Pediococcus pentosaceus high-density fermentation medium and fermentation process

Abstract

The invention relates to the technical field of microbial fermentation, and particularly discloses a pediococcus pentosaceus high-density fermentation medium and a fermentation process. The invention relates to a pediococcus pentosaceus high-density fermentation medium which comprises the following components in mass concentration: a carbon source, a nitrogen source, beef extract powder, yeast extract powder, dipotassium phosphate, ammonium hydrogen citrate, sodium acetate, magnesium sulfate, manganese sulfate, tween-80, L-cysteine hydrochloride and growth factors. The growth factor is selected from a celery juice extract, a potato juice extract, a green pepper juice extract or a tomato juice extract. The pediococcus pentosaceus high-density fermentation culture medium can be used for high-density fermentation of pediococcus pentosaceus, the yield and viable bacterium yield of pediococcus pentosaceus can be remarkably increased, and the industrial production scale and application of pediococcus pentosaceus are greatly expanded.

Description

Technical Field

[0001] This application relates to the field of microbial fermentation technology, and more specifically, to a high-density fermentation medium for Pediococcus pentosaceus and a fermentation process. Background Technology

[0002] Pediococcus pentosaceus is a Gram-positive lactic acid bacterium belonging to the genus Pediococcus in the family Streptococciaceae. Its core biological characteristics and probiotic value have gained widespread recognition in academia and industry. This strain ferments glucose to produce lactic acid, playing multiple key roles in the host's gut. On one hand, it competitively inhibits the adhesion and growth of pathogenic microorganisms such as Escherichia coli and Salmonella through rapid reproduction, effectively maintaining the balance of the host's gut microbiota. On the other hand, it enhances intestinal barrier function, improves the host's systemic and local immune defense capabilities, and reduces the risk of infectious diseases and inflammatory bowel disease. In recent years, the potential of Pediococcus pentosaceus in the intervention of specific diseases has been further explored, and the market demand for related products continues to grow, making the research and development of its industrial production technology a key focus of the industry.

[0003] In the production of Pediococcus pentosacchari, the formulation of the culture medium and the control of fermentation conditions are crucial, directly affecting the growth density and yield of Pediococcus pentosacchari. However, most mainstream cultivation processes for Pediococcus pentosacchari on the market currently use traditional MRS medium or simple modified mediums, and the viable cell count in the fermentation broth is generally only 1×10⁻⁶. 9 -1×10 10 The CFU / mL concentration was low, resulting in a low final viable cell yield. This was due to factors such as an unsuitable culture medium composition, imprecise control of culture conditions, and the accumulation of metabolic byproducts like lactic acid and acetic acid in the later stages of fermentation, leading to a decrease in cell viability. A low viable cell yield directly necessitates the addition of large amounts of microbial powder to downstream products to achieve the desired efficacy. This not only increases raw material costs but may also negatively impact the product's taste, stability, and other physicochemical properties due to excessive powder addition, limiting its application in high-end functional foods and pharmaceuticals. Furthermore, it presents challenges in low-cost conversion, large-scale production, and industrialization during the biotechnology transformation process.

[0004] Therefore, developing a cultivation method that can effectively improve the viable yield of Pediococcus pentosaccharis is of great practical significance for overcoming the problems of high production costs and complex fermentation technology processes encountered in the large-scale industrialization of Pediococcus pentosaccharis, and for further expanding the application fields of Pediococcus pentosaccharis. Summary of the Invention

[0005] To improve the yield and viable cell rate of Pediococcus pentosus, this application provides a high-density fermentation medium and fermentation process for Pediococcus pentosus.

[0006] In a first aspect, this application provides a high-density fermentation medium for *Pediococcus pentosaceus*, employing the following technical solution: A high-density fermentation medium for *Pediococcus pentosaceus*, comprising, by mass concentration, 20-30 g / L carbon source, 10-20 g / L nitrogen source, 5-10 g / L beef extract, 10-15 g / L yeast extract, 2-5 g / L dipotassium hydrogen phosphate, 2-5 g / L ammonium citrate, 2-5 g / L sodium acetate, 0.2-0.5 g / L magnesium sulfate, 0.1-0.2 g / L manganese sulfate, 1-2 g / L Tween-80, 0.1-0.2 g / L L-cysteine ​​hydrochloride, and 5-15 g / L growth factor, wherein the growth factor is selected from celery juice extract, potato juice extract, green pepper juice extract, or tomato juice extract.

[0007] By adopting the above technical solution, this application adds L-cysteine ​​hydrochloride and tomato juice extract growth factors as formulation components. Pediococcus pentosaceus, as a Gram-positive lactic acid bacterium, has a cell structure and metabolic process that is highly sensitive to oxidative stress. Even trace amounts of dissolved oxygen in the fermentation system can generate free radicals such as superoxide anions and hydrogen peroxide through oxidation reactions. These free radicals attack the phospholipid bilayer of the bacterial cell membrane, leading to membrane structure damage, and also inactivate key enzymes in the glycolysis pathway.

[0008] L-cysteine ​​hydrochloride is a thiol-containing amino acid derivative. Its thiol group can act as a free radical scavenger, reacting with oxidative free radicals in the system to undergo redox reactions, converting them into harmless water or small molecules, eliminating oxidative stress, and thus reducing the damage of free radicals to cells and metabolic enzymes. At the same time, it can also participate in the synthesis of glutathione in bacterial cells, enhancing the antioxidant capacity of the bacteria and prolonging the logarithmic growth phase of the bacteria.

[0009] Furthermore, in the later stages of fermentation, *Pediococcus pentosaceus* continuously produces metabolic byproducts such as lactic acid and acetic acid, leading to a rapid decrease in the pH of the fermentation broth. This results in a decline in bacterial activity and affects the yield of viable cells. The amino group in L-cysteine ​​hydrochloride can neutralize the lactic acid in the fermentation broth. Together with buffers such as dipotassium hydrogen phosphate and sodium acetate in the culture medium, it helps maintain the stability of the environmental pH, preventing metabolic stagnation caused by a sudden drop in pH. This ensures efficient intake of nutrients such as glucose and amino acids, thereby increasing the bacterial reproduction rate.

[0010] As a natural fruit and vegetable extract, tomato juice extract not only provides abundant vitamins and minerals, but also acts as a natural antioxidant. It works synergistically with L-cysteine ​​to further reduce oxidative stress in the fermentation system, protect cell membranes and biomolecules from oxidative damage, and maintain cell membrane integrity and cell vitality.

[0011] Furthermore, the inventors discovered that the addition of tomato juice extract, compared to other fruit and vegetable juice extracts, is more effective in increasing the viable count and viable yield of Pediococcus pentosaceus. This may be because the unique lycopene, polyphenols, and other natural active substances in tomato juice extract act as signaling molecules to activate genes related to the proliferation of Pediococcus pentosaceus. Under the same nutritional conditions, the expression level of proliferation genes can be significantly increased, thus acting as a specific growth factor to overcome the bottleneck of incomplete nutrient supply in traditional culture media and accelerate the proliferation of bacteria.

[0012] Optionally, the growth factor is tomato juice extract.

[0013] Optionally, the method for preparing the tomato juice extract includes the following steps: Wash fresh tomatoes, juice them, centrifuge them, collect the supernatant, concentrate it to 1 / 5 of the original volume using a rotary evaporator at 40℃, mix it evenly, adjust the pH value to 6.0, sterilize it, and cool it to obtain the final product.

[0014] Optionally, the carbon source is selected from glucose, trehalose, sucrose, or lactose.

[0015] Optionally, the carbon source is glucose.

[0016] Optionally, the nitrogen source is selected from soybean peptone, trypsin, fish peptone, or casein peptone.

[0017] Optionally, the nitrogen source is tryptone.

[0018] By adopting the above technical solution, the inventors discovered that glucose is the best carbon source and tryptone is the best nitrogen source. Pediococcus pentosus NSS0402 has the shortest metabolic pathway for glucose and tryptone, and the highest absorption efficiency, which is superior to other carbon and nitrogen sources. It is more easily absorbed by the bacteria and promotes the proliferation and survival of the bacteria.

[0019] Optionally, the high-density fermentation medium for Pediococcus pentosaceus comprises, by mass concentration, 25 g / L glucose, 20 g / L tryptone, 5 g / L beef extract, 10 g / L yeast extract, 4 g / L dipotassium hydrogen phosphate, 4 g / L ammonium citrate, 5 g / L sodium acetate, 0.5 g / L magnesium sulfate, 0.1 g / L manganese sulfate, 1.5 g / L Tween-80, 0.1 g / L L-cysteine ​​hydrochloride, and 10 g / L tomato extract.

[0020] By adopting the above technical solution, the above formula is the optimal culture medium formula for high-density fermentation culture of Pediococcus pentosaccharis NSS0402. At this formula concentration, Pediococcus pentosaccharis NSS0402 has the highest fermentation density and the best fermentation efficiency.

[0021] Secondly, this application provides a fermentation process for Pediococcus pentosaceus, employing the following technical solution: A fermentation process for Pediococcus pentosaceus includes the following steps: After activating and expanding *Pediococcus pentosaceus*, a seed culture of *Pediococcus pentosaceus* was obtained. This seed culture was then inoculated at an inoculation rate of 2-5% into the high-density fermentation medium of *Pediococcus pentosaceus* as described in any one of claims 1-5. The initial pH of the medium was 5-6.5. Fermentation was carried out at a temperature of 31-37°C with stirring. A neutralizing agent was added during the fermentation process to maintain the pH of the environment at 5-6.5. Fermentation was terminated when the absorbance of the fermentation broth at a wavelength of 600 nm no longer increased.

[0022] Optionally, the Pediococcus pentosaceus is Pediococcus pentosaceus NSS0402, which was deposited on July 1, 2024 at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, and named Pediococcus pentosaceus NSS0402, with accession number CCTCC NO:M 20241421.

[0023] Optionally, the inoculum size of *Pediococcus pentosaceus* seed culture is 4%, the fermentation temperature in the high-density fermentation medium is 33°C, and the pH of the medium is 6.0.

[0024] By adopting the above technical solution, the inventors discovered that the optimal metabolic temperature for Pediococcus pentosaceus NSS0402 is 33℃. Temperatures that are too high or too low will affect the metabolic activity of the bacteria. The optimal pH for bacterial proliferation is 6.0, and the bacterial fermentation density is highest when the inoculum size is 4%.

[0025] In summary, this application has the following beneficial effects: 1. This application optimizes the composition of traditional MRS broth culture medium by adding L-cysteine ​​hydrochloride and tomato juice extract as formulation components. This effectively eliminates the oxidative stress that may occur during the fermentation of Pediococcus pentosus, protects the cell membrane and metabolic enzyme activity, reduces environmental pH fluctuations, and ensures the survival and proliferation environment of the bacteria. The tomato juice extract supplements the culture medium with specific growth factors, breaking through the bottleneck of incomplete nutrient supply in traditional culture media and accelerating the reproduction of bacteria.

[0026] 2. The fermentation process of Pediococcus pentosaceus NSS0402 using high-density fermentation medium in this application can increase the viable count of the fermentation suspension to ≥7×10⁻⁶. 10 With a CFU / mL concentration and a sludge yield exceeding 2.5%, it significantly improves the yield and viable cell rate of Pediococcus pentosaceus.

[0027] 3. The high-density fermentation process of this application greatly expands the industrial production scale of Pediococcus pentosaceus NSS0402 and the production application of this strain in the fields of food, beverages and pharmaceuticals. Detailed Implementation

[0028] The present application will be further described in detail below with reference to embodiments and comparative examples.

[0029] Unless otherwise specified, the experimental methods used in the embodiments of this application are all conventional methods, and the materials and reagents used can be purchased from biological or chemical reagent companies.

[0030] Preparation Example Preparation Example 1 A high-density fermentation medium for Pediococcus pentosaceus, by mass concentration, comprises the following components: 20 g / L glucose, 15 g / L tryptone, 5 g / L beef extract, 10 g / L yeast extract, 4 g / L dipotassium hydrogen phosphate, 4 g / L ammonium citrate, 5 g / L sodium acetate, 0.5 g / L magnesium sulfate, 0.1 g / L manganese sulfate, 1.5 g / L Tween-80, 0.1 g / L L-cysteine ​​hydrochloride, and 10 g / L tomato juice extract, sterilized at 115°C for 20 min; The preparation method of tomato juice extract is as follows: wash fresh tomatoes, juice them, centrifuge at 5000 r / min for 10 min, collect the supernatant, concentrate it to 1 / 5 of the original volume at 40℃ using a rotary evaporator, mix the above components evenly, adjust the pH value to 6.0, sterilize at 115℃ for 20 min, and obtain the extract after cooling.

[0031] Preparation Example 2 A high-density fermentation medium for Pediococcus pentosaceus, differing from Preparation Example 1 only in that its formulation includes the following components: It includes 30 g / L glucose, 10 g / L tryptone, 10 g / L beef extract, 15 g / L yeast extract, 5 g / L dipotassium hydrogen phosphate, 5 g / L ammonium citrate, 2 g / L sodium acetate, 0.5 g / L magnesium sulfate, 0.2 g / L manganese sulfate, 2 g / L Tween-80, 0.2 g / L L-cysteine ​​hydrochloride, and 5 g / L tomato juice extract.

[0032] Preparation Example 3 A high-density fermentation medium for Pediococcus pentosaceus, differing from Preparation Example 1 only in that its formulation includes the following components: It includes 25 g / L glucose, 20 g / L tryptone, 8 g / L beef extract, 12 g / L yeast extract, 2 g / L dipotassium hydrogen phosphate, 2 g / L ammonium citrate, 3 g / L sodium acetate, 0.3 g / L magnesium sulfate, 0.15 g / L manganese sulfate, 1 g / L Tween-80, 0.15 g / L L-cysteine ​​hydrochloride, and 8 g / L tomato juice extract. Example

[0033] The Pediococcus pentosaceus mentioned in this application is Pediococcus pentosaceus NSS0402, which was deposited on July 1, 2024 at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, and named Pediococcus pentosaceus NSS0402, with accession number CCTCC NO:M 20241421.

[0034] The culture medium in the embodiments of this application: 1. Basic culture medium formula: 20 g / L glucose, 10 g / L peptone, 10 g / L beef extract, 10 g / L yeast extract, 3 g / L dipotassium hydrogen phosphate, 3 g / L ammonium hydrogen citrate, 5 g / L sodium acetate, 0.5 g / L magnesium sulfate, 0.1 g / L manganese sulfate, 1.5 g / L Tween-80; 2. High-density fermentation medium formula is the same as the high-density fermentation medium for preparing Pediococcus pentosaceus in Example 1.

[0035] Example 1: Preparation of Pediococcus pentosaceus NSS0402 seed culture S1: Strain activation: After thawing and resuscitating the frozen Pediococcus pentosus NSS0402 strain, the strain was inoculated into the basal culture medium at an inoculation amount of 4% and the liquid volume of the shake flask was 60%. The culture was statically incubated for 18 hours at 37℃, 0.02MPa, and 50rpm with stirring to obtain activated seed liquid. The colony morphology was uniform and there were no contaminants. S2: Strain expansion: Re-inoculate the activated seed culture into the basal medium and culture as above to obtain *Pediococcus pentosaceus* seed culture. The viable count of the *Pediococcus pentosaceus* seed culture is ≥1.0 × 10⁻⁶. 10 CFU / mL.

[0036] Example 2: High-density fermentation process of Pediococcus pentosaceus NSS0402 S1: Primary fermentation: The *Pediococcus pentosaceus* seed culture obtained in Example 1 was inoculated into a high-density fermentation medium at an inoculation rate of 2-5%, with a liquid volume ≤80% and an initial pH of 5-6.5. The culture was carried out at a temperature of 31-37℃, an air pressure of 0.02MPa, and a stirring rate of 60rpm with zero aeration. During the fermentation process, the pH of the bacterial culture was monitored, and a neutralizing agent (food-grade NaOH) was added to maintain the environmental pH at 5-6.5. Fermentation began after 6 hours of cultivation, and the OD value of the bacterial culture was monitored every 30 minutes. Fermentation was stopped when the difference between two consecutive OD values ​​was ≤0.1 or showed a negative increase, thus obtaining the primary fermentation broth. S2: Final Fermentation: After primary fermentation, the primary fermentation broth is re-inoculated into the final fermenter of high-density fermentation medium at an inoculation rate of 2-5%, with a liquid volume ≤80%. The fermentation is carried out at a temperature of 31-37℃, an air pressure of 0.02-0.05MPa, and agitated at 50rpm. The pH of the bacterial suspension is monitored during fermentation. The resulting fermentation suspension is then analyzed using the plate count method to determine the viable cell count, which must be ≥1×10⁻⁶. 10 CFU / mL, the fermentation bacterial suspension was centrifuged at 8000 rpm and the bacterial sludge was collected. The viable count in the bacterial sludge was ≥6×10⁻⁶. 10 CFU / mL, sludge yield ≥2.5%. Sludge yield = sludge weight (kg) / fermentation suspension volume (L) × 100%.

[0037] Example 3: Effects of different culture conditions on high-density fermentation of Pediococcus pentosaceus NSS0402 The Pediococcus pentosaceus seed culture obtained in Example 1 was used as an activated strain and inoculated into the basic culture medium in a 5L fermenter. The culture was stirred at 50 rpm and pressurized with nitrogen to investigate the effect of culture conditions on the growth of Pediococcus pentosaceus NSS0402 cells.

[0038] 3.1 Effect of different temperatures on the viable cell count of Pediococcus pentosaceus NSS0402 in high-density fermentation. Constant: pH 5.5, inoculum size 4%, culture medium is basal medium; Variables: 31, 33, 35, and 37℃ were selected as fermentation temperatures, respectively; The viable count of the bacterial strains was determined, with three parallel samples prepared for each group. The results are shown in Table 1.

[0039] Table 1 It can be seen that, under the same conditions of pH and inoculum amount, the highest number of viable bacteria and the best fermentation effect are achieved when the fermentation temperature is 33℃.

[0040] 3.2 Effect of different initial pH on viable cell count of Pediococcus pentosaceus NSS0402 in high-density fermentation. Constant: fermentation temperature 37℃, inoculum size 4%, culture medium basal medium; Variables: Initial pH values ​​were 5.0, 5.5, 6.0, and 6.5. The viable count of the bacterial strains was determined, with three parallel samples prepared for each group. The results are shown in Table 2.

[0041] Table 2 It can be seen that, under the same conditions of fermentation temperature and inoculum amount, the highest number of viable bacteria and the best fermentation effect are achieved when the initial pH is 6.0.

[0042] 3.3 Effect of different inoculum sizes on viable cell count of Pediococcus pentosaceus NSS0402 in high-density fermentation. Constants: fermentation temperature 37℃, pH 5.5, culture medium basal medium; Variables: Inoculation rates of 2%, 3%, 4%, and 5% respectively; The viable count of the bacterial strains was determined, with three parallel samples prepared for each group. The results are shown in Table 3.

[0043] Table 3 It can be seen that, under the conditions of maintaining the same fermentation temperature and initial pH, the highest number of viable bacteria and the best fermentation effect are achieved when the inoculum size is 4%.

[0044] 3.4 Effect of different carbon sources on viable cell count of Pediococcus pentosaceus NSS0402 in high-density fermentation. Constants: fermentation temperature 37℃, inoculum size 4%, pH 5.5, culture medium basal medium; Variables: The carbon sources in the basal culture medium are glucose, trehalose, sucrose, and lactose; The viable count of the bacterial strains was determined, with three parallel samples prepared for each group. The results are shown in Table 4.

[0045] Table 4 It can be seen that, under the same conditions, when glucose is the carbon source, Pediococcus pentosaceus NSS0402 has the highest viable cell count and the best fermentation effect.

[0046] 3.5 Effect of different nitrogen sources on viable cell count of Pediococcus pentosus NSS0402 in high-density fermentation. Constants: fermentation temperature 37℃, inoculum size 4%, pH 5.5, culture medium basal medium; Variables: The nitrogen sources in the basal culture medium were soybean peptone, tryptone, fish peptone, and casein peptone, respectively; The viable count of the bacterial strains was determined, with three parallel samples prepared for each group. The results are shown in Table 5.

[0047] Table 5 It can be seen that, under the same conditions, when the nitrogen source is tryptone, Pediococcus pentosacchari NSS0402 has the highest viable cell count and the best fermentation effect.

[0048] 3.6 Effects of different growth factors on viable cell count of Pediococcus pentosaceus NSS0402 in high-density fermentation. Constants: fermentation temperature 37℃, inoculum size 4%, pH 5.5, culture medium basal medium; Variables: 10 g / L of growth factors were added to the basal culture medium. The growth factors were celery juice extract, potato juice extract, green bell pepper juice extract, and tomato juice extract. The viable count of the bacterial strains was determined, with three parallel samples prepared for each group. The results are shown in Table 6.

[0049] Table 6 It can be seen that, under the same conditions, when the growth factor is tomato juice extract, Pediococcus pentosaceus NSS0402 has the highest viable count and the best fermentation effect.

[0050] In summary, based on the test results in Tables 1-6, the optimal conditions for high-density fermentation of Pediococcus pentosaceus NSS0402 are: inoculum size of 4%, fermentation temperature of 33℃, initial pH of 6, carbon source of glucose, nitrogen source of tryptone, and growth factor of tomato juice extract.

[0051] Example 4: Effects of different culture media on high-density fermentation of Pediococcus pentosaceus NSS0402 The *Pediococcus pentosaceus* seed culture obtained in Example 1 was used as an activated strain and inoculated into a 5L fermenter high-density fermentation medium at an inoculation rate of 4%. The medium was stirred at 50 rpm and pressurized with nitrogen to investigate the effect of culture conditions on the components of the high-density fermentation medium on the growth of *Pediococcus pentosaceus* NSS0402 cells.

[0052] 4.1 Effect of different temperatures on the viable cell count of Pediococcus pentosaceus NSS0402 in high-density fermentation. Constants: pH 6, inoculum size 4%, culture medium is high-density fermentation medium; Variables: 31, 33, 35, and 37℃ were selected as fermentation temperatures, respectively; The viable count of the bacterial strains was determined, with three parallel samples prepared for each group. The results are shown in Table 7.

[0053] Table 7 As can be seen, compared with the basal culture medium (Examples 3-3.1), the overall viable cell rate is increased by about 13% when using the high-density fermentation culture medium of this application.

[0054] 4.2 Effect of different initial pH on viable cell count of Pediococcus pentosaceus NSS0402 in high-density fermentation. Constant: fermentation temperature was 33℃, inoculum size was 4%, and culture medium was high-density fermentation medium. Variables: Initial pH values ​​were 5.0, 5.5, 6.0, and 6.5. The viable count of the bacterial strains was determined, with three parallel samples prepared for each group. The results are shown in Table 8.

[0055] Table 8 As can be seen, compared with the basal culture medium (Examples 3-3.2), the overall viability is increased by about 15% when using the high-density fermentation culture medium of this application.

[0056] 4.3 Effect of different inoculum amounts on viable cell count of Pediococcus pentosaceus NSS0402 in high-density fermentation. Constants: fermentation temperature was 33℃, pH was 6, and the culture medium was high-density fermentation medium. Variables: Inoculation rates of 2%, 3%, 4%, and 5% respectively; The viable count of the bacterial strains was determined, with three parallel samples prepared for each group. The results are shown in Table 9.

[0057] Table 9 As can be seen, compared with the basal culture medium (Examples 3-3.3), the overall viability is increased by about 19% when using the high-density fermentation medium of this application.

[0058] 4.4 Effect of different carbon sources on viable cell count of Pediococcus pentosus NSS0402 in high-density fermentation. Constants: fermentation temperature 33℃, inoculum size 4%, pH 6, culture medium is high-density fermentation medium; Variables: The carbon sources in the basal culture medium are glucose, trehalose, sucrose, and lactose; The viable count of the bacterial strains was determined, with three parallel samples prepared for each group. The results are shown in Table 10.

[0059] Table 10 As can be seen, compared with the basal culture medium (Examples 3-3.4), the overall viability is increased by about 24% when using the high-density fermentation medium of this application.

[0060] 4.5 Effect of different nitrogen sources on viable cell count of Pediococcus pentosus NSS0402 in high-density fermentation. Constants: fermentation temperature 33℃, inoculum size 4%, pH 6, culture medium is high-density fermentation medium; Variables: The nitrogen sources in the basal culture medium were soybean peptone, tryptone, fish peptone, and casein peptone, respectively; The viable count of the bacterial strains was determined, with three parallel samples prepared for each group. The results are shown in Table 11.

[0061] Table 11 As can be seen, compared with the basal culture medium (Examples 3-3.5), the overall viable cell rate is increased by about 19% when using the high-density fermentation culture medium of this application.

[0062] 3.6 Effects of different growth factors on viable cell count of Pediococcus pentosaceus NSS0402 in high-density fermentation. Constants: fermentation temperature 33℃, inoculum size 4%, pH 6, culture medium is high-density fermentation medium; Variables: growth factors were celery juice extract, potato juice extract, green pepper juice extract, and tomato juice extract; viable bacterial counts were measured, with three replicates for each group. The results are shown in Table 12.

[0063] Table 12 As can be seen, compared with the basal culture medium (Examples 3-3.6), the overall viability is increased by about 22% when using the high-density fermentation medium of this application.

[0064] In summary, when using a high-density fermentation medium at a fermentation temperature of 33℃, an inoculum size of 4%, a pH of 6, and with glucose as the carbon source, tryptone as the nitrogen source, and tomato juice extract as the growth factor, the high-density fermentation of Pediococcus pentosaceus NSS0402 resulted in the highest number of viable cells and the best fermentation effect.

[0065] Example 5: Optimal High-Density Fermentation Process for Pediococcus Pentosacchari NSS0402 Based on the comparative analysis of Examples 3-4, the optimal culture conditions for *Pediococcus pentosaceus* NSS0402 were selected. Using the high-density fermentation medium for *Pediococcus pentosaceus* obtained in Example 1 as a basis, orthogonal experimental design was employed to conduct high-density fermentation culture of *Pediococcus pentosaceus* NSS0402. The carbon source is glucose, the nitrogen source is tryptone, and the growth factor is tomato juice extract; Glucose: 15g / L (A1), 20g / L (A2), 25g / L (A3); Tryptone: 10 g / L (B1), 15 g / L (B2), 20 g / L (B3); Tomato juice extract: 5 g / L (C1), 10 g / L (C2), 15 g / L (C3); The viable count of Pediococcus pentosaceus NSS0402 was determined, and the results are shown in Table 13. The K / value is shown in Table 14.

[0066] Table 13 Table 14 K1 6.57 6.51 6.78 K2 6.26 6.25 6.76 K3 6.30 7.07 6.30 R 0.31 0.82 0.48 In summary, the optimal high-density fermentation medium formulation is: 25 g / L glucose, 20 g / L tryptone, 5 g / L beef extract, 10 g / L yeast extract, 4 g / L dipotassium hydrogen phosphate, 4 g / L ammonium citrate, 5 g / L sodium acetate, 0.5 g / L magnesium sulfate, 0.1 g / L manganese sulfate, 1.5 g / L Tween-80, 0.1 g / L L-cysteine ​​hydrochloride, and 10 g / L tomato extract, with a maximum bacterial count ≥ 7.74 × 10⁻⁶. 10 The cfu / mL count is about 50% higher than the maximum bacterial count in the initial (Example 3) case, indicating that the high-density fermentation process of Pediococcus pentosacchari NSS0402 of this application can significantly improve the yield and viable cell yield of Pediococcus pentosacchari, greatly expanding the industrial production scale of Pediococcus pentosacchari NSS0402 and the production application of this strain in the fields of food, beverages and pharmaceuticals.

[0067] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A high-density fermentation medium for Pediococcus pentosaceus, characterized in that, The product, by mass concentration, includes 20-30 g / L carbon source, 10-20 g / L nitrogen source, 5-10 g / L beef extract, 10-15 g / L yeast extract, 2-5 g / L dipotassium hydrogen phosphate, 2-5 g / L ammonium citrate, 2-5 g / L sodium acetate, 0.2-0.5 g / L magnesium sulfate, 0.1-0.2 g / L manganese sulfate, 1-2 g / L Tween-80, 0.1-0.2 g / L L-cysteine ​​hydrochloride, and 5-15 g / L growth factor, wherein the growth factor is selected from celery juice extract, potato juice extract, green bell pepper juice extract, or tomato juice extract.

2. The high-density fermentation medium for Pediococcus pentosaceus according to claim 1, characterized in that, The growth factor is a tomato juice extract.

3. The high-density fermentation medium for Pediococcus pentosaceus according to claim 2, characterized in that, The method for preparing the tomato juice extract includes the following steps: Wash fresh tomatoes, juice them, centrifuge them, collect the supernatant, concentrate it to 1 / 5 of the original volume using a rotary evaporator at 40℃, mix it evenly, adjust the pH value to 6.0, sterilize it, and cool it to obtain the final product.

4. The high-density fermentation medium for Pediococcus pentosaceus according to claim 1, characterized in that, The carbon source is selected from glucose, trehalose, sucrose, or lactose.

5. The high-density fermentation medium for Pediococcus pentosaceus according to claim 1, characterized in that, The nitrogen source is selected from soybean peptone, trypsin, fish peptone, or casein peptone.

6. A high-density fermentation process for Pediococcus pentosaceus, characterized in that, Includes the following steps: After activating and expanding *Pediococcus pentosaceus*, a seed culture of *Pediococcus pentosaceus* was obtained. This seed culture was then inoculated at an inoculation rate of 2-5% into the high-density fermentation medium of *Pediococcus pentosaceus* as described in any one of claims 1-5. The initial pH of the medium was 5-6.

5. Fermentation was carried out at a temperature of 31-37°C with stirring. A neutralizing agent was added during the fermentation process to maintain the pH of the environment at 5-6.

5. Fermentation was terminated when the absorbance of the fermentation broth at a wavelength of 600 nm no longer increased.

7. The high-density fermentation process for Pediococcus pentosaceus according to claim 6, characterized in that, The Pediococcus pentosaceus mentioned is Pediococcus pentosaceus NSS0402, which was deposited on July 1, 2024 at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, and named Pediococcus pentosaceus NSS0402, with accession number CCTCC NO: M 20241421.

8. The high-density fermentation process for Pediococcus pentosaceus according to claim 6, characterized in that, The inoculum size of *Pediococcus pentosaceus* seed culture was 4%, the fermentation temperature in the high-density fermentation medium was 33℃, and the initial pH was 6.0.

Images