A novel lactic acid bacteria culture medium, culture method and application

Abstract

The application discloses a novel lactic acid bacteria culture medium, a culture method and application. The application systematically optimizes a culture medium formula and fermentation process parameters through single factor tests, Plackett-Burman design and response surface analysis, and provides a DYH culture medium suitable for lactic acid bacteria, which can significantly improve the fermentation density of lactic acid bacteria, the lactic acid content in a fermentation liquor and the L-lactic acid proportion; in addition, the DYH culture medium effectively enhances the stress resistance of lactic acid bacteria in resisting bile salts and artificial intestinal fluid, and significantly improves the antioxidant capacity of the fermentation liquor. The DYH culture medium is a novel lactic acid bacteria culture medium integrating high-density fermentation, metabolic regulation, stress resistance enhancement and antioxidant function, and can be widely applied to various lactic acid bacteria strains, has good universality and industrialization prospect, provides strong support for development and industrialization of lactic acid bacteria functional products, and has wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of food microbiology, specifically relating to a novel lactic acid bacteria culture medium, culture method, and application. Background Technology

[0002] Probiotics are live microorganisms that, when ingested in sufficient quantities, can produce beneficial effects on the health of the host. They mainly include lactic acid bacteria, bifidobacteria, and some yeasts, with lactic acid bacteria being the most widely used. Lactic acid bacteria (LAB) are a group of microorganisms whose core metabolic characteristic is fermentation and acid production. They are Gram-positive, catalase-negative, and do not produce spores. These microorganisms are widely distributed in nature, not only in various fermented foods but also commonly found in the gastrointestinal tracts of humans and animals. As an important class of functional microorganisms, lactic acid bacteria have a long history of application in human production and daily life, and are one of the core microbial groups in traditional fermentation industries and modern probiotic research. With the rapid development of microbial molecular biology and omics technologies, the systematic classification, functional mechanisms, and roles of lactic acid bacteria in health regulation are being increasingly revealed, making them an important research subject in food science, nutrition, and biomedicine.

[0003] Lactic acid bacteria, as an important probiotic resource, have broad application prospects in functional food development, microecological preparation production, and metabolic disease intervention research. However, lactic acid bacteria have relatively strict requirements for nutritional conditions, and their growth rate, viable cell count, and metabolite production are all significantly affected by the composition of the culture medium. Therefore, constructing a culture system with a reasonable nutrient ratio, controllable cost, and effective promotion of lactic acid bacteria growth and functional expression is a key step in achieving efficient fermentation and industrial application of lactic acid bacteria strains.

[0004] Currently, most lactic acid bacteria products on the market exist in the form of fermented bacterial powder or inoculum. However, these products generally face bottlenecks such as low viable cell counts and insufficient product stability, severely restricting the full realization of the comprehensive probiotic properties of lactic acid bacteria. While traditional MRS medium is a classic choice for lactic acid bacteria culture, it still has limitations in achieving high-density fermentation, improving strain resistance, and increasing the yield of functional metabolites. Therefore, developing a novel lactic acid fermentation medium to achieve high-density fermentation and improve strain efficacy is particularly necessary. Summary of the Invention

[0005] This invention addresses the limitations of existing technologies by providing a novel lactic acid bacteria culture medium, a method for cultivating lactic acid bacteria based on this medium, and its applications. By combining single-factor experiments, Plackett-Burman design, and response surface methodology, this invention systematically optimizes the culture medium formulation and fermentation process parameters, providing a DYH culture medium suitable for lactic acid bacteria. This medium, with animal and plant protein resources and growth-promoting factors as its core components, not only significantly promotes lactic acid growth and increases the fermentation density of lactic acid bacteria, but also significantly increases the total lactic acid content and the proportion of L-lactic acid in the fermentation broth. Furthermore, this medium effectively enhances the stress resistance of lactic acid bacteria strains in terms of bile salt tolerance and artificial intestinal fluid tolerance, and significantly improves the antioxidant capacity of the fermentation broth, particularly in reducing power, superoxide anion scavenging capacity, and hydroxyl radical scavenging capacity. It can be widely applied to various different lactic acid bacteria strains, providing strong support for the development and industrialization of functional lactic acid bacteria products, and has broad application prospects.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a novel lactic acid bacteria culture medium, the composition of which includes: 5-50 g / L soy protein isolate, 2-30 g / L oat flour, 2-40 g / L maltose, 5-100 g / L carrot, 2-4 g / L anhydrous sodium acetate, 2-4 g / L K₂HPO₄, 4-5 g / L ammonium citrate, 0.05-1.0 g / L magnesium sulfate heptahydrate, 0.01-0.2 g / L manganese sulfate monohydrate, 0.01-0.1 g / L phenylalanine, and 0.01-0.05 g / L vitamin B5.

[0007] Further, the culture medium comprises: 40-50 g / L soy protein isolate, 10-30 g / L oat flour, 20-40 g / L maltose, 60-100 g / L carrot, 2-4 g / L anhydrous sodium acetate, 2-4 g / L K₂HPO₄, 4-5 g / L ammonium citrate, 0.56-0.79 g / L magnesium sulfate heptahydrate, 0.058-0.082 g / L manganese sulfate monohydrate, 0.03-0.05 g / L phenylalanine, and 0.01-0.03 g / L vitamin B5.

[0008] Furthermore, the culture medium comprises: 48.2 g / L soy protein isolate, 10 g / L oat flour, 29 g / L maltose, 60 g / L carrot, 2 g / L anhydrous sodium acetate, 2.37 g / L K₂HPO₄, 4 g / L ammonium citrate, 0.56 g / L magnesium sulfate heptahydrate, 0.058 g / L manganese sulfate monohydrate, 0.03 g / L phenylalanine, and 0.01 g / L vitamin B5.

[0009] Furthermore, the particle size of the soy protein isolate is ≤200 mesh.

[0010] Further, the carrots were juiced with water and filtered before being added to the culture medium to achieve a final concentration of 60 g / L.

[0011] Furthermore, the preparation method of the culture medium is as follows: weigh each component according to the above ratio, add it to distilled water and stir to dissolve, sterilize it by high-pressure steam at 115℃~121℃, and then cool it for later use.

[0012] This invention also provides the application of the above-mentioned novel lactic acid bacteria culture medium in any of the following A1)-A8): A1) Application in promoting the growth of lactic acid bacteria; A2) Application in the preparation of products that promote the growth of lactic acid bacteria; A3) Application in increasing the lactic acid content and / or L-lactic acid ratio in lactic acid bacteria fermentation broth; A4) Application in the preparation of products with high lactic acid bacteria fermentation broth and / or high lactic acid content and / or L-lactic acid ratio; A5) Application in improving the bile salt and / or artificial intestinal fluid tolerance of lactic acid bacteria; A6) Application in the preparation of products that enhance the bile salt and / or artificial intestinal fluid tolerance of lactic acid bacteria; A7) Application in improving the antioxidant capacity of lactic acid bacteria fermentation broth; A8) Application in the preparation of products that enhance the antioxidant capacity of lactic acid bacteria fermentation broth.

[0013] Furthermore, the antioxidant capacity includes one or more of the following: reducing capacity, superoxide anion scavenging capacity, and hydroxyl radical scavenging capacity.

[0014] The present invention also provides a method for fermenting lactic acid bacteria, comprising: inoculating lactic acid bacteria into the above-mentioned novel lactic acid bacteria culture medium for fermentation culture.

[0015] Furthermore, the fermentation conditions include: an inoculum size of 4% to 10%, a temperature of 35 to 39°C, a fermentation time of more than 18 hours, and the addition of a pH adjuster during the fermentation process to control the pH of the fermentation broth to be 5.5 to 6.5.

[0016] Furthermore, the fermentation culture conditions include: an inoculum size of 6%, a temperature of 37°C, a fermentation culture time of more than 18 hours, and the addition of a pH adjuster during the fermentation process to control the pH value of the fermentation broth to 6.

[0017] Furthermore, the pH adjuster is selected from Na2CO3 or NaHCO3, preferably NaHCO3.

[0018] Furthermore, the lactic acid bacteria are selected from one or more of the following: Lactobacillus paracasei, Lactobacillus casei, Lactobacillus pentosus, and Lactobacillus plantarum.

[0019] Further, the lactic acid bacteria are selected from one or more of the following: Lacticaseibacillus paracasei LP33, Lacticaseibacillus paracasei LPCWU, Lacticaseibacillus casei SHIROTA, Lacticaseibacillus casei L. Zhang, Lactiplantibacillus pentosus JS1, Lactiplantibacillus plantarum LPHS, Lactiplantibacillus plantarum ST-III, and Lactiplantibacillus plantarum GL-2.

[0020] Compared with the prior art, the present invention has the following outstanding advantages: This invention employs single-factor experiments, Plackett-Burman design, and response surface methodology to systematically optimize the culture medium formulation and fermentation process parameters, successfully developing a culture medium suitable for lactic acid bacteria, which is named DYH culture medium. Compared with traditional MRS culture medium, the universal compatibility of DYH culture medium is systematically evaluated using in vitro probiotic performance parameters such as viable cell count, stress resistance, and antioxidant capacity as indicators.

[0021] 1. The DYH culture medium provided by this invention is composed of animal and plant protein resources and growth-promoting factors. By optimizing its ratio, the fermentation density of lactic acid bacteria can be increased by about 2.5 to 4 times, realizing high-density culture of strains. This is beneficial to reduce production costs, improve production efficiency, meet the needs of large-scale industrial fermentation, and also provide new ideas for the utilization of animal and plant protein resources.

[0022] 2. The DYH culture medium of the present invention can significantly increase the lactic acid content in the same lactic acid bacteria fermentation broth and increase the proportion of L-lactic acid, thereby obtaining higher quality lactic acid fermentation products, which is beneficial to improving the nutritional value and functional characteristics of the products.

[0023] 3. Lactic acid bacteria cultured on DYH medium exhibit significantly enhanced resistance to bile salts and artificial intestinal fluid, which is beneficial for the survival and colonization of probiotics in the gastrointestinal environment, thereby improving the in vivo efficacy and stability of probiotic preparations.

[0024] 4. DYH medium can also significantly improve the antioxidant properties of lactic acid bacteria fermentation broth, especially in terms of reducing power, superoxide anion scavenging ability and hydroxyl radical scavenging ability, giving fermentation products stronger potential for free radical scavenging and oxidative damage protection.

[0025] 5. The DYH culture medium of the present invention has shown good applicability and stable promoting effect on a variety of different lactic acid bacteria strains such as GL-2, ST-III, LP33, JS1, and LPH, and has broad application value.

[0026] In summary, this invention provides a novel lactic acid bacteria culture medium that integrates high-density fermentation, metabolic regulation, stress resistance enhancement, and antioxidant functions. It significantly improves the overall performance of lactic acid bacteria, provides strong support for the development and industrialization of functional lactic acid bacteria products, is suitable for large-scale promotion and use, and has broad application prospects. Attached Figure Description

[0027] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This is a single-factor optimization experiment of DYH culture medium in Example 1 of the present invention, wherein: A is the effect of different nitrogen sources on fermentation results; B is the optimization of soybean protein isolate concentration; C is the effect of compound nitrogen source on fermentation results; D is the optimization of the compound nitrogen source ratio of soybean protein and oat protein; E is the effect of different carbon sources on fermentation results; F is the optimization of maltose concentration; and G is the effect of different carbon-nitrogen ratios on fermentation results.

[0029] Figure 2 This is a single-factor optimization experiment of DYH culture medium in Example 1 of the present invention, wherein: A is the effect of dipotassium hydrogen phosphate concentration on fermentation results; B is the effect of sodium acetate concentration on fermentation results; C is the effect of ammonium citrate concentration on fermentation results; D is the effect of magnesium sulfate heptahydrate concentration on fermentation results; and E is the effect of manganese sulfate monohydrate concentration on fermentation results.

[0030] Figure 3This is a single-factor optimization experiment of DYH culture medium in Example 1 of the present invention, wherein: A is the effect of different vegetable juices on fermentation results; B is the optimization of carrot juice concentration; C is the effect of different amino acids on fermentation results; D is the optimization of phenylalanine concentration; E is the effect of different vitamins on fermentation results; and F is the optimization of VB5 concentration.

[0031] Figure 4 The figures are the half-normal distribution diagram (A) and Pareto diagram (B) of the PB experiment in Embodiment 2 of the present invention.

[0032] Figure 5 This is a diagram showing the results of the steepest hill climb experiment in Embodiment 3 of the present invention.

[0033] Figure 6 The diagram shows the pairwise response surface interaction diagrams in Embodiment 3 of the present invention, where: A is the interaction diagram between K2HPO4 and maltose; B is the interaction diagram between soy protein isolate and maltose; and C is the interaction diagram between soy protein isolate and K2HPO4.

[0034] Figure 7 For the optimization of the fermentation process in Example 4 of the present invention, wherein: A is the effect of temperature on fermentation results; B is the effect of inoculum size on fermentation results; C is the effect of fermentation time on fermentation results; D is the pH value change of each group; E is the change of fermentation bacteria number of each group; F is the change of residual sugar content in fermentation broth of each group; and G is the change of lactic acid content of each group.

[0035] Figure 8 This illustrates the effect of DYH culture medium on the fermentation density of eight different lactic acid bacteria strains in Example 5 of this invention.

[0036] Figure 9 This figure illustrates the effect of DYH culture medium on the acid-producing capacity of eight different lactic acid bacteria in Example 5 of the present invention. A represents the lactic acid content of different strains; B represents the relative content of L-lactic acid and D-lactic acid in the fermentation broth of each strain cultured on MRS medium, with the numbers in the figure indicating the relative content of L-lactic acid; C represents the relative content of L-lactic acid and D-lactic acid in the fermentation broth of each strain cultured on DYH medium, with the numbers in the figure indicating the relative content of L-lactic acid.

[0037] Figure 10 The figure shows the effect of DYH culture medium on the stress resistance of eight different lactic acid bacteria in Example 5 of this invention. A represents the bile salt tolerance of lactic acid bacteria; B represents the tolerance of lactic acid bacteria to artificial intestinal fluid. The vertical axis in the figure represents the survival rate (%).

[0038] Figure 11The antioxidant effect of DYH culture medium on eight different lactic acid bacteria fermentation products in Example 5 of this invention is shown, where: A is the reducing power of lactic acid bacteria fermentation broth; B is the hydroxyl radical scavenging power of lactic acid bacteria fermentation broth; and C is the superoxide anion scavenging power of lactic acid bacteria fermentation broth. Detailed Implementation

[0039] The following embodiments are only used to more clearly illustrate the technical solutions of the present invention, and are therefore merely examples and should not be used to limit the scope of protection of the present invention. It should be noted that, unless otherwise stated, the technical or scientific terms used in this application should have the ordinary meaning understood by those skilled in the art. Unless specifically stated, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless specifically stated, the reagents and materials used in the following embodiments are commercially available.

[0040] Example 1: Preparation of DYH Culture Medium - Single-Factor Experiment In the preparation of DYH culture medium, *Lactobacillus plantarum* GL-2 was mainly used as the experimental strain, and the number of viable fermentation cells was used as the reference indicator. The preservation information for *Lactobacillus plantarum* GL-2 is as follows: Preservation name: *Lactobacillus plantarum*; Preservation institution: China Center for Type Culture Collection; Preservation address: Bayi Road, Wuchang District, Wuhan City, Hubei Province; Preservation number: CCTCCM 2023328; Preservation date: March 15, 2023 (see patent CN120718808A for details). This embodiment optimizes the components of DYH culture medium through single-factor experiments, as detailed below.

[0041] (1) Single-factor optimization of carbon and nitrogen sources: First, the nitrogen source for DYH medium was determined by selecting oat flour (A), soy protein isolate (B), corn flour (C), skim milk powder (D), and whey protein powder (E), with MRS medium as a control, to determine the optimal nitrogen source and optimize its concentration. Then, the nitrogen source with the best effect was used as the main nitrogen source and combined with the other nitrogen sources to determine the final nitrogen source of the system.

[0042] Table 1 Nitrogen source combination scheme

[0043] After determining that the optimal nitrogen source mixture was soy protein isolate and oats, the ratios of soy protein isolate and oats were mixed at 1:1, 2:1, 3:1, 4:1, and 5:1 (mass ratio) (Table 2), with other components remaining constant. Each group was tested in triplicate. After strain GL-2 was cultured to the end of the logarithmic growth phase, the culture was adjusted to one OD, and each experimental group was inoculated at a 1% inoculum rate. The fermentation volume was 100 mL, and fermentation was carried out at 37 ℃ for 20 h. The viable cell count in each group was then determined using the dilution-spreading method. The optimal nitrogen source mixture ratio was determined by comparing the viable cell counts in each group.

[0044] Table 2. Blend ratio of soy protein isolate and oat protein

[0045] Next, nine different monosaccharides and polysaccharides—xylose (A), mannose (B), mannitol (C), lactose (D), erythritol (E), glucose (F), maltose (G), sucrose (H), and trehalose (I)—were added to the system at the same concentration, while keeping other components constant. The optimal carbon source and its optimal concentration were determined using the viable cell count as a reference indicator. The nitrogen source concentration was kept constant, and the optimal carbon-to-nitrogen ratio in the system was determined by varying the carbon source concentration.

[0046] The results of the single-factor optimization experiment of carbon and nitrogen sources are as follows: Figure 1 As shown. Nitrogen and carbon sources are the core nutrients in the entire culture medium, playing a crucial role in energy supply, cell construction, and metabolite synthesis for the strain. It can be seen that in studies of different plant proteins used as nitrogen sources in DYH, soybean protein isolate showed the best promoting effect on the growth of GL-2. Figure 1 A), showing a significant difference from other groups (p<0.05), the optimal addition amount was 4% (w / v), i.e., 40 g / L. Figure 1 B); Soy protein isolate was used as the main nitrogen source and combined with several other nitrogen sources, as shown in Table 1. When soy protein isolate and oats were combined (Group F) as the nitrogen source, the cell fermentation density reached its maximum value and showed a significant difference compared with other groups (p<0.05). Figure 1 C); and when the mass ratio of soy protein isolate to oats was 4:1 (group D), the number of viable bacteria reached its maximum value, significantly better than other ratio groups (p<0.05), indicating that the nitrogen source in this ratio had the best growth-promoting effect on strain GL-2. Figure 1 D). Among the several small-molecule sugars tested, maltose was used as the carbon source and GL-2 reached its maximum fermentation density. Figure 1 E), followed by trehalose; when the maltose concentration exceeded 20 g / L, the number of viable fermentation bacteria in GL-2 showed a decreasing trend ( Figure 1 (F) Based on comprehensive analysis, the optimal addition amount of maltose is 2% (w / v), i.e., 20 g / L. Reasonable control of the carbon-to-nitrogen ratio (C / N) is crucial for high-density fermentation and metabolite synthesis, and also determines the economy and efficiency of the fermentation process. The optimization results for the C / N ratio of DYH are shown in […]. Figure 1 G. As the carbon-nitrogen ratio increases, the number of viable fermentation bacteria fluctuates. When the carbon-nitrogen ratio reaches 1:2, the number of viable bacteria reaches its maximum value, which is statistically significant (p<0.05).

[0047] (2) Single-factor optimization of inorganic salts: Further optimization of inorganic salts in the system. Using MRS medium, dipotassium hydrogen phosphate, anhydrous sodium acetate, ammonium citrate, and Mg were sequentially selected and optimized. 2+ and Mn 2+ The optimal concentration of ions and the concentration of all buffers were determined in accordance with the national standard GB2760-2024.

[0048] The results are as follows Figure 2 As shown, although inorganic salts account for a small proportion of the content in the culture medium, they are indispensable nutrients. Their role runs through the entire process of microbial growth and product synthesis, involving multiple dimensions such as cell structure and function construction, enzyme activity regulation, environmental homeostasis and product synthesis. Figure 2 As shown in Figure A, when the concentration of dipotassium hydrogen phosphate (K2HPO4) was 2 g / L, the fermentation density of GL-2 reached its maximum value and was significantly different from that of the control group (p<0.05); the cell density showed a trend of first increasing and then decreasing with the increase of anhydrous sodium acetate concentration, reaching its maximum value when the concentration was 2 g / L. Figure 2 B); 4 g / L ammonium citrate is the optimal addition level, at which the viable bacteria count reaches its maximum and is statistically significant (p<0.05). Figure 2 C); by Figure 2 From D, we can know that Mg 2+ The viable cell count reached its maximum at an addition level of 0.79 g / L, compared to the control group (i.e., no Mg added). 2+ There was a significant difference compared to the blank control (p<0.05), and when Mg 2+ There was no significant difference in viable bacterial count between 0.56 g / L and 0.79 g / L of Mg (p>0.05), therefore Mg 2+ The optimal addition amount of (i.e., magnesium sulfate heptahydrate) is 0.56 g / L; in Mn 2+ The investigation into the amount added shows that Mn 2+ The optimal addition level of manganese sulfate monohydrate (Mn) is 0.058 g / L, at which point the viable bacterial count of GL-2 reaches its maximum value, and is significantly higher than that of the control group (i.e., no Mn added). 2+ The difference was statistically significant (p<0.05) compared to the blank control group. Figure 2 E).

[0049] (3) Optimization of other trace elements: Further optimization of vegetable juice, amino acids and vitamins in the system. Different vegetables were juiced and filtered with water. Carrot juice (A), white radish juice (B), cabbage juice (C), tomato juice (D), cucumber juice (E) and celery juice (F) were added to the system respectively. Other components remained unchanged. The blank control CK without additional vegetable juice was used. The optimal vegetable juice and its optimal addition amount were determined with the number of viable fermentation bacteria as the reference index. Thirteen different amino acids, namely tyrosine (A), glycine (B), aspartic acid (C), tryptophan (D), citrulline (E), threonine (F), cysteine ​​(G), glutamic acid (H), leucine (I), alanine (J), phenylalanine (K), isoleucine (L) and valine (M), and six different vitamins, namely VC, VM, VB1, VB2, VB5 and VB6, were added to the system respectively. Other components remained unchanged. The blank control CK without additional amino acids was used. The optimal amino acids and vitamins in the system and their addition amounts were determined with the number of viable fermentation bacteria as the reference index.

[0050] The results are as follows Figure 3 As shown, vegetable juice provides essential nutrients and energy, and contains natural sugars, vitamins, and minerals. Figure 3 A showed that carrot juice had the most significant promoting effect on strain GL-2, and the difference was statistically significant compared with other experimental groups and the control group (p<0.05). The optimal addition amount was a final concentration of carrot juice of 6% (w / v), i.e., 60 g / L. Figure 3 B); from Figure 3 As shown in Figure C, the fermentation density of GL-2 reached its maximum value after adding phenylalanine to the culture medium, and the difference was significant compared with other experimental groups (p<0.05). With the increase of phenylalanine concentration, the fermentation density showed a trend of first increasing and then decreasing, and the cell density reached its maximum value at 0.03 g / L. Figure 3 D); After supplementing with different vitamins, the bacterial cell density increased to varying degrees, with VB5 showing the best promoting effect. Figure 3 E), its optimal addition amount is 0.01 g / L ( Figure 3 F).

[0051] Example 2: Preparation of DYH medium - Plackett-Burman experiment Plackett-Burman statistical design is frequently used in studies on the optimization of bacterial culture media. It is a two-factor (-1 and +1) design that identifies key production variables by screening "n" variables in an "n+1" experiment. This study investigated factors such as carbon and nitrogen sources, buffers, trace elements, amino acids, vitamins, and fruit and vegetable juices in DYH culture medium. All 10 selected factors were tested at both Plackett-Burman levels. The factors and design levels are shown in Table 3. Since the main effect nitrogen source in the compound nitrogen source study was soy protein isolate, the nitrogen source study primarily focused on soy protein isolate. The effects of the 10 factors in DYH culture medium on the viable count of GL-2 bacteria were examined, and the three variables with the greatest impact were selected for subsequent RSM experimental design.

[0052] Table 3. PB Experimental Design Table of Factor Levels

[0053] Based on the results of the previous single-factor optimization, a 10-factor, 3-level experimental design was conducted using Design-Expert 12 software. Each group was replicated in triplicate, and the average viable count of each group was used as the response value. The results are shown in Table 4. Plackett-Burman analysis of variance was performed on the data in Table 3, as shown in Table 5.

[0054] Table 4. Plackett-Burman Experimental Design and Results

[0055] Table 5. Results of the Plackett-Burman Experimental Analysis of Variance

[0056] The ANOVA table shows that the fitted model is significant (p<0.05) and the lack-of-fit term is not significant (p>0.05), thus having practical reference value. Using viable cell count as an indicator to screen for significant influencing factors, the results show that maltose, carrot juice, soy protein isolate, K2HPO4, MnSO4, and phenylalanine all have significant effects on viable cell count (p<0.05).

[0057] Through multiple regression analysis, the fitting equations between the response values ​​and each factor are obtained as follows: .

[0058] And draw the half-normal distribution plot and Pareto plot ( Figure 4 ),in Figure 4A is a semi-normal distribution plot. The distance of a factor from the baseline reflects the magnitude of its influence on the response value. The distances from the baseline, from farthest to closest, are maltose, K2HPO4, soy protein isolate, manganese sulfate, phenylalanine, carrot, and magnesium sulfate. Orange represents positive effects, and blue represents negative effects. Among the positive effects, maltose is farthest from the baseline, and among the negative effects, manganese sulfate is farthest from the baseline. Figure 4 B is the Pareto chart; the colors represent... Figure 4 A is consistent (orange is positive, blue is negative). Factors are arranged from highest to lowest column height to reflect the magnitude of their influence. The top three factors are: maltose, K2HPO4, and soy protein isolate.

[0059] Example 3: Preparation of DYH medium - Response surface methodology (RSM) experiment The top three effectors identified in the Plackett-Burman experiment—maltose, K2HPO4, and soy protein isolate—were subjected to a steepest ramp experiment to identify the center point for subsequent RSM experiments and narrow down the range of each factor. The ramp step size for each factor ( According to the coefficients of the fitting equation in the PB experiment ( ) and the original step size of the PB experiment ( Calculate using the following formula:

[0060] In the formula, : Coefficients of the fitting equation for significant factors : PB experimental step size; The steepest incline test step length, :maltose, K2HPO4; Soy protein isolate.

[0061] The experimental design and results are shown in Table 6 and Figure 5 As shown in the figure. The results show that when the experiment reached level 1 (i.e., maltose concentration 2.91% (w / v), K2HPO4 concentration 2.33 g / L, and soy protein isolate 4.26% (w / v)), the viable count reached its peak, indicating that this condition was near the optimal region in the current parameter space. Therefore, this point was determined as the center point of the response surface methodology experiment.

[0062] Table 6. Results of the Climbing Scheme Design for Each Factor

[0063] Based on the results of the steepest climbing experiment, maltose, K2HPO4 and soy protein isolate were used as independent variables, and viable cell count was used as the response value. The contents of other components in the culture medium were kept constant. The response surface methodology was optimized using Design-Expert software. A 3-factor, 3-level design was carried out. The level settings of each factor are shown in Table 7. The experimental design and results are shown in Table 8.

[0064] Table 7 Response Surface Experimental Design Level Table

[0065] Table 8 Response Surface Experimental Design and Results

[0066] The results of the analysis of variance are shown in Table 9 below. The results show that the effects of maltose, K2HPO4, and soy protein isolate on the viable cell count were all highly significant (p<0.01). Interaction analysis showed that the interactions between maltose and K2HPO4 (AB) and between maltose and soy protein isolate (AC) were significant (p<0.05), indicating a non-linear effect of their synergistic effect on the viable cell count. The interaction between K2HPO4 and soy protein isolate (BC) was not significant (p>0.05), indicating that their individual effects on the viable cell count were relatively independent. All quadratic terms showed highly significant effects (p<0.01), and the fitted regression equation is as follows: .

[0067] Table 9 Results of Response Surface Experimental Variance Analysis

[0068] Further, the interaction diagrams of the factors in the response surface model were plotted, such as... Figure 6 As shown, the interaction between K2HPO4 and maltose exhibits a convex surface, with the optimal response value located near the center point, indicating a synergistic effect between the two in the central region. The interaction between soy protein isolate and maltose shows a parabolic characteristic, with significant extreme points on the surface, indicating a significant nonlinear interaction effect of pairwise interactions on viable cell count. The surface morphology results are consistent with the significant AB and AC interactions (p<0.05) in the analysis of variance, verifying the crucial role of interactions in influencing viable cell count.

[0069] According to Design-Expert 12 software analysis, response surface methodology optimization yielded the optimal addition levels of maltose at 2.9% (w / v), i.e., 29 g / L; K₂HPO₄ at 2.37 g / L; and soy protein isolate at 4.82% (w / v), i.e., 48.2 g / L. Three replicate validation experiments were conducted under these conditions, and the actual viable cell counts were measured. The results are shown in Table 10. The relative error between the actual measured viable cell counts and the software predictions was approximately 1.4%, indicating good model prediction accuracy and reliable optimization results.

[0070] Table 10 Response Surface Experiment Validation Results .

[0071] Based on the results of the single-factor experiments, Plackett-Burman experiments, and with reference to the steepest ascent experiment and response surface methodology optimization, the final composition of the DYH culture medium was determined to be: 48.2 g / L soy protein isolate (200 mesh), 10 g / L oat flour, 29 g / L maltose, 60 g / L carrot juice, 2 g / L anhydrous sodium acetate, 2.37 g / L K₂HPO₄, 4 g / L ammonium citrate, 0.56 g / L magnesium sulfate heptahydrate, 0.058 g / L manganese sulfate monohydrate, 0.03 g / L phenylalanine, and 0.01 g / L vitamin B5.

[0072] Example 4: Optimization of fermentation process conditions in DYH medium Based on the previous research, the DYH medium composition was used for the cultivation and fermentation of strain GL-2. After strain GL-2 was cultured to the end of the logarithmic growth phase, the OD of the bacterial culture was measured. 600 and diluted to OD 600 = 1.0, used as seed culture for inoculation, was inoculated into DYH medium, with a fermentation volume of 100 mL. Different fermentation temperatures, inoculation amounts, and fermentation times were set, with the viable cell count as a reference indicator to determine the optimal fermentation conditions. Furthermore, during fermentation, the pH of the system was adjusted to 6 every 3 hours using either Na2CO3 or NaHCO3. A control group was used with DYH medium undergoing normal fermentation (i.e., without artificial pH adjustment). Changes in pH, viable cell count, sugar content, and lactic acid content in the fermentation broth were measured at each sampling point. The results are as follows: Figure 7 As shown.

[0073] The effect of temperature on GL-2 growth, such as Figure 7 As shown in Figure A, the fermentation density initially increased and then decreased with increasing temperature, reaching a peak at 37℃, and showing a significant difference compared to other experimental groups (p<0.05); Figure 7As shown in Figure B, with the increase of inoculum size, the fermentation density of GL-2 generally showed a trend of first increasing and then stabilizing. When the inoculum size was 6% (v / v), the cell density reached its peak and was statistically significant (p<0.05). Furthermore, compared to MRS medium, DYH medium could prolong the cell growth time and significantly increase the fermentation density. The optimal fermentation time for strain GL-2 in DYH medium was 18 h (…). Figure 7 C).

[0074] After adjusting the pH of the experimental groups at intervals with different buffers, the measurement results are as follows: Figure 7 As shown in Figure D: In the original solution control group, the pH rapidly decreased from approximately 6.8 to 4.0 within 0-9 hours and continued to decline, indicating that the accumulation of large amounts of organic acids such as lactic acid led to acidification of the system. In the Na2CO3 and NaHCO3 groups, the pH was adjusted to approximately 6.0 every 3 hours to maintain the system within a suitable growth range, with the pH value exhibiting periodic fluctuations. Within 3-9 hours, the fermentation density of all three groups of strains increased rapidly, climbing from 26 log CFU / mL to over 30 log CFU / mL. The control group entered a plateau phase after 9 hours, reaching a final value of approximately 31.5 log CFU / mL, while the two experimental groups maintained a high growth rate, reaching a final value of 32.2-32.4 log CFU / mL, an increase of approximately 12% compared to the control group. The NaHCO3 group was slightly higher, indicating that a suitable pH is beneficial for prolonging the logarithmic growth phase and increasing cell density. Figure 7 E).

[0075] The residual sugar content in each group gradually decreased over time. At 18 h, the residual sugar content in the original solution control group was approximately 35 mg / mL, while the Na2CO3 and NaHCO3 groups decreased to approximately 12 mg / mL and 10 mg / mL, respectively. Figure 7 F); Lactic acid levels increased synchronously, with the highest level at the endpoint in the NaHCO3 group (approximately 80 g / L), followed by the Na2CO3 group (approximately 75 g / L), both higher than the control group (approximately 58 g / L). Figure 7 G).

[0076] In summary, acidification is a key factor limiting the growth and metabolism of lactic acid bacteria. Periodic pH adjustment can significantly improve cell density, substrate utilization, and lactic acid production, with NaHCO3 showing the best effect. Therefore, the optimal fermentation process for GL-2 cultured on DYH medium was determined to be as follows: inoculate the strain into DYH medium at an inoculum volume of 6% (v / v), and culture at 37°C for at least 18 hours. During this period, the pH of the system is adjusted to 6 using Na2CO3 or NaHCO3 (for example, every 3 hours). This prolongs the cell growth time, significantly increases the fermentation density, and ultimately significantly increases the lactic acid production (up to approximately 80 g / L).

[0077] Example 5: Effect of DYH culture medium on lactic acid bacteria The lactic acid bacteria used in this experiment include: *Lactaseibacillus paracasei* LP33, *Lactaseibacillus paracasei* LPCWU, *Lactaseibacillus casei* SHIROTA, *Lactaseibacillus casei* L. Zhang, *Lactobacillus pentosus* JS1, *Lactaseibacillus plantarum* LPHS, *Lactaseibacillus plantarum* ST-III, and *Lactaseibacillus plantarum* GL-2. *Lactaseibacillus plantarum* ST-III was sourced from Bright Dairy & Food Co., Ltd., and *Lactaseibacillus casei*... The strain L. Zhang was derived from Professor Zhang Heping's team at Inner Mongolia Agricultural University, while the other six strains were isolated and preserved in our laboratory.

[0078] 1. The effect of DYH medium on increasing the fermentation density of lactic acid bacteria Both the commonly used MRS medium and the DYH medium of this invention were fermented under the same conditions (OD). 600 =1.0, inoculum size 6% (v / v), cultured at 37℃ for 18 h, a variety of different lactic acid bacteria including: *Lactobacillus paracasei* LP33, LPCWU, *Lactobacillus casei* SHIROTA, L. Zhang, *Lactobacillus pentosaccharide* JS1, and *Lactobacillus plantarum* LPHS, ST-III, GL-2, totaling eight species. The viable cell count was used as a reference index, and the dilution plating method was employed to determine the viable cell count in each group, in order to analyze the effect of MRS and DYH media on the fermentation density of lactic acid bacteria. The results are as follows: Figure 8As shown, the viable cell counts at the fermentation endpoints of the eight lactic acid bacteria strains (ST-III, SHIROTA, GL-2, JS1, LPHS, LP33, LPCWU, and L. Zhang) differed significantly between MRS and DYH media (p<0.05), with all strains showing significantly higher counts in DYH medium compared to MRS. This indicates that the DYH medium of this invention, through optimized carbon-nitrogen ratios and growth factor supply, constructs a superior nutrient system, significantly increasing the fermentation density of multiple lactic acid bacteria strains. Experiments confirm that this formulation has good broad-spectrum adaptability and industrialization potential.

[0079] 2. Effect of DYH culture medium on the acid-producing capacity of lactic acid bacteria Following the acid-base indicator titration method in GB 12456-2021, the lactic acid content of the fermentation broths of eight lactic acid bacteria—LP33 and LPCWU, SHIROTA and L. Zhang, Lactobacillus pentosaccharide JS1, and LPHS, ST-III, and GL-2—cultured in MRS and DYH media was determined. Simultaneously, the contents of L-lactic acid and D-lactic acid in each experimental group were relatively quantified using an enzymatic method. The results are as follows: Figure 9 As shown.

[0080] The results showed that the lactic acid production of each strain in MRS medium ranged from 55 to 65 g / L, with *Lactobacillus pentosus* JS1 having the highest lactic acid content, reaching approximately 65 g / L, while strain *L. zhang* had the lowest content, only about 57 g / L. In contrast, the acid production capacity of each strain was significantly improved in the DYH medium of this invention (p<0.05), with an overall lactic acid content ranging from 65 to 80 g / L. The strain *SHIROTA* had the highest acid production, reaching 77 g / L. The acid production of each strain in DYH medium was significantly higher than that in MRS medium. Figure 9 A).

[0081] Humans lack efficient D-lactic acid-metabolizing enzymes, resulting in slow D-lactic acid metabolism. Excessive accumulation can lead to D-lactic acid poisoning. Therefore, the proportions of L-lactic acid and D-lactic acid in each experimental group were measured. The results showed that both L-lactic acid and D-lactic acid were detected in the fermentation broths of all strains on both MRS and DYH media, but their proportions varied significantly among strains and were influenced by the type of culture medium. Specifically, in MRS media, the proportions of L-lactic acid and D-lactic acid were relatively similar across strains. Except for *Lactobacillus pentosus* JS1, where the D-lactic acid proportion (51.14%) was slightly higher than the L-lactic acid proportion, the remaining strains were predominantly L-lactic acid, ranging from 51.32% to 64.79%, with GL-2 having the highest proportion (64.79%). In DYH medium, most strains showed a significant increase in L-lactic acid and a significant decrease in D-lactic acid. Specifically, ST-III and L. zhang showed L-lactic acid ratios of 96% and 90%, respectively; GL-2 and LPHS also had L-lactic acid ratios above 80%; LP33 and LPCWU had L-lactic acid ratios of approximately 70%; and strains SHIROTA and JS1, although showing relatively smaller changes, still exhibited an increase in L-lactic acid ratios. Figure 9 BC).

[0082] Comprehensive analysis shows that the composition of the culture medium has a significant impact on the conformation of lactic acid produced by the strain. The commonly used MRS medium in this field has a relatively balanced nutritional profile, resulting in a near-uniform ratio of the two isomers. In contrast, the DYH medium of this invention, by optimizing the carbon and nitrogen source structure and ratio, improves the carbon metabolic flux distribution of lactic acid bacteria, while simultaneously regulating the intracellular redox state and enhancing the metabolic flux of the L-lactic acid dehydrogenase (L-LDH) pathway. This significantly increases the yield of L-lactic acid in the fermentation broth and effectively reduces the accumulation of the undesirable product D-lactic acid, thereby obtaining a higher quality lactic acid fermentation product.

[0083] 3. Effects of DYH culture medium on the stress resistance of lactic acid bacteria Eight types of lactic acid bacteria, namely *Lactobacillus paracasei* LP33 and LPCWU, *Lactobacillus casei* SHIROTA and L. Zhang, *Lactobacillus pentosacchari* JS1, and *Lactobacillus plantarum* LPHS, ST-III, and GL-2, which were cultured in MRS and DYH media respectively, were adjusted to a bacterial concentration of 1×10⁻⁶ with physiological saline. 9 The bacterial suspension was inoculated at a concentration of CFU / mL at a volume fraction of 3% into MRS broth medium containing 3 g / L ox bile salts. After static incubation for a certain period, the viable bacterial count was determined using the plate count method, and the survival rate was calculated to evaluate the bile salt tolerance of the strain. Separately, 100 μL of the bacterial suspension cultured in both media was inoculated into 900 μL of artificial gastric fluid and cultured for 3 h. Then, 100 μL of the suspension was transferred to 900 μL of artificial intestinal fluid and cultured for 3 h. Afterward, the bacterial suspension was plate-swabbed and counted at the initial time and after incubation in artificial intestinal fluid, and the survival rate was calculated.

[0084] like Figure 10 As shown in Figure A, the bile salt tolerance of different strains varied significantly. After culture in MRS medium, only STIII showed strong bile salt tolerance (survival rate >90%), while the survival rates of most other strains were below 30%, with LPHS below 10%. However, after culture in DYH medium, the survival rates and bile salt tolerance of strains SHIROTA, GL-2, LP33, and LPCWU were significantly improved (p<0.05), with GL-2 showing the most significant improvement. These results indicate that there are inherent differences in the stress resistance among different lactic acid bacteria strains, and the composition of the culture medium can significantly regulate their bile salt tolerance characteristics. The DYH medium of this invention can effectively improve the bile salt tolerance of various lactic acid bacteria.

[0085] The tolerance of 8 strains of lactic acid bacteria in artificial intestinal fluid as follows Figure 10 As shown in Figure B, the survival rates of different strains varied significantly, and the type of culture medium had a significant impact on some strains. Under MRS culture conditions, the survival rate ranged from approximately 13% to 85%. LPHS and LPCWU had the highest survival rates (approximately 82% and 85%, respectively); ST-III, SHIROTA, and GL-2 had moderate survival rates (approximately 56%-65%); LP33 and JS1 had the lowest survival rates (approximately 13%-15%); and L. zhang was in the middle range. Compared with MRS medium, the tolerance of strains showed a differentiated regulatory trend after culture in DYH medium. The survival rates of ST-III, SHIROTA, GL-2, and LPHS were significantly improved (p<0.05), indicating that DYH medium can effectively enhance the survival ability of these strains in the intestinal environment. The above results indicate that the composition of the culture medium can regulate the survival ability of bacteria by affecting their physiological state. The DYH medium of this invention can significantly improve the artificial intestinal fluid tolerance of most strains. Overall, DYH medium is superior to traditional MRS medium in improving the adaptability of lactic acid bacteria to the intestinal environment, demonstrating its superiority as a functional culture system.

[0086] 4. The antioxidant effect of DYH culture medium on lactic acid bacteria fermentation products Fermentation broths of eight lactic acid bacteria (LP33, LPCWU, SHIROTA, L. Zhang, Lactobacillus pentosus JS1, LPHS, ST-III, and GL-2) were collected after being cultured in MRS and DYH media at 37 ℃ for 18 h. The reducing power of the fermentation broth was determined by the potassium ferricyanide (K3[Fe(CN)6]) method, and the superoxide anion (O3) content was determined by the pyrogallol method. 2- Scavenging ability and determination of OH in fermentation broth using salicylic acid method -The free radical scavenging capacity was assessed, with the culture medium without the added strain serving as a control (original sample), to comprehensively evaluate the antioxidant effect of DYH culture medium on lactic acid bacteria fermentation products. Results are as follows: Figure 11 As shown.

[0087] All strains exhibited some reducing ability in both MRS and DYH fermentation broths, showing significant strain-specific differences. Overall, the reducing ability of the DYH medium fermentation broth was significantly higher than that of the MRS medium fermentation broth (p<0.05). The reducing ability of the MRS fermentation broth was generally low, ranging from approximately 20% to 30%, while the reducing ability of the DYH fermentation broth was significantly improved. The differences between strains were small, indicating that the strain type had a relatively limited impact on the final reducing ability of the fermentation broth, while the medium type had a more significant impact. Figure 11 A).

[0088] MRS of different strains and OH of DYH fermentation broth - The free radical scavenging abilities showed significant differences, with strains ST-III, GL-2, JS-1, LPHS, and L. Zhang exhibiting the highest OH content in their DYH fermentation broths. - The free radical scavenging ability of DYH medium was significantly higher than that of MRS medium fermentation broth (p<0.05), while the differences among the other three strains were not significant. Overall, DYH medium is beneficial for enhancing the antioxidant capacity of the fermentation broth. Figure 11 B).

[0089] Superoxide ion scavenging capacity of different strains of MRS and DYH fermentation broth, such as Figure 11 As shown in C, the superoxide anion (O2) content in the DYH fermentation broth of most strains... 2- The superoxide ion scavenging ability of the DYH fermentation broth of strain JS1 was slightly higher than or close to that of the MRS medium fermentation broth. Specifically, the superoxide ion scavenging ability of the DYH fermentation broth of strain LPCWU was significantly higher than that of the MRS medium fermentation broth (p<0.05); while the superoxide ion scavenging ability of the DYH fermentation broth of strain LPCWU was significantly lower than that of the MRS fermentation broth (p<0.05). The scavenging abilities of the other strains in the two media fermentation broths did not differ significantly (p>0.05). These results indicate that the composition of the culture medium may affect the metabolic pathways of the strains, thereby altering the synthesis and accumulation patterns of antioxidant substances. Considering the above-mentioned reducing power and OH... - The results of free radical scavenging ability indicate that the composition of the culture medium is one of the key factors affecting the antioxidant properties of the fermentation broth. The DYH culture medium of the present invention can improve the antioxidant performance of fermentation broths of different strains to a certain extent.

[0090] In summary, this invention, employing single-factor experiments, Plackett-Burman design, and response surface methodology, systematically optimized the culture medium formulation and fermentation process parameters, successfully obtaining a novel fermentation medium widely applicable to lactic acid bacteria, which is named DYH medium. Further, using in vitro probiotic performance parameters such as viable cell count, stress resistance, and antioxidant capacity as evaluation indicators, a comprehensive comparison was conducted with traditional MRS medium. The results showed that DYH medium significantly increased the fermentation density of lactic acid bacteria, approximately 2.5 to 4 times higher than traditional MRS medium, and significantly increased the total lactic acid content and the proportion of L-lactic acid in the fermentation broth, achieving high-density culture of the strains, which is beneficial for reducing production costs and improving production efficiency. Furthermore, strains cultured in DYH medium exhibited significantly enhanced stress resistance, including tolerance to bile salts and artificial intestinal fluid, which is beneficial for the survival and colonization of probiotics in the gastrointestinal environment. It also improved the antioxidant properties of the lactic acid bacteria fermentation broth, particularly in reducing power, superoxide anion scavenging capacity, and hydroxyl radical scavenging capacity, endowing the fermentation products with stronger potential for free radical scavenging and oxidative damage protection. The DYH medium provided by this invention is a novel lactic acid bacteria culture medium that integrates high-density fermentation, metabolic regulation, stress resistance enhancement and antioxidant functions. It significantly improves the overall performance of lactic acid bacteria and can be widely used in various lactic acid bacteria strains, including GL-2, LP33 and ST-III. It has good universality and industrialization prospects, is suitable for large-scale promotion and use, and has broad application prospects.

[0091] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A novel lactic acid bacteria culture medium, characterized in that, The culture medium comprises: 5-50 g / L soy protein isolate, 2-30 g / L oat flour, 2-40 g / L maltose, 5-100 g / L carrot, 2-4 g / L anhydrous sodium acetate, 2-4 g / L K₂HPO₄, 4-5 g / L ammonium citrate, 0.05-1.0 g / L magnesium sulfate heptahydrate, 0.01-0.2 g / L manganese sulfate monohydrate, 0.01-0.1 g / L phenylalanine, and 0.01-0.05 g / L vitamin B5.

2. The novel lactic acid bacteria culture medium according to claim 1, characterized in that, The culture medium comprises: 40-50 g / L soy protein isolate, 10-30 g / L oat flour, 20-40 g / L maltose, 60-100 g / L carrot, 2-4 g / L anhydrous sodium acetate, 2-4 g / L K₂HPO₄, 4-5 g / L ammonium citrate, 0.56-0.79 g / L magnesium sulfate heptahydrate, 0.058-0.082 g / L manganese sulfate monohydrate, 0.03-0.05 g / L phenylalanine, and 0.01-0.03 g / L vitamin B5.

3. The novel lactic acid bacteria culture medium according to claim 1, characterized in that, The culture medium consists of: 48.2 g / L soy protein isolate, 10 g / L oat flour, 29 g / L maltose, 60 g / L carrot, 2 g / L anhydrous sodium acetate, 2.37 g / L K2HPO4, 4 g / L ammonium citrate, 0.56 g / L magnesium sulfate heptahydrate, 0.058 g / L manganese sulfate monohydrate, 0.03 g / L phenylalanine, and 0.01 g / L vitamin B5.

4. The use of the novel lactic acid bacteria culture medium according to any one of claims 1-3 in any of the following A1)-A8): A1) Application in promoting the growth of lactic acid bacteria; A2) Application in the preparation of products that promote the growth of lactic acid bacteria; A3) Application in increasing the lactic acid content and / or L-lactic acid ratio in lactic acid bacteria fermentation broth; A4) Application in the preparation of products with high lactic acid bacteria fermentation broth and / or high lactic acid content and / or L-lactic acid ratio; A5) Application in improving the bile salt and / or artificial intestinal fluid tolerance of lactic acid bacteria; A6) Application in the preparation of products that enhance the bile salt and / or artificial intestinal fluid tolerance of lactic acid bacteria; A7) Application in improving the antioxidant capacity of lactic acid bacteria fermentation broth; A8) Application in the preparation of products that enhance the antioxidant capacity of lactic acid bacteria fermentation broth.

5. The application according to claim 4, characterized in that, The antioxidant capacity includes one or more of the following: reducing capacity, superoxide anion scavenging capacity, and hydroxyl radical scavenging capacity.

6. A method for fermenting and culturing lactic acid bacteria, characterized in that, include: The lactic acid bacteria are inoculated into the novel lactic acid bacteria culture medium of any one of claims 1-3 for fermentation culture.

7. The method according to claim 6, characterized in that, The fermentation conditions include: an inoculum size of 4% to 10%, a temperature of 35 to 39°C, a fermentation time of more than 18 hours, and the addition of a pH adjuster during the fermentation process to control the pH of the fermentation broth to be 5.5 to 6.

5.

8. The method according to claim 7, characterized in that, The fermentation conditions include: an inoculum size of 6%, a temperature of 37°C, a fermentation time of more than 18 hours, and the addition of a pH adjuster during fermentation to control the pH of the fermentation broth to 6.

9. The method according to claim 6, characterized in that, The lactic acid bacteria are selected from one or more of the following: Lactobacillus paracasei, Lactobacillus casei, Lactobacillus pentosus, and Lactobacillus plantarum.

10. The method according to claim 9, characterized in that, The lactic acid bacteria are selected from one or more of the following: Lacticaseibacillus paracasei LP33, Lacticaseibacillus paracasei LPCWU, Lacticaseibacillus casei SHIROTA, Lacticaseibacillus casei L. Zhang, Lactiplantibacillus pentosus JS1, Lactiplantibacillus plantarum LPHS, Lactiplantibacillus plantarum ST-III, and Lactiplantibacillus plantarum GL-2.

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