Lactobacillus plantarum P6 and static high-density culture method thereof

Abstract

The invention relates to the technical field of microorganisms, and provides a lactobacillus plantarum P6 and a static high-density culture method thereof.Under a static culture system without material supplementation and precise pH regulation and control, a fermentation medium containing specific components is optimized, and ultrasonic physical field stimulation is applied in a thallus logarithmic phase in a time-sequence mode, so that the lactobacillus plantarum P6 is obtained. The lactobacillus plantarum P6 has the advantages that the density and activity of the lactobacillus plantarum P6 are obviously improved, the viable count of the lactobacillus plantarum P6 can reach CFU / mL or above, meanwhile, the tolerance of the lactobacillus plantarum P6 to alimentary canal stress such as cholate is enhanced, the requirements of low-cost and large-scale production of the probiotic industry are met, and the bottleneck that the viable count of existing lactobacillus plantarum in static culture is increased is broken through.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and specifically relates to a strain of Lactobacillus plantarum P6 and its static high-density culture method. Background Technology

[0002] Lactobacillus plantarum is an important probiotic, belonging to the phylum Firmicutes and the genus Lactobacillus. It is a Gram-positive bacterium that converts sugars into lactic acid through homofermentation. Within the host, Lactobacillus plantarum possesses multiple physiological functions, including regulating intestinal flora, enhancing immune responses, lowering serum cholesterol, and maintaining intestinal barrier integrity. It is widely used in fermented foods, dietary supplements, and pharmaceuticals. With the increasing market demand for highly active probiotic preparations, achieving high-density, low-cost industrial cultivation of Lactobacillus plantarum has become a key technological bottleneck in the industry.

[0003] The core of achieving high-density culture lies in overcoming nutrient limitations and metabolic product inhibition during the culture process. High-density culture is key to improving the efficiency of the probiotic industry, aiming to obtain higher cell density with a shorter cycle and lower cost, thereby reducing production costs and improving product competitiveness. In existing technologies, optimization strategies mainly fall into two categories: one is complex nutrient optimization and feeding control, and the other is seeking simpler and more efficient culture modes. For example, invention patent CN120349933A discloses a high-density culture method for *Lactobacillus plantarum*, which optimizes the ratio of carbon source (trehalose), nitrogen source (beef extract powder), and manganese sulfate in the culture medium through artificial neural networks and genetic algorithms, and adopts a complex process of dynamic fermentation, constant pH control, and exponential feeding to achieve a high cell density. However, when statically cultured in its unoptimized MRS liquid medium, the number of viable cells is only... The CFU / mL capacity has significant room for improvement. Although this method can achieve extremely high cell density through a complex feeding process, it relies on sophisticated fermenter equipment, continuous acid-base neutralization, and complex feeding calculations and control. The process is complex, energy-intensive, and has stringent operational requirements, which is not conducive to simplification and cost control for large-scale production.

[0004] Another type of research focuses on developing more economical and simpler static high-density culture methods. Current optimization techniques mostly concentrate on the combination of nutrients in static culture, which, while having some effect, often fails to effectively alleviate the severe metabolic inhibition caused by lactic acid accumulation, resulting in limited increases in viable cell counts and generally failing to achieve breakthroughs. The concentration was in the CFU / mL range, and it failed to effectively improve the stress tolerance of the bacteria.

[0005] Therefore, developing a new method for culturing *Lactobacillus plantarum* that is low in raw material cost, simple in process (such as static culture), and can effectively alleviate lactic acid inhibition and simultaneously improve cell density and functional tolerance has important industrial demand and application value. Summary of the Invention

[0006] To overcome the problems of existing technologies, this invention provides a strain of Lactobacillus plantarum P6 and develops a static high-density culture method that does not require complex dynamic feeding and precise pH control. By optimizing the fermentation medium containing specific components and coupling it with the sequential application of ultrasonic physical field stimulation during the logarithmic growth phase of the bacteria, the density and activity of Lactobacillus plantarum P6 cells are significantly improved, while its tolerance to digestive tract adversity such as bile salts is enhanced, meeting the needs of the probiotic industry for low-cost, large-scale production.

[0007] The objective of this invention is achieved as follows: The first aspect of this invention provides a strain of Lactiplantibacillus plantarum P6, which was deposited on September 24, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 36056.

[0008] The second aspect of this invention provides a method for static high-density culture of *Lactobacillus plantarum* P6 as described in the first aspect, comprising the following steps: The *Lactobacillus plantarum* P6 was inoculated into a fermentation medium and cultured at 37°C. The fermentation medium consisted of: 5-15 g / L sugarcane bagasse, 5-15 g / L beef extract, 1-5 g / L anhydrous sodium acetate, and 40-80 mg / L histidine. By applying ultrasonic physical field stimulation during the logarithmic growth phase of the bacterial cells and continuing the culture until the end, high-density bacterial cells are obtained.

[0009] Furthermore, the power of the ultrasonic physical field stimulation is 50-250 W, the processing time is 10-30 minutes, and the processing temperature is 37℃.

[0010] Furthermore, the power of the ultrasonic physical field stimulation is 150 W, and the processing time is 20 minutes.

[0011] Furthermore, the fermentation medium consists of: 7.5 g / L sugarcane bagasse, 12.5 g / L beef extract, 5 g / L anhydrous sodium acetate, and 60 mg / L histidine.

[0012] A third aspect of the present invention provides a *Lactobacillus plantarum* P6 culture obtained by the aforementioned culture method, wherein the viable count of the culture is ≥ CFU / mL, and the survival rate after treatment with 0.3% (m / v) bile salts for 4 hours is not less than 80%.

[0013] The fourth aspect of this invention provides the application of Lactobacillus plantarum P6 as described in the first aspect in the preparation of probiotic agents.

[0014] The fifth aspect of this invention provides the application of the Lactobacillus plantarum P6 culture described in the third aspect in the preparation of probiotic agents.

[0015] The sixth aspect of the present invention provides the application of ultrasonic physical fields in improving the viable count of Lactobacillus plantarum P6, bile salt tolerance, and antioxidant and / or antibacterial activity of metabolites as described in the first aspect.

[0016] Furthermore, the power of the ultrasonic physical field stimulation is 50-250 W, the processing time is 10-30 minutes, and the processing temperature is 37°C; preferably, the power of the ultrasonic physical field stimulation is 150 W, and the processing time is 20 minutes.

[0017] Information on strain preservation: The strain of *Lactiplantibacillus* was named P6 and is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Its classification name is *Lactiplantibacillus plantarum*, accession number is CGMCC NO. 36056, and the deposit date is September 24, 2025.

[0018] The advantages and beneficial effects of this invention are: 1. The present invention screened a strain of Lactobacillus plantarum P6 with high bile salt tolerance from kimchi. After treatment with 0.3% (m / v) bile salt for 4 hours, the survival rate exceeded 80%, which is a high-quality strain resource for developing gastrointestinal probiotic preparations. 2. This invention, targeting the characteristics of *Lactobacillus plantarum* P6, achieves a viable cell count of up to [a certain number] by optimizing the culture medium and the synergistic effect of ultrasonic stimulation during the logarithmic growth phase, in a static culture system without supplemental feeding or precise pH control. With a CFU / mL or higher, it breaks through the current bottleneck of increasing the number of viable bacteria in static culture of Lactobacillus plantarum; 3. Multi-omics analysis confirmed that ultrasonic physical field treatment can specifically induce the accumulation of key metabolites related to cell membrane integrity, antioxidant defense and antibacterial activity. It not only increases bacterial density, but also simultaneously enhances cell membrane integrity, antioxidant defense capacity and antibacterial activity, and further improves bile salt tolerance, realizing the leap of bacterial cells from "high density" to "high density and high function". 4. The static high-density culture method described in this invention is a static mode that does not require precision fermentation tank equipment, complex feeding calculations, or continuous acid-base neutralization. It greatly simplifies the operation process, reduces energy consumption and production operation requirements, and is more conducive to industrial-scale production and cost control, thus meeting the needs of the large-scale development of the probiotic industry. Attached Figure Description

[0019] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0020] Figure 1 : Results of single-factor screening of carbon source (A) and metal ion (B); Figure 2 : Results of single-factor screening of nitrogen source (A) and dietary fiber (B); Figure 3 : Results of single-factor screening of growth factors; Figure 4 : Results of single-factor screening of buffer salts; Figure 5 : Result diagram of step-by-step optimization; Figure 6 Response surface methodology for optimizing the interactions of key culture medium components and its contour plots; Figure 7 : Graph showing the effect of ultrasonic power on viable bacterial count; Figure 8 : Graph showing the effect of ultrasound on bacterial growth curve; Figure 9 Physiological performance characterization diagrams of different treatment groups (Con, Nut, ULT); including pH change curves (A), bile salt tolerance (B), antibacterial activity (C), and scanning electron microscopy morphology (D). Figure 10 Untargeted metabolomics OPLS-DA score map (A) (a) negative ion mode, (b) positive ion mode, and differential metabolite heatmap (B); Figure 11 Map of the number of differentially expressed metabolites in non-targeted metabolomics (A), map of KEGG pathway enrichment (B); Figure 12 Box plot of key metabolites in non-targeted metabolomics; Figure 13 : Prokaryotic transcriptomics gene expression abundance distribution map (A), PCA analysis map (B), differential gene statistics map (C); Figure 14 : KEGG pathway enrichment map in prokaryotic transcriptomics. Detailed Implementation

[0021] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0022] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0023] The present invention will be described in detail below through embodiments. It should be understood that the following embodiments are only used to exemplify and further explain and illustrate the content of the present invention, and are not intended to limit the present invention.

[0024] Example 1: Screening and optimization of key components of culture medium 1. Preparation of seed liquid Lactobacillus plantarum P6 with preservation number CGMCC No.36056 was streaked onto MRS solid plates and incubated in an anaerobic incubator at 37℃ for 48 h. Uniform single colonies on the plates were picked and inoculated into MRS liquid medium and incubated statically at 37℃ for 18 h. After two generations of continuous activation, a stable seed culture of Lactobacillus plantarum P6 was obtained for later use.

[0025] 2. Single-factor screening of key components of culture medium Using MRS basal medium as a control, systematic single-factor concentration gradient experiments were conducted on the following six types of culture medium components. All cultures were incubated at 37℃ for 24 h before the viable cell count (CFU / mL) was determined.

[0026] (1) Optimization of carbon source screening Using sucrose, glucose, low-galactose, and maltose as carbon sources, five concentrations of 10, 20, 30, 40, and 50 g / L were set up. Figure 1 (A). The results showed that the above-mentioned single sugars, when used as carbon sources, had limited effect on promoting bacterial growth, and the viable cell count did not exceed the target. CFU / mL level.

[0027] (2) Optimization of metal ion screening The effects of adding manganese sulfate (0.05, 0.1, 0.2, 0.3, 0.4 g / L) and magnesium sulfate (0.2, 0.4, 0.6, 0.8, 1.0 g / L) were investigated respectively. Figure 1 (B). The results showed that in this study system, the addition of a single metal ion did not significantly promote the increase in viable bacterial count.

[0028] (3) Nitrogen source screening and optimization Using yeast extract, tryptone, beef extract, and soy peptone as nitrogen sources, tests were conducted at different concentration gradients. Figure 2 (A). The results showed that beef extract powder had the most significant growth-promoting effect, with a viable bacterial count reaching [value missing] at a concentration of 7.5 g / L. CFU / mL.

[0029] (4) Optimization of dietary fiber screening The tests were conducted using fructooligosaccharides, raffinose, bagasse, and oat beta-glucan as dietary fiber sources, respectively. Figure 2 (Medium B). The results showed that sugarcane bagasse exhibited a unique growth-promoting advantage, with the viable bacterial count reaching [value missing] at a concentration of 7.5 g / L. CFU / mL, significantly better than other dietary fibers.

[0030] (5) Growth factor screening: Leucine, serine, arginine, histidine, glycine, and other amino acids were tested as growth factors. Figure 3 The results showed that histidine had the most significant promoting effect, with the viable bacterial count reaching [value missing] at 60 mg / L. CFU / mL.

[0031] (6) Optimization of buffer salt screening Various buffer salts, including anhydrous sodium acetate, ammonium sulfate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, and triammonium citrate, were tested. Figure 4 The results showed that anhydrous sodium acetate had the best buffering and growth-promoting effects, with the highest viable bacterial count at 3 g / L. CFU / mL.

[0032] In summary, sugarcane bagasse (dietary fiber), beef extract (nitrogen source), anhydrous sodium acetate (buffer salt), and histidine (growth factor) were identified as the four key components affecting the high-density culture of Lactobacillus plantarum P6. Subsequent optimization will be carried out based on these components.

[0033] 3. Stepwise optimization of key components Based on the results of the single-factor screening above, the four key factors (sugarcane bagasse, beef extract powder, anhydrous sodium acetate, and histidine) were added and optimized step by step: (1) With the sugarcane bagasse concentration fixed at 7.5 g / L, the effect of anhydrous sodium acetate concentration (1-5 g / L) was re-examined under this condition. Figure 5 (A) It was found that when the concentration of anhydrous sodium acetate was 5 g / L, the combination with sugarcane bagasse had the best effect, and the viable bacteria count reached [value missing]. CFU / mL. This indicates that the optimal buffer salt concentration of the system changed after the introduction of bagasse, revealing a synergistic effect between the components.

[0034] (2) Results of optimizing the concentration of added beef extract (5-15 g / L) by fixing sugarcane bagasse at 7.5 g / L and anhydrous sodium acetate at 5 g / L. Figure 5 (B), determined that 12.5 g / L was optimal, achieving a viable bacterial count of [value missing]. CFU / mL.

[0035] (3) Fix sugarcane bagasse at 7.5 g / L, anhydrous sodium acetate at 5 g / L, and beef extract at 12.5 g / L, and optimize histidine concentration (40-80 mg / L) ( Figure 5 (C), determined that 60 mg / L is optimal, achieving a viable bacterial count of [value missing]. CFU / mL.

[0036] The final optimized culture medium formula was obtained: sugarcane bagasse 7.5 g / L, anhydrous sodium acetate 5 g / L, beef extract powder 12.5 g / L, and histidine 60 mg / L. Under this formula, the viable count of *Lactobacillus plantarum* P6 reached [value missing]. CFU / mL.

[0037] 4. Optimization of key component interactions using response surface methodology After completing the single-factor screening and stepwise optimization of key components, in order to further explore the interaction between key factors and determine their optimal combination, response surface methodology was used to systematically optimize four key factors (sugarcane bagasse concentration, beef extract concentration, anhydrous sodium acetate concentration, and histidine concentration).

[0038] (1) Experimental design A Box-Behnken design (BBD) was used, with viable cell count (CFU / mL) as the response value. A three-level design was implemented for four factors: bagasse concentration (A), anhydrous sodium acetate concentration (B), beef extract concentration (C), and histidine concentration (D), resulting in 29 experiments (including 5 center-point replicates). The experimental design and results are shown in Table 1. Multiple regression fitting was performed using Design-Expert software, yielding the quadratic polynomial regression equation:

[0039] The response function was fitted to the experimental data using regression analysis, and the simultaneous influence of both on the selected variables was tested. The results of the ANOVA of the regression equation are shown in Table 2. The ANOVA results show that the F-value of the model is 56.63, corresponding to a P-value < 0.0001, indicating that the model is highly significant and the selected response surface methodology is reliable. The P-value for the model's lack of fit is 0.3984 (> 0.05), indicating that the lack of fit is not significant, meaning that unknown factors have little interference with the model fit, and the experimental error mainly comes from random error. Furthermore, the coefficient of determination of this model... The value of 0.9705 indicates that the model can explain 97.05% of the variation in response values, and there is a high correlation between the predicted and measured values, proving that the established model can effectively predict and optimize the high-density culture conditions of Lactobacillus plantarum P6.

[0040]

[0041]

[0042] Figure 2 The diagram displays a three-dimensional response surface plot and a corresponding two-dimensional contour plot of the interactions between factors in the regression model, visually reflecting the combined effect of the two factors on the viable cell count. A steeper slope on the response surface indicates a more significant impact of the interaction on the response value. The shape of the two-dimensional contour lines also indicates the strength of the interaction: the closer the contour lines are to an ellipse, the more significant the interaction; the closer they are to a circle, the less significant the interaction.

[0043] Based on the ANOVA results in Table 2, the order of influence of the four factors on viable cell count is as follows: sugarcane bagasse concentration > anhydrous sodium acetate concentration > histidine concentration > beef extract concentration. The model predicts the optimal culture medium composition as: sugarcane bagasse 7.5 g / L, beef extract 12.5 g / L, anhydrous sodium acetate 5 g / L, and histidine 60 mg / L. Under these conditions, the predicted viable cell count is [value missing]. CFU / mL. Verified through three repeated experiments, the actual average viable count was [value missing]. The CFU / mL value, with a relative error of less than 1% compared to the predicted value, indicates that the regression model fits well and the prediction results are reliable.

[0044] Example 2: Static high-density culture and optimization of sonication conditions for Lactobacillus plantarum P6 This embodiment optimizes the ultrasonic conditions based on the optimized culture medium obtained in Example 1. Specifically, 3% (v / v) of Lactobacillus plantarum P6 seed culture is inoculated and statically cultured at 37°C. At the 6th hour of culture (logarithmic growth phase), ultrasonic treatment with different powers (50, 100, 150, 200, 250 W) is applied for 20 minutes, while maintaining the temperature at 37°C, and then culture continues for 24 hours.

[0045] The results showed that the 150 W ultrasonic treatment group had the best effect on promoting bacterial growth. At this point, the viable bacterial count reached its highest value. CFU / mL Figure 7 ), OD 600 Reached 1.673 ( Figure 8 The promoting effect is weakened when the power is below or above this value.

[0046] Based on the above optimization results, the optimal process for static high-density culture of Lactobacillus plantarum P6 was determined as follows: (1) Prepare fermentation culture medium according to the formula: sugarcane bagasse 7.5 g / L, beef extract powder 12.5 g / L, anhydrous sodium acetate 5 g / L, histidine 60 mg / L, sterilize and set aside; (2) Seed culture inoculation: The activated Lactobacillus plantarum P6 seed culture was inoculated into the fermentation medium at an inoculation rate of 3% (v / v); (3) Static culture: Static culture at 37℃ for 6 h until the cells enter the logarithmic growth phase; (4) Ultrasonic stimulation: At the 6th hour of culture, an ultrasonic physical field with a power of 150 W was applied for 20 min, and the treatment temperature was maintained at 37℃. (5) Continued culture: After ultrasonic treatment, continue static culture at 37℃ for 24 h to obtain high-density Lactobacillus plantarum P6 culture.

[0047] Example 3: Performance characterization and application performance verification of Lactobacillus plantarum P6 culture To verify the effectiveness of the static high-density culture method described in Example 2, three control experiments were set up for performance characterization: After determining the optimal sonication conditions (150 W, 20 min treatment at 6 h), static culture was performed using an optimized culture medium (7.5 g / L sugarcane bagasse, 12.5 g / L beef extract, 5 g / L anhydrous sodium acetate, 60 mg / L histidine), and the following three groups were established for systematic comparison: Control group (Con): Static culture in basal MRS medium; Optimized culture medium group (Nut): The optimized culture medium obtained in Example 1 was incubated statically; Ultrasonic treatment group (ULT): The optimized culture medium obtained in Example 1 was subjected to ultrasonic treatment at 150 W (6 h, 20 min).

[0048] After cultivation, the following performance characteristics were performed on each group of samples: (1) pH change curve ( Figure 9 (A) During the culture process, the pH value of the Con group decreased the fastest, dropping below 4.0 after 6 hours of culture, indicating significant lactic acid accumulation. The Nut group, containing a buffer system (anhydrous sodium acetate), experienced a more gradual pH decrease. In contrast, the pH of the ULT group remained between 5.0 and 5.5 after sonication treatment until the end of the culture, demonstrating that sonication effectively alleviated metabolic acid inhibition and maintained a more stable extracellular environment.

[0049] (2) Bile salt tolerance ( Figure 9 (B) After collecting the bacterial cells from each group, they were treated in 0.3% (m / v) bile salt solution at 37℃ for 4 h, and the survival rate was measured. The survival rate of the Con group was 75.52%, the Nut group increased to 78.36%, and the ULT group had the highest survival rate, reaching 80.67%. The results indicate that ultrasonic treatment not only did not impair the inherent bile salt tolerance of the strains, but also further enhanced their survival ability under bile salt stress.

[0050] (3) Antibacterial activity ( Figure 9 (C) The inhibitory effects of each culture group on pathogenic bacteria were determined using the agar diffusion method. The inhibition zone diameters of the ULT group against Staphylococcus aureus and Escherichia coli were 19.91 mm and 19.13 mm, respectively, significantly larger than those of the Con group (16.43 mm, 16.15 mm) and the Nut group (17.93 mm, 18.95 mm). This indicates that ultrasonic treatment enhanced the production or secretion of antibacterial metabolites by Lactobacillus plantarum P6.

[0051] (4) Observation of bacterial cell morphology (scanning electron microscopy, Figure 9 (D) Scanning electron microscopy results showed that the cells in the Con group were shrunken; the cells in the Nut group were relatively intact; and the cells in the ULT group were the most plump, with smooth cell surfaces and typical bacillus morphology, showing no obvious damage. This indicates that ultrasonic treatment at appropriate power can promote cell membrane integrity and help maintain the physiological activity of the cells.

[0052] Conclusion: The above results indicate that the synergistic effect of optimized culture medium and ultrasonic treatment significantly increased the viable count of *Lactobacillus plantarum* P6 (reaching [a certain number]). (CFU / mL), and also enhances its bile salt tolerance, antibacterial activity and cell membrane permeability, demonstrating the comprehensive advantages of this invention in improving bacterial function.

[0053] Example 4: Investigation of the molecular mechanism of high-density culture of Lactobacillus plantarum P6 To elucidate the synergistic promotion mechanism of culture medium optimization and ultrasonic treatment on the high-density growth of Lactobacillus plantarum P6, non-targeted metabolomics and prokaryotic transcriptomics were used to analyze the following three groups of samples: Control group (CON): Static culture in basal MRS medium; Optimized culture medium group (NUT): The optimized culture medium obtained in Example 1 was incubated statically; Ultrasonic treatment group (ULT): The culture medium was optimized in combination with ultrasonic treatment as described in Example 2 (150 W, 6 h, 20 min).

[0054] 1. Non-targeted metabolomics analysis After cultivation, accurately transfer an appropriate amount of bacterial culture into a 2 mL centrifuge tube, add 240 µL of extraction buffer containing internal standard, shake for 60 s to mix thoroughly, then add 60 µL of water and mix thoroughly. Centrifuge at 12000 r / min, 4℃ for 10 min. Filter the supernatant through a 0.22 μm membrane and use a UPLC HSS T3 column at a flow rate of 0.3 mL / min, a column temperature of 40℃, and an injection volume of 5 μL. The positive ion mobile phase consisted of 0.1% formic acid and acetonitrile, and the negative ion mobile phase consisted of acetonitrile and 5 mM ammonium formate in aqueous solution, with gradient elution. A Thermo Orbitrap Exploris 120 mass spectrometer detector acquired data in both positive and negative ion modes. The positive ion spray voltage was 3.50 kV, the negative ion spray voltage was -2.50 kV, the sheath gas was 40 arb, and the auxiliary gas was 10 arb. The capillary temperature was 325℃, and a first-stage full scan was performed at a resolution of 60,000 Hz. The first-stage ion scan range was 100-1000 m / z. Second-stage fragmentation was performed using HCD with a collision energy of 30% and a second-stage resolution of 15,000 Hz. The first four ions acquired were fragmented, and unnecessary MS / MS information was removed using dynamic exclusion. The raw data were imported into the metabolomics processing software Progenesis QI for data preprocessing. The processed data were analyzed using PCA, PLS-DA, and OPLS-DA. Significantly differentially expressed metabolites were screened using VIP≥1, P<0.05, and FC>1.2 as criteria. KEGG pathway enrichment analysis was used to screen key metabolic pathways for significantly differentially expressed metabolites.

[0055] The score plots of orthogonal partial least squares discriminant analysis (OPLS-DA) under positive and negative ion modes show ( Figure 10 (A) The three groups of samples (Con, Nut, and ULT) were clearly separated, and the samples within each group were tightly clustered, indicating that different treatment conditions led to global and reproducible changes in the metabolome. Compared with the Con group, the Nut group identified 540 significantly different metabolites (VIP≥1, p<0.05); while compared with the Nut group, the ULT group only identified 180 significantly different metabolites (…). Figure 11 (A). This data indicates that culture medium optimization is the main cause of the drastic changes in the metabolic profile, while the subsequent sonication treatment is a fine-tuning based on this new metabolism.

[0056] KEGG pathway enrichment analysis ( Figure 11 (B) The results showed that, compared with the control group, the differentially metabolites in the optimized culture medium group (Nut) were significantly enriched in core pathways such as ABC transporters, the TCA cycle, amino acid metabolism, and purine metabolism, confirming that nutritional optimization comprehensively enhanced the cell's material transport, energy production, and biosynthetic basis. Quantitative analysis of key metabolites ( Figure 12 Further analysis revealed that in the Nut group, the levels of 1-palmitoyl lysophosphatidylcholine, which is closely related to membrane lipid synthesis, histidine, which participates in amino acid metabolism, and inosine monophosphate (IMP), an important precursor to nucleotides, were significantly upregulated. In the ULT group, which received additional ultrasound treatment, the levels of some of these anabolic metabolites showed an adaptive regression. However, simultaneously, the glutathione metabolic pathway, which is closely related to oxidative stress response, was specifically activated, and the levels of its related metabolites were significantly increased. This in-depth analysis of the metabolic profile reveals that ultrasound stimulation does not simply continue the anabolic trend of nutrient fortification, but rather guides a precise redirection of metabolic flux. On the one hand, it promotes the accumulation of membrane phospholipid precursors such as 1-palmitoyl lysophosphatidylcholine, providing a direct material basis for the stability and repair of cell membrane structure, thereby enhancing the physical tolerance of the bacteria. On the other hand, it systematically enhances the bacteria's ability to cope with endogenous and exogenous oxidative stress by activating metabolic hubs of antioxidant defense systems such as glutathione. Furthermore, the regulation of amino acid metabolic networks such as histidine may also provide precursor support for the synthesis of stress-response proteins or antibacterial active molecules.

[0057] Therefore, ultrasonic treatment has achieved a leap from "high-density" culture that simply pursues biomass accumulation to "high-functionality" properties such as enhanced membrane integrity, antioxidant capacity and potential antibacterial activity of bacteria. It has profoundly elucidated the mechanism by which it improves the intrinsic quality and probiotic potential of bacteria at the metabolite level.

[0058] 2. Transcriptomics analysis Bacterial culture was centrifuged at 12000 r / min for 2 min at 4℃ to collect the bacterial pellet. Total RNA was extracted from the pellet using the RNAprep Pure Cell / Bacteria Kit. RNA purity and quantification were determined using a NanoDrop 2000 spectrophotometer, and RNA integrity was assessed using an Agilent 2100 Bioanalyzer. Samples that passed quality control were used for subsequent library construction. Ribosomal RNA was removed using the TIANSeq rRNA Depletion Kit, and transcriptome libraries were constructed using the VAHTS UniversalV6 RNA-seq Library Prep kit according to the manufacturer's instructions. Library concentration was quantified using Qubit, and library purity was detected using an Agilent 2100 Bioanalyzer. HTSeq-count was used to count the read counts of each gene, and gene expression levels were evaluated based on the Fragments Per Kilobase Million (FPKM) algorithm. DESeq2 was used for differentially expressed gene screening, based on gene expression levels, combined with fold change (FC) and statistical analysis methods to screen for differentially expressed genes. The screening criteria were set as |log2FC|≥1 and FDR≤0.05. Subsequently, bioinformatics analyses such as GO functional annotation and KEGG pathway enrichment were performed on these genes.

[0059] Principal component analysis (PCA) results showed that ( Figure 13 (B) The transcription profiles of the three groups of samples were clearly separated along the first principal component, indicating that treatment conditions have a decisive influence on global gene expression. Statistical analysis of differentially expressed genes ( Figure 13 The results from the (C) study quantified this effect: compared to the Con group, the ULT group had the most differentially expressed genes (654), followed by the Nut group (554); while the Nut group had the fewest differentially expressed genes compared to the ULT group (98). This trend is consistent with the results of metabolomics analysis, further confirming the dominant role of nutrient factors and the fine-tuning characteristics of ultrasound treatment.

[0060] KEGG pathway enrichment analysis of differentially expressed genes ( Figure 14The results showed that, compared with the control group (Con), the differentially expressed genes in the optimized culture medium (Nut) treatment group were significantly enriched in core metabolic pathways such as the phosphotransferase system (PTS), ribosomes, the TCA cycle, and amino acid biosynthesis. This reflects that nutrient factors simultaneously activated core biological processes such as carbohydrate uptake, protein translation, and central carbon metabolism at the transcriptional level. In the sonication treatment group (ULT), while continuing the activation of the above-mentioned basic metabolic pathways, the differentially expressed genes were also specifically enriched in the bacterial secretion system and the HIF-1 signaling pathway, indicating that ultrasound additionally stimulated transcriptional programs related to efflux, environmental perception, and stress adaptation. Particularly noteworthy is the comparison of the effects of ultrasound treatment on top of nutrient optimization (Nut vs ULT). The enriched pathways shifted to fatty acid degradation, glycolysis / gluconeogenesis, and glutathione metabolism. This clearly indicates that ultrasound treatment guided the cellular transcriptional focus from simply enhancing anabolic metabolism to optimizing energy metabolism patterns and finely maintaining redox homeostasis.

[0061] Transcriptomic analysis revealed at the gene expression level that the method of this invention fully activated the transcriptional program related to growth and synthesis through culture medium optimization; while temporal sonication further induced the expression of genes related to stress resistance, metabolic flux redistribution, and cell protection. This provides a transcriptional-level mechanistic explanation for the physiological characterization that sonication can enhance bacterial stress resistance and assist in achieving metabolic homeostasis.

[0062] 3. Integration of transcriptional and metabolic multi-omics and mechanism inference Based on the integrated analysis of metabolomics and transcriptomics data, a molecular response model was constructed to synergistically promote the high-density growth and high tolerance of *Lactobacillus plantarum* using the method of this invention. This model first elucidates the core driving role of nutrient optimization: culture medium optimization simultaneously upregulates genes in pathways such as the phosphotransferase system (PTS) and ribosomes at the transcriptional level, and promotes the accumulation of biosynthetic metabolites such as amino acids and nucleotide precursors at the metabolic level. These two factors synergistically constitute the core driving force for the burst of cell anabolic metabolism and rapid biomass accumulation. Secondly, the model reveals the balancing and enhancing effects of sonication: while inducing widespread transcriptional reprogramming to activate stress responses and global regulatory networks, sonication at the metabolic level manifests as a fine-tuning of the over-activated state of nutrients and specifically enhances the antioxidant defense system centered on glutathione metabolism, forming a unique pattern of "broad transcriptional early warning and precise metabolic fine-tuning," which helps cells maintain metabolic homeostasis and membrane structural integrity during rapid growth. Finally, pathway consistency analysis validated the effectiveness of the model: core hubs such as the TCA cycle, alanine-aspartate-glutamate metabolism, and purine metabolism showed consistent synergistic changes at the transcriptional and metabolic levels. For example, the accumulation of the key metabolite IMP and the upregulation of genes in the purine metabolism pathway directly corroborated each other, thus confirming the effective biological transmission from gene expression regulation to the final metabolic function output and systematically elucidating the molecular basis of the synergistic regulation of nutrition and physical fields.

[0063] The "optimized culture medium combined with logarithmic growth phase sonication" strategy of this invention achieves systematic optimization from gene expression, metabolic flow, to the final phenotype by synergistically regulating anabolic metabolism and stress defense networks at multiple omics levels. This enables the method to simultaneously achieve high-density culture (viable count ≥ ) of *Lactobacillus plantarum* P6 in a simple static culture system. The CFU / mL ratio and high stress resistance (bile salt survival rate ≥80%) provide a solid molecular mechanism basis.

[0064] Finally, it should be noted that the above description is only used to illustrate the technical solutions of the present invention and is not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention.

Claims

1. A strain of *Lactobacillus plantarum* P6, characterized in that, The Lactiplantibacillus plantarum P6 strain was deposited on September 24, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 36056.

2. A static high-density culture method for *Lactobacillus plantarum* P6 as described in claim 1, characterized in that, The static high-density culture method includes the following steps: The *Lactobacillus plantarum* P6 was inoculated into a fermentation medium and cultured at 37°C. The fermentation medium consisted of: 5-15 g / L sugarcane bagasse, 5-15 g / L beef extract, 1-5 g / L anhydrous sodium acetate, and 40-80 mg / L histidine. By applying ultrasonic physical field stimulation during the logarithmic growth phase of the bacterial cells and continuing the culture until the end, high-density bacterial cells are obtained.

3. The static high-density culture method according to claim 2, characterized in that, The power of the ultrasonic physical field stimulation is 50-250 W, the processing time is 10-30 minutes, and the processing temperature is 37℃.

4. The static high-density culture method according to claim 3, characterized in that, The power of the ultrasonic physical field stimulation is 150 W, and the processing time is 20 minutes.

5. The static high-density culture method according to claim 2, characterized in that, The fermentation medium consisted of: 7.5 g / L sugarcane bagasse, 12.5 g / L beef extract, 5 g / L anhydrous sodium acetate, and 60 mg / L histidine.

6. A culture of *Lactobacillus plantarum* P6 obtained by the culture method according to any one of claims 2 to 5, characterized in that, The viable bacterial count of the culture is ≥ CFU / mL, and the survival rate after treatment in 0.3% bile salts for 4 hours is not less than 80%.

7. The application of Lactobacillus plantarum P6 as described in claim 1 in the preparation of probiotic agents.

8. The use of the Lactobacillus plantarum P6 culture as described in claim 6 in the preparation of probiotic agents.

9. The application of ultrasonic physical fields in improving the viable count, bile salt tolerance, and antioxidant and / or antibacterial activity of the metabolites of Lactobacillus plantarum P6 as described in claim 1.

10. The application according to claim 9, characterized in that, The power of the ultrasonic physical field stimulation is 50-250 W, the processing time is 10-30 minutes, and the processing temperature is 37℃.

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