation to obtain primary activation solution; S2. The primary activation solution is inoculated into an optimized culture medium for secondary expansion to obtain a secondary expansion solution, wherein the optimized culture medium contains yeast peptone FP103, beef extract, yeast extract FM902, glucose, dipotassium hydrogen phosphate, citric acid monohydrate, sodium acetate, magnesium sulfate, manganese sulfate and Tween 80. S3. The secondary expansion broth is inoculated into the optimized culture medium, and nitrogen gas is introduced into a fermenter at a pressure of 0.01~0.04 MPa for tertiary fermentation to obtain fermented Lactobacillus mucinus MSJK0025 culture medium.

2. The high-density fermentation method for *Lactobacillus mucilaginosus* according to claim 1, characterized in that, In step S3, the flow rate of nitrogen gas introduced into the fermenter is 0.001~0.004 VVM.

3. The high-density fermentation method for *Lactobacillus mucilaginosus* according to claim 1, characterized in that, In the optimized culture medium, the content of yeast peptone FP103 is 18-25 g / L, the content of beef extract is 2-6 g / L, the content of yeast extract FM902 is 4-8 g / L, the content of glucose is 28-32 g / L, the content of dipotassium hydrogen phosphate is 1-4 g / L, the content of citric acid monohydrate is 1-4 g / L, the content of sodium acetate is 3-8 g / L, the content of magnesium sulfate is 0.2-0.8 g / L, the content of manganese sulfate is 0.01-0.05 g / L, and the content of Tween 80 is 0.1-0.4 g / L.

4. The high-density fermentation method for *Lactobacillus mucilaginosus* according to claim 1, characterized in that, In step S1, the inoculation amount of the fermenting Lactobacillus mucin MSJK0025 strain is 3% of the volume of the broth culture medium; in step S2, the inoculation amount of the primary activation solution is 3% of the volume of the optimized culture medium; in step S3, the inoculation amount of the secondary expansion solution is 3% of the volume of the optimized culture medium.

5. The high-density fermentation method for *Lactobacillus mucilaginosus* according to claim 1, characterized in that, The culture conditions for the primary activation, secondary expansion and tertiary fermentation include: a culture temperature of 37 ℃ and a culture time of 10~16 h; In step S3, the initial pH value of the tertiary fermentation is 7.

0.

6. A method for preparing lyophilized powder of fermented Lactobacillus mucilaginosus, characterized in that, The preparation method includes the following steps: (1) Centrifuge the fermented Lactobacillus mucinus MSJK0025 culture medium obtained by any one of the fermentation methods described in claims 1 to 5 to obtain bacterial sludge; (2) The bacterial mud is mixed with a freeze-drying protectant and then freeze-dried under vacuum to obtain a freeze-dried powder of fermented Lactobacillus mucilaginosus, wherein the freeze-drying protectant includes trehalose, sodium ascorbate, monosodium glutamate and glycerol.

7. The method for preparing lyophilized Lactobacillus fermentum powder according to claim 6, characterized in that, Based on the total mass of the freeze-drying protectant as 100%, the freeze-drying protectant comprises: 10-14% trehalose, 0.1-0.5% sodium ascorbate, 1-5% monosodium glutamate, 1-5% glycerol, and the balance being water.

8. The method for preparing lyophilized Lactobacillus fermentum powder according to claim 7, characterized in that, Based on the total mass of the freeze-drying protectant as 100%, the freeze-drying protectant comprises: 12-13% trehalose, 0.1-0.2% sodium ascorbate, 1-2% monosodium glutamate, 1-2% glycerol, and the balance being water.

9. The method for preparing lyophilized Lactobacillus fermentum powder according to claim 6, characterized in that, In step (2), the mass ratio of the bacterial sludge to the freeze-drying protectant is 1:12~18.

10. A freeze-dried powder of fermented Lactobacillus mucinus obtained by the preparation method according to any one of claims 6 to 9.

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