Lactobacillus plantarum high-density fermentation medium and fermentation method

Abstract

The invention discloses a lactobacillus plantarum high-density fermentation medium and a fermentation method, and belongs to the field of microbial fermention.The high-density fermentation medium comprises 1-2% of peptone, 1-2% of glucose, 1-2% of beef extract, self-made yeast powder, Tween, sodium acetate, diammonium hydrogen citrate, dipotassium phosphate and the like. According to the method, a lactobacillus plantarum seed solution is inoculated into a high-density fermentation culture medium for fermentation, in the fermentation process, the fermentation temperature is kept at 37 DEG C, the pH value is controlled to be 6.5-7.0 through 10% ammonia water, the dissolved oxygen content is kept to be 6-9 mg / L, a continuous fed-batch feeding mode is adopted, and the feeding speed is regulated in real time according to the biomass and nutrient substance concentration of fermentation liquor; and carrying out high-density fermentation with the total fermentation time of 12 hours. According to the method, high-density fermentation of lactobacillus plantarum is realized, the production cost is reduced, and the production efficiency is improved.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, specifically relating to a high-density fermentation culture medium and fermentation method for Lactobacillus plantarum. Background Technology

[0002] Probiotics are "live microorganisms that, when ingested in sufficient quantities, provide health benefits to the host." Lactic acid bacteria (LAB), as the main type of probiotic microorganism, are classified as Generally Recognized As Safe (GRAS) by the U.S. Food and Drug Administration (FDA). In the digestive system, probiotics can synthesize digestive enzymes, participate in the digestion of nutrients in the intestines, stimulate the body to secrete digestive enzymes, and promote nutrient absorption by improving intestinal structure. In terms of immune regulation, their structural components can activate or stimulate the host's immune system. In maintaining intestinal health, they can maintain the balance of the gut microbiota and inhibit inflammation. In addition, they also possess antioxidant properties and protect the intestinal mucosal barrier. Driven by modern technology, probiotics are widely used in cosmetics, food, pharmaceuticals, and other fields.

[0003] *Lactobacillus plantarum*, an important member of the probiotic family, belongs to the kingdom Bacteria, phylum Firmicutes, class Bacillus, order Lactobacilli, family Lactobacillus, and genus *Lactobacillus*. It is a Gram-positive lactic acid bacterium, presenting as a straight, round-ended rod-shaped organism, occurring singly or in chains. Its optimal growth temperature is 30℃-37℃, and it is anaerobic or facultatively anaerobic, with an optimal pH of approximately 6.5. It is a homofermentative lactic acid bacterium. Through glycolysis, it converts sugars into lactic acid. After colonizing the human gut, it can maintain host health through various mechanisms, playing a particularly important role in the prevention and treatment of intestinal diseases such as ulcerative colitis.

[0004] The industrial production of *Lactobacillus plantarum* currently faces numerous challenges. Existing traditional culture media have unreasonable nutrient ratios, with imbalances in carbon, nitrogen, and trace elements, and lack key growth-promoting substances, resulting in slow cell growth and low biomass. While yeast extract is rich in nutrients, existing culture media are poorly utilized and formulated, failing to fully leverage its advantages. In terms of fermentation methods, traditional processes suffer from imprecise control of fermentation conditions and unscientific feeding strategies, failing to meet the high efficiency and stability requirements of large-scale industrial production. Therefore, there is an urgent need to develop optimized *Lactobacillus plantarum* yeast extract culture medium formulations and efficient, high-density fermentation methods. Summary of the Invention

[0005] Purpose of the invention: To address the problems existing in the prior art, this invention provides a high-density fermentation culture medium and fermentation method for Lactobacillus plantarum. By integrating high-protein yeast hydrolysis technology, precise nutrient factor formulation, and intelligent fermentation process control, it breaks through the limitations of traditional culture media on bacterial density, reduces fermentation costs, and achieves efficient and stable production of Lactobacillus plantarum.

[0006] Technical Solution: To achieve the above objectives, the present invention provides a high-density culture medium for *Lactobacillus plantarum*, comprising high-protein yeast powder, compounding factors, and basic components; the compounding factors include one or more of aspartic acid, adenine, uracil, vitamin B2, vitamin B3, vitamin B5, and ferric sulfate; the basic components include one or more of peptone, glucose, beef extract, sodium acetate, Tween, dipotassium hydrogen phosphate, manganese sulfate, diammonium hydrogen citrate, and magnesium sulfate.

[0007] The high-protein yeast powder is obtained by hydrolyzing yeast milk as a hydrolysis substrate, centrifuging the yeast milk, taking the supernatant and freeze-drying it. The yeast milk is fermented by a yeast strain and then centrifuged to collect the precipitate. The yeast strain is CGMCC No. 30663.

[0008] The hydrolysis substrate is hydrolyzed by papain and ethyl acetate. The amount of hydrolysis substrate added is 8-10% of the total mass of the hydrolysis system, the amount of papain added is 0.5-1% of the total mass of the hydrolysis system, and the amount of ethyl acetate added is 0.5-1% of the total mass of the hydrolysis system. The hydrolysis is carried out at a temperature of 50-53℃ and a pH of 6.0-6.5 for 6-7 hours.

[0009] Preferably, the hydrolysis conditions are as follows: hydrolysis time 6h, hydrolysis temperature 50℃, hydrolysis substrate concentration 10% (10g yeast milk added to 100ml water), hydrolysis pH 6.0, papain addition 0.5%, enzymatic hydrolysis time 30h, and ethyl acetate addition 0.5%. After hydrolysis according to the above hydrolysis process, the supernatant is centrifuged at 8000rpm for 5min. The supernatant is then frozen into a solid state in a -80℃ freezer and then freeze-dried into powder in a laboratory freeze dryer to obtain the homemade yeast powder.

[0010] The culture medium includes high-protein yeast powder, aspartic acid, adenine, uracil, vitamin B2, vitamin B3, vitamin B5, ferric sulfate, peptone, glucose, beef extract, sodium acetate, Tween, dipotassium hydrogen phosphate, manganese sulfate, diammonium hydrogen citrate, and magnesium sulfate.

[0011] The culture medium comprises the following components by weight percentage: 1-3% peptone, 1-3% glucose, 1-3% beef extract, 0.2-0.8% high-protein yeast extract, 0.2-0.8% sodium acetate, 0.1-0.5% Tween, 0.2-0.5% dipotassium hydrogen phosphate, 0.025-0.05% manganese sulfate, 0.2-0.5% diammonium hydrogen citrate, 0.058-0.1% magnesium sulfate, 0.0185-0.05% aspartic acid, 0.003%-0.01% adenine, 0.009-0.03% uracil, 0.003-0.008% vitamin B2, 0.003-0.008% vitamin B3, 0.003-0.008% vitamin B5, 0.004-0.008% FeSO4, with the balance being water.

[0012] Preferably, the culture medium comprises the following components by weight percentage: 2% peptone, 2% glucose, 1% beef extract, 0.5% high-protein yeast extract, 0.5% sodium acetate, 0.1% Tween, 0.2% dipotassium hydrogen phosphate, 0.025% manganese sulfate, 0.2% diammonium hydrogen citrate, 0.058% magnesium sulfate, 0.0185% aspartic acid, 0.003% adenine, 0.009% uracil, 0.003% vitamin B2, 0.003% vitamin B3, 0.003% vitamin B5, and 0.004% FeSO4.

[0013] The high-density fermentation culture method of Lactobacillus plantarum described in this invention includes the following steps:

[0014] (1) Treatment of bacterial strains: Streak Lactobacillus plantarum on a culture medium plate, culture, and select plates with good single colony growth as working plates;

[0015] (2) Preparation of fermentation tank seed liquid: Select a single colony of Lactobacillus plantarum on the working plate and inoculate it into the culture medium. Incubate at constant temperature to obtain the primary seed liquid. Take the primary seed liquid and inoculate it into the culture medium. Incubate at constant temperature to obtain the secondary seed liquid. Transfer the secondary seed liquid into the culture medium and incubate at constant temperature to obtain the fermentation tank seed liquid.

[0016] (3) Fermentation culture: The seed liquid in the fermenter is centrifuged and the supernatant is discarded. The Lactobacillus plantarum is collected and introduced into the fermenter for fermentation. During the fermentation process, the mixture is stirred, the temperature is controlled, the pH value is adjusted, and feed is added intermittently. The fermentation medium and the feed medium are both the Lactobacillus plantarum high-density medium.

[0017] In step (1), the plate is placed in an incubator at 35-37℃ and incubated for 20-24 hours.

[0018] In step (2), a single colony of Lactobacillus plantarum is inoculated into 30-50 mL of MRS liquid culture medium, and the primary seed culture is cultured on a constant temperature shaker at 35-37℃ at 180-220 rpm for 8-12 hours.

[0019] In step (2), 1-2 mL of primary seed culture is placed in 30-50 mL of MRS liquid culture medium, and 30-50 mL of secondary seed culture is transferred into 200-300 mL of MRS liquid culture medium. The culture is placed on a constant temperature shaker and cultured at 35-37℃ and 180-220 rpm for 20-24 h.

[0020] In step (3), centrifugation is performed at 8000-9000 rpm and 4-6℃ for 5-10 minutes. During fermentation, the temperature is controlled at 35-37℃, the pH is adjusted to maintain at 6.0-7.0, the dissolved oxygen is maintained at 5-8 mg / L, the stirring speed is 180-220 rpm, and the total fermentation time is 12 hours. Feed is added every 2-4 hours.

[0021] The yeast powder prepared in this invention, through an optimized hydrolysis process, has a high protein content and is rich in various vitamins and trace elements, further enhancing the nutritional value of the culture medium. The high-density culture medium prepared using the self-made yeast powder significantly promotes the growth of Lactobacillus plantarum: the concentration of Lactobacillus plantarum reaches 2.78 at 4 hours and 5.47 at 12 hours, which are 10.95%, 17.38%, 21.02%, and 37.78% higher than other culture media, respectively, fully demonstrating that this formula has a significant promoting effect on the growth of Lactobacillus plantarum.

[0022] This invention involves inoculating *Lactobacillus plantarum* onto a culture medium plate for strain activation. A seed culture is prepared from a single colony, and after stepwise cultivation, it is used in a 5L fermenter for scale-up cultivation. During fermentation, a continuous feedstock is employed to maintain the concentration of nutrients in the fermentation system, promoting the continuous growth of *Lactobacillus plantarum* and the accumulation of its metabolites, until the culture reaches OD (Organic Degree). 600 Stop fermentation when no more is added.

[0023] This invention clarifies the optimal ranges for key parameters such as temperature, pH, dissolved oxygen, and rotation speed during fermentation. It employs 10% ammonia solution to adjust the pH and monitors and adjusts the feed rate in real time, ensuring stable and controllable fermentation conditions and guaranteeing the consistency and stability of product quality. This provides a reliable guarantee for large-scale industrial production. The 5L high-density fermentation culture system is a mature technology. After 12 hours of cultivation in a 5L fermenter, the concentration of *Lactobacillus plantarum* reached 20.1, and the viable count reached 3.02 × 10⁻⁶. 17 With a CFU / mL fermentation capacity, its fermentation conditions and operating methods can be directly scaled up to larger-scale industrial production, exhibiting good scalability and facilitating the large-scale industrial production of Lactobacillus plantarum.

[0024] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0025] 1. Improved production efficiency and product quality: The optimized culture medium formula and fermentation process significantly increased the biomass of *Lactobacillus plantarum*. Excellent results were achieved in both shake-flask culture and fermentation in tanks, ensuring the production of high-quality *Lactobacillus plantarum* products and meeting market demand for high-quality probiotic products.

[0026] 2. Reduce production costs: By utilizing waste yeast raw materials and optimizing fermentation processes, raw material costs and energy consumption are reduced, fermentation cycles are shortened, and production efficiency is improved, thereby effectively reducing overall production costs and improving the company's economic benefits.

[0027] 3. Promoting large-scale industrial production: The technical solution of this invention has good scalability and practicality, providing a feasible technical path for the large-scale industrial production of Lactobacillus plantarum, which helps to promote the development of the probiotic industry and promote technological progress and product upgrading in related food, pharmaceutical and other industries. Attached Figure Description

[0028] Figure 1 Line graphs showing the biomass of *Lactobacillus plantarum* cultured in alternative and MRS media;

[0029] Figure 2 Line graphs showing the biomass of *Lactobacillus plantarum* cultured on five different culture media;

[0030] Figure 3 Line graph showing data for Lactobacillus plantarum in tanks. Detailed Implementation

[0031] The present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0032] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent companies.

[0033] The yeast strain CGMCC No. 30663 used in the examples was provided by Huaiyin Institute of Technology and has been publicly deposited in the applicant's prior application CN118620753A.

[0034] Papain (SC20137148207585, enzyme activity 800,000 u / g, Nanning Pangbo Bioengineering Co., Ltd.).

[0035] The yeast powder is OXOID LP0021.

[0036] Lactobacillus plantarum is CGMCC 1.12732.

[0037] Example 1

[0038] Preparation of high-protein yeast powder

[0039] Yeast strain (CGMCC No. 30663) was fermented to a biomass of 220 g / L using conventional fermentation methods. The fermentation broth was centrifuged at 8000 rpm for 5 min, the supernatant was removed, and 10 g of the precipitate was weighed as yeast milk, which was used as the hydrolysis substrate. Through single-factor experiments under different conditions, the optimal hydrolysis time was determined to be 6 h, the optimal hydrolysis temperature was 50 °C, the optimal hydrolysis pH was 6.0, and the optimal enzymatic hydrolysis time was 30 h. In the hydrolysis system, 100 mL of water was added at a mass ratio of 10% yeast milk, 0.5% papain, and 0.5% ethyl acetate. After hydrolysis according to the above process, the supernatant was centrifuged at 8000 rpm for 5 min, frozen into a solid state at -80 °C, and then freeze-dried into powder to obtain high-protein yeast powder.

[0040] Example 2

[0041] Alternative culture media for high-density culture of Lactobacillus plantarum

[0042] A substitute culture medium was prepared by replacing the conventional yeast powder in the MRS medium with an equal amount of high-protein yeast powder. The substitute culture medium consisted of: 1% peptone, 1% beef extract, 0.5% high-protein yeast powder, 0.2% diammonium citrate, 0.1% Tween 80, 2% glucose, 0.2% dipotassium hydrogen phosphate, 0.058% MgSO4, 0.025% MnSO4, and 0.5% sodium acetate. (The percentages of the culture medium are by weight, and the remaining water is by weight of the whole system.) The remainder was water. The medium was sterilized at 115°C for 20 minutes.

[0043] Example 3

[0044] Alternative culture medium for Lactobacillus plantarum

[0045] MRS liquid medium: 1% peptone, 1% beef extract, 0.5% yeast extract (OXOID LP0021), 0.2% diammonium citrate, 0.1% Tween, 2% glucose, 0.2% dipotassium hydrogen phosphate, 0.058% MgSO4, 0.025% MnSO4, 0.5% sodium acetate, with the remainder being water. Sterilization conditions are the same as for alternative media.

[0046] Lactobacillus plantarum (CGMCC1.12732) cultured to the logarithmic phase was inoculated at 2% of the total liquid volume of the culture medium into the alternative culture medium prepared in Example 2 and the above-mentioned MRS liquid culture medium. Both were cultured at the same conditions of 37°C and 180 rpm for 12 h. The bacterial culture was taken every 2 h to detect the concentration of Lactobacillus plantarum at OD600.

[0047] The results are as follows Figure 1 As shown, under the same initial inoculum conditions, the concentration of *Lactobacillus plantarum* in the alternative culture medium was significantly higher than that in the MRS medium, and the concentration of *Lactobacillus plantarum* in the alternative culture medium was 23.5% higher than that in the MRS medium after 12 hours.

[0048] Example 4

[0049] Optimization and preparation of high-density culture medium for Lactobacillus plantarum

[0050] Based on the alternative culture medium of Example 2, additional compound factors were added. Seven factors were designed: aspartic acid, adenine, uracil, vitamin B2, vitamin B3, vitamin B5 and ferrous sulfate. An orthogonal experiment was conducted with seven factors and three levels (the proportions are by mass percentage, added on the basis of the alternative culture medium, with the remainder being water). A high-density culture medium was prepared according to the orthogonal table, which is shown in Table 1.

[0051] Table 1 Orthogonal array

[0052]

[0053]

[0054] The culture media prepared according to the orthogonal array were inoculated with *Lactobacillus plantarum* (CGMCC 1.12732) cultured to the logarithmic growth phase at an inoculation rate of 2% of the total liquid volume of the culture medium. The culture was then cultured at 37℃ and 180 rpm for 12 h. The bacterial culture was taken every 2 h, and the concentration of *Lactobacillus plantarum* was measured by OD600. The corresponding substances with greater growth-promoting effects on *Lactobacillus plantarum* were screened. The orthogonal results are shown in Table 2.

[0055] Table 2 Orthogonal Results

[0056]

[0057] The factor levels obtained by the range analysis of the orthogonal array are shown in Table 3. Among the seven significant compound component factors affecting the growth of Lactobacillus plantarum, the order of influence is as follows: aspartic acid > B2 > adenine > B5 > uracil > FeSO4 > B3.

[0058] Table 3. Factor Levels

[0059]

[0060] Therefore, the composition and content of the high-density culture medium for *Lactobacillus plantarum* were determined based on the following mass percentages: 2% peptone, 2% glucose, 1% beef extract, 0.5% yeast powder (the yeast powder prepared in Example 1), 0.5% sodium acetate, 0.1% Tween, 0.2% dipotassium hydrogen phosphate, 0.025% manganese sulfate, 0.2% diammonium hydrogen citrate, 0.058% magnesium sulfate, 0.0185% aspartic acid, 0.003% adenine, 0.009% uracil, 0.003% B2, 0.003% B3, 0.003% B5, 0.004% FeSO4, with the balance being water.

[0061] Example 5

[0062] Determination of Lactobacillus plantarum concentration

[0063] Alternative culture medium composed of FM985 yeast powder: The yeast powder in the MRS liquid culture medium in Example 3 was replaced with an equal amount of Angel Yeast's FM985 yeast powder, and the other components and contents were the same as those in the MRS liquid culture medium.

[0064] Alternative culture medium composed of FM503 yeast powder: The yeast powder in the MRS liquid culture medium in Example 3 was replaced with an equal amount of FM503 yeast powder from Angel Yeast Co., Ltd., while other components and contents were the same as those in the MRS liquid culture medium.

[0065] The high-density culture medium prepared in Example 4, the alternative culture medium prepared in Example 2, the alternative culture medium composed of FM985 yeast powder, the alternative culture medium composed of FM503 yeast powder, and the MRS liquid culture medium were inoculated with Lactobacillus plantarum (CGMCC 1.12732) at an inoculation rate of 2% of the total liquid volume of the culture medium and cultured at 37°C and 180 rpm for 12 h. The bacterial culture was taken every 2 h and the concentration of Lactobacillus plantarum was measured by OD600.

[0066] Test results as follows Figure 2 As shown, the concentration of *Lactobacillus plantarum* in the high-density medium was higher than that in the other four media within 12 hours, and the concentration reached 2.78 at 4 hours, indicating that the addition of amino acids, purines, bases, vitamins, and trace elements significantly promoted the growth of *Lactobacillus plantarum*. At 12 hours, the concentrations of *Lactobacillus plantarum* in the five media were 5.47, 4.93, 4.66, 4.52, and 3.97, respectively. It can be seen that the concentration of *Lactobacillus plantarum* in the high-density medium increased by 10.95%, 17.38%, 21.02%, and 37.78% respectively compared with these media.

[0067] Example 6

[0068] High-density culture medium for Lactobacillus plantarum

[0069] The high-density culture medium was adjusted to pH 7.5 with phosphate. At 37°C, with 2% glucose added to the total culture medium mass, and a rotation speed of 180 rpm, 2% of the total liquid volume of *Lactobacillus plantarum* (CGMCC 1.12732) was inoculated and cultured to the logarithmic growth phase. At 4 hours, the bacterial concentration exceeded OD600 (4). The bacterial solution after 12 hours of fermentation was then taken and diluted 10-fold. 100 μL of the diluted solution was evenly spread onto the surface of MRS solid culture medium using a sterilized spreader. The medium was incubated at 37°C for 12 hours to allow colony formation. Plates with colony counts between 30 and 50 were selected, and the viable count (CFU / mL) was calculated. The calculation formula is: viable count + single colony count / (dilution factor × inoculum volume). After dilution and plating, the final viable count was 5.2 × 10^13 CFU / mL, which is higher than the viable count (2.797 × 10^11 CFU / mL) reported in existing literature (Wang Xinyu. Study on high-density fermentation, lyophilization protectant screening and microencapsulation process of Lactobacillus plantarum [D]. Master's thesis, Jilin University, 2023).

[0070] Example 7

[0071] High-density fermentation culture method of Lactobacillus plantarum

[0072] Strain treatment: Streak Lactobacillus plantarum at -80℃ onto MRS solid medium plates, incubate at 37℃ for 24 hours, select plates with good single colony growth on MRS solid medium plates, and place them in a 4℃ refrigerator as working plates for subsequent experiments.

[0073] Preparation of fermenter seed culture: A single colony of *Lactobacillus plantarum* from the experimental working plate was inoculated into 50 mL of MRS liquid medium and then placed on a constant temperature shaker at 37℃ and cultured at 180 rpm for 12 h as the primary seed culture. 2 mL of the primary seed culture was taken and inoculated into 50 mL of MRS liquid medium and placed on a constant temperature shaker at 37℃ and cultured at 180 rpm for 12 h as the secondary seed culture. In a clean bench, 50 mL of the secondary seed culture was directly transferred into 300 mL of MRS liquid medium and placed on a constant temperature shaker at 37℃ and 180 rpm for 24 h as the fermenter seed culture.

[0074] Preparation of culture in a 5L fermenter: The culture media consisted of a substrate medium and a fed-batch medium. The substrate medium was the high-density culture medium for *Lactobacillus plantarum* prepared in Example 4; the fed-batch medium was three times the concentration of the substrate medium to support high-density growth of a large number of cells. Simultaneously, a 10% ammonia solution was prepared to adjust the pH.

[0075] Fermentation Culture: All the seed culture from the fermenter was centrifuged at 8000 rpm and 4℃ for 10 min. The supernatant was discarded, and all *Lactobacillus plantarum* were collected in a glass bottle using sterile water. Flame inoculation was then performed and the mixture was transferred to a 5L fermenter with 1.8L of fermentation broth for fermentation. During fermentation, the temperature was controlled at 37℃, and the pH was adjusted with 10% ammonia to maintain it at 6.5-7.0. Aeration and rotation speed were also controlled to maintain dissolved oxygen at 5-8 mg / L. Specific fermentation conditions and feed rates are shown in Table 4. The total fermentation time was 12 hours, with feed added every 4 hours, for a total of 2.2L of fed-batch culture medium.

[0076] Table 4 Fermentation conditions and feed amounts

[0077]

[0078] During the period, bacterial concentration OD was measured 600 pH results as follows Figure 3 As shown, at the end of fermentation, the concentration of *Lactobacillus plantarum* in the product reached 20.1 (OD200). 600 The viable count was 3.02 × 10⁻⁶. 17 CFU / mL, all indicators met expectations.

[0079] Compared to traditional fermentation methods (referring to the method in the literature on high-density fermentation conditions optimization of Lactobacillus plantarum ZJ316), using the Lactobacillus plantarum (CGMCC 1.12732) of this invention as the fermentation strain according to the method in the literature, after 24 hours of fermentation, the OD600 value of Lactobacillus plantarum was less than 5, and the viable cell count was also significantly lower than 4.45 × 10⁻⁶. 10 CFU / mL. This invention, through precise control of fermentation parameters and optimized feeding strategy, achieved a *Lactobacillus plantarum* concentration of 20.1 and a viable count of 3.02 × 10⁻⁶ CFU / mL in a 5L fermenter after 12 hours of cultivation. 17 CFU / mL, viable count can reach 5.2 × 10⁻⁶ after 12 h of shake-flask incubation. 13 The CFU / mL concentration significantly increases biomass, and the yield of the key metabolite lactic acid also increases accordingly, greatly improving fermentation efficiency and product quality. The culture medium and method of this invention can reach the ideal bacterial concentration within 12 hours through fermentation in a tank, improving production efficiency and reducing production costs.

Claims

1. A high-density culture medium for Lactobacillus plantarum, characterized in that, It includes high-protein yeast powder, compounding factors, and basic ingredients; the compounding factors include one or more of aspartic acid, adenine, uracil, vitamin B2, vitamin B3, vitamin B5, and ferrous sulfate; the basic ingredients include one or more of peptone, glucose, beef extract, sodium acetate, Tween, dipotassium hydrogen phosphate, manganese sulfate, diammonium hydrogen citrate, and magnesium sulfate.

2. The high-density culture medium for *Lactobacillus plantarum* according to claim 1, characterized in that, The high-protein yeast powder is obtained by hydrolyzing yeast milk as a hydrolysis substrate, centrifuging the yeast milk, taking the supernatant and freeze-drying it. The yeast milk is fermented by a yeast strain and then centrifuged to collect the precipitate. The yeast strain is CGMCC No. 30663.

3. The high-protein yeast powder according to claim 2, characterized in that, The hydrolysate is hydrolyzed by papain and ethyl acetate. The amount of hydrolysate added is 8-10% of the total mass of the hydrolysis system, the amount of papain added is 0.5-1% of the total mass of the hydrolysis system, and the amount of ethyl acetate added is 0.5-1% of the total mass of the hydrolysis system. The hydrolysis is carried out at a temperature of 50-53℃ and a pH of 6.0-6.5 for 6-7 hours.

4. The high-density culture medium for *Lactobacillus plantarum* according to claim 1, characterized in that, It includes high-protein yeast powder, aspartic acid, adenine, uracil, vitamin B2, vitamin B3, vitamin B5, ferric sulfate, peptone, glucose, beef extract, sodium acetate, Tween, dipotassium hydrogen phosphate, manganese sulfate, diammonium hydrogen citrate, and magnesium sulfate.

5. The high-density culture medium for *Lactobacillus plantarum* according to claim 4, characterized in that, The composition includes the following components by mass percentage: 1-3% peptone, 1-3% glucose, 1-3% beef extract, 0.2-0.8% high-protein yeast powder, 0.2-0.8% sodium acetate, 0.1-0.5% Tween, 0.2-0.5% dipotassium hydrogen phosphate, 0.025-0.05% manganese sulfate, 0.2-0.5% diammonium hydrogen citrate, 0.058-0.1% magnesium sulfate, 0.0185-0.05% aspartic acid, 0.003%-0.01% adenine, 0.009-0.03% uracil, 0.003-0.008% vitamin B2, 0.003-0.008% vitamin B3, 0.003-0.008% vitamin B5, 0.004-0.008% FeSO4, with the balance being water.

6. A method for high-density fermentation culture of Lactobacillus plantarum, characterized in that, Includes the following steps: (1) Treatment of bacterial strains: Streak Lactobacillus plantarum on a culture medium plate, culture, and select plates with good single colony growth as working plates; (2) Preparation of fermentation tank seed liquid: Select a single colony of Lactobacillus plantarum on the working plate and inoculate it into the culture medium. Incubate at constant temperature to obtain the primary seed liquid. Take the primary seed liquid and inoculate it into the culture medium. Incubate at constant temperature to obtain the secondary seed liquid. Transfer the secondary seed liquid into the culture medium and incubate at constant temperature to obtain the fermentation tank seed liquid. (3) Fermentation culture: The seed liquid in the fermenter is centrifuged and the supernatant is discarded. The plant lactobacillus is collected and introduced into the fermenter for fermentation. During the fermentation process, the mixture is stirred, the temperature is controlled, the pH value is adjusted, and feed is added intermittently. The fermentation culture medium and the feed culture medium are both the plant lactobacillus high-density culture medium as described in claim 1.

7. The method for high-density fermentation culture of *Lactobacillus plantarum* according to claim 6, characterized in that, In step (1), the plate is placed in an incubator at 35-37℃ and incubated for 20-24 hours.

8. The method for high-density fermentation culture of *Lactobacillus plantarum* according to claim 6, characterized in that, Step (2) Inoculate a single colony of Lactobacillus plantarum into 30-50 mL of MRS liquid medium. Incubate the primary seed culture at 180-220 rpm for 8-12 h on a constant temperature shaker at 35-37℃.

9. The method for high-density fermentation culture of *Lactobacillus plantarum* according to claim 6, characterized in that, In step (2), take 1-2 mL of primary seed culture into 30-50 mL of MRS liquid culture medium, take 30-50 mL of secondary seed culture into 200-300 mL of MRS liquid culture medium, place on a constant temperature shaker, and culture at 35-37℃ and 180-220 rpm for 20-24 h.

10. The method for high-density fermentation culture of *Lactobacillus plantarum* according to claim 6, characterized in that, Step (3) Centrifuge at 8000-9000 rpm and 4-6℃ for 5-10 min. During fermentation, control the temperature at 35-37℃, adjust the pH value to maintain at 6.0-7.0, maintain the dissolved oxygen at 5-8 mg / L, stir at 180-220 rpm, and the total fermentation time is 12 hours. Feed is added every 2-4 hours.

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