A method for rejuvenation and high-density culture of paracasei

Abstract

The application discloses a rejuvenation and high-density culture method of paracasei. The application optimizes through single-factor test and response surface test, explores the influence of medium components such as carbon source, nitrogen source and inorganic salt, inoculation amount, initial pH and culture temperature on the cell density of paracasei, takes the absorbance of fermentation liquor as a main reference index, and determines the optimal medium process conditions which promote the growth of paracasei. The optimal medium formula is as follows: lactose 40 g / L, yeast extract powder 60 g / L, manganese sulfate monohydrate 0.15 g / L, anhydrous sodium acetate: citric acid hydrogen diammonium: potassium hydrogen phosphate = 1:1:1 (each 3 g / L), and Tween 80: 1 g / L. The optimal culture conditions are as follows: temperature 38.5 DEG C, initial pH 7, and inoculation amount 5%, and at this time, the viable cell count is 4.6 x 10 9 CFU / mL, which lays a solid foundation for the industrialized fermentation production of paracasei.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, specifically relating to a method for the rejuvenation and high-density culture of Lactobacillus paracasei. Background Technology

[0002] Lactobacillus paracasei is a lactic acid bacterium with various probiotic properties, often used in the production of probiotic preparations and in the field of intestinal microecological regulation. Studies have shown that Lactobacillus paracasei can regulate the immune system, improve lipid metabolism, and has antioxidant and antibacterial effects. It can also regulate intestinal flora function, enhance immunity, and promote intestinal motility.

[0003] Strain degeneration refers to the phenomenon where, during subculturing, a strain loses some or all of its desirable or typical traits due to genetic variation. Inadequate nutrient control during cultivation, such as insufficient intake of carbohydrates, proteins, and growth factors, can lead to slow growth and weakened metabolism. Similarly, improper environmental control, such as temperature and humidity, can also affect growth and metabolism, as well as the quantity and quality of secondary metabolites, causing strain degeneration. This can result in significant economic losses for strain cultivation companies. Causes of strain degeneration include excessive subculturing, gene mutations, improper culture medium composition and environment, and unsuitable preservation methods. Rejuvenation measures for degenerated strains include: timely removal of degenerated cells, regular purification and isolation of the strain, creating favorable environmental conditions for strain cultivation, controlling the number of subculturing cycles, and using appropriate preservation methods. For example, after the degeneration of *Volvariella volvacea* strains such as those by Kong Zixuan, the content of various amino acids in their fruiting bodies showed varying degrees of decline. The strains were rejuvenated by altering the carbon source in the culture medium and adding exogenous amino acids.

[0004] This invention addresses the problem of Lactobacillus paracasei degeneration. Summary of the Invention

[0005] In order to overcome the shortcomings and deficiencies of the prior art, the purpose of this invention is to provide a method for the rejuvenation and high-density culture of Lactobacillus paracasei, so as to improve the bacterial density of Lactobacillus paracasei.

[0006] This invention determines the optimal culture medium formula and optimal culture conditions for *Lactobacillus paracasei* through single-factor experiments and response surface methodology to improve the bacterial density of *Lactobacillus paracasei* during production. The rejuvenated strain shows a significantly higher bacterial density during room temperature storage compared to the unrejuvenated strain, meeting the requirements for stable production, reducing production costs, and satisfying market demand.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A method for revitalizing and high-density culturing Lactobacillus paracasei includes the following steps:

[0009] S1. Strain activation: Select a single colony of Lactobacillus paracasei and place it in MRS liquid medium for activation to obtain fermentation seed liquid;

[0010] S2. Rejuvenation and High-Density Culture of Strains: The fermentation seed liquid was inoculated into MRS modified liquid medium at an inoculation rate of 1-7% (v / v) and fermented to obtain rejuvenated and high-density Lactobacillus paracasei culture.

[0011] Preferably, in step S1, the activation is performed twice;

[0012] Furthermore, the conditions for the two activations are: 34–39°C, 180–220 rpm shaking culture for 10–12 h (preferably 37±1°C, 200 rpm shaking culture for 11±1 h), and activation is performed twice.

[0013] Preferably, in step S2, the inoculation amount is 2-7% (v / v); further, it is 3-7% (v / v); and even further, it is 5% (v / v).

[0014] Preferably, in step S2, the formula of the MRS modified liquid culture medium is: lactose 30-50 g / L, yeast extract 50-70 g / L, manganese sulfate monohydrate 0.05-0.3 g / L, Tween-80 1 g / L, anhydrous sodium acetate 2-6 g / L, diammonium hydrogen citrate 2-6 g / L, dipotassium hydrogen phosphate 2-6 g / L, pH 6.5-7.5.

[0015] Further preferred formulation of MRS modified liquid culture medium is: lactose 40 g / L, yeast extract 60 g / L, manganese sulfate monohydrate 0.15 g / L, Tween-80 1 g / L, anhydrous sodium acetate 2–6 g / L, diammonium hydrogen citrate 2–6 g / L, dipotassium hydrogen phosphate 2–6 g / L, pH 7.0.

[0016] The mass ratio of anhydrous sodium acetate: diammonium hydrogen citrate: dipotassium hydrogen phosphate is 1:1:1.

[0017] Preferably, in step S2, the fermentation temperature is 34–38.5°C; more preferably, it is 38.5°C.

[0018] Preferably, in step S2, the fermentation culture time is 20-48 hours; more preferably, it is 20 hours.

[0019] This invention uses *Lactobacillus paracasei* as the strain, which is activated and then inoculated into MRS liquid medium for cultivation. The growth curve was measured, and the effects of different types and levels of carbon sources, nitrogen sources, inorganic salts, buffer salts, and other medium components, as well as inoculation amount, initial pH, and culture temperature, on the bacterial density of *Lactobacillus paracasei* were investigated through single-factor experiments and response surface methodology. The optimal medium conditions that promote the growth of *Lactobacillus paracasei* were determined using the absorbance of the fermentation broth as the main reference indicator.

[0020] After optimization through the above experiments, the optimal culture medium formula was determined to be: lactose 40 g / L, yeast extract 60 g / L, manganese sulfate monohydrate 0.15 g / L, anhydrous sodium acetate: diammonium hydrogen citrate: dipotassium hydrogen phosphate = 1:1:1 (3 g / L each), and Tween 80: 1 g / L. The optimal culture conditions were: temperature 38.5℃, initial pH 7, inoculum size 5%, and culture for 20 h, at which point the viable cell count was 4.6 × 10⁻⁶. 9 The CFU / mL is 2.88 times that of the original (MRS liquid medium).

[0021] The present invention has the following advantages and effects compared with the prior art:

[0022] *Lactobacillus paracasei* is a lactic acid bacterium with various probiotic properties, commonly used in the production of probiotic preparations and in the field of intestinal microecological regulation. However, after multiple passages, the strain is prone to degeneration. Therefore, it is necessary to optimize its culture medium formulation and culture conditions to enhance the physiological activity of the rejuvenated *Lactobacillus paracasei* and make the strain more stable, in order to meet the requirements of stable production and lay a solid foundation for the industrial fermentation production of *Lactobacillus paracasei*. Attached Figure Description

[0023] Figure 1 This is the growth curve of Lactobacillus paracasei.

[0024] Figure 2 It is a single-factor optimization of culture medium: screening for the optimal carbon source.

[0025] Figure 3 It is a single-factor optimization of the culture medium: screening for the optimal nitrogen source.

[0026] Figure 4 It is a single-factor optimization of culture medium: screening for optimal trace elements.

[0027] Figure 5 It is a single-factor optimization of the culture medium: screening for the optimal buffer salt.

[0028] Figure 6 The optimal carbon source: single-factor optimization of lactose addition amount.

[0029] Figure 7The optimal nitrogen source: single-factor optimization of yeast extract addition amount.

[0030] Figure 8 The optimal amount of manganese sulfate monohydrate as a trace element was determined by single-factor optimization.

[0031] Figure 9 The optimal buffer salt ratio is: anhydrous sodium acetate: diammonium hydrogen citrate: dipotassium hydrogen phosphate = 1:1:1. The optimal addition amount is determined by a single factor.

[0032] Figure 10 It is a 3D surface plot and contour plot showing the relationship between lactose and yeast extract.

[0033] Figure 11 These are 3D surface plots and contour plots showing the relationship between manganese sulfate and lactose.

[0034] Figure 12 These are 3D surface plots and contour plots showing the relationship between manganese sulfate and yeast extract.

[0035] Figure 13 It involves optimizing the concentration of seed liquid addition.

[0036] Figure 14 It's pH optimization.

[0037] Figure 15 It involves optimizing the culture temperature. Detailed Implementation

[0038] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0039] The following are some of the culture media involved in this invention and its embodiments:

[0040] MRS medium: 1% peptone (m / v), 1% beef extract (m / v), 0.5% yeast extract (m / v), 2% glucose (m / v), 0.1% Tween-80 (m / v), 0.2% K2HPO4 (m / v), 0.5% sodium acetate (m / v), 0.2% diammonium citrate (m / v), 0.02% magnesium sulfate heptahydrate (m / v), and 0.0054% manganese sulfate monohydrate (m / v). If using solid medium, add 2% agar (m / v), adjust the pH to 6.5, and sterilize at 121°C for 15 min.

[0041] The *Lactobacillus paracasei* mentioned in the examples is *Lactobacillus paracasei* 22-HM, purchased from Guangzhou Zhujiang Meileduo Beverages (Hong Kong) Co., Ltd. For experiments not specifically described in the examples, the procedures or conditions described in the literature in this field can be followed. All raw materials and instruments used are commercially available conventional products, including but not limited to those used in the examples of this application.

[0042] The *Lactobacillus paracasei* in this invention was obtained by streaking activation of freeze-dried bacterial powder provided by Guangzhou Pearl River Meileduo Beverages (Hong Kong) Co., Ltd., and this strain is a strain that has undergone a certain degree of degeneration.

[0043] Example 1

[0044] 1. Determination of growth curve of Lactobacillus paracasei

[0045] The bacterial culture was stored in glycerol at -80℃ and directly streaked onto MRS solid medium. After incubation at 37℃ inverted for 48 h, single colonies were picked and transferred to MRS liquid medium at 37℃ and 200 rpm. -1 After shaking culture for 11 hours and activation twice, the fermentation seed liquid is obtained.

[0046] Activated second-generation *Lactobacillus paracasei* was inoculated into 100-well plates containing MRS liquid medium at an inoculum of 2% (v / v). The absorbance of the bacterial culture was measured every 1 hour using an automated growth curve analyzer. A growth curve of *Lactobacillus paracasei* was plotted with time on the x-axis and absorbance (OD600nm) on the y-axis. Figure 1 The growth characteristics and optimal culture time of the strain were determined by analyzing the growth curve. The results showed that *Lactobacillus paracasei* was in the lag phase from 0 to 4 h, in the logarithmic growth phase from 4 to 20 h, and entered the stationary phase after 20 h. No decline phase was observed within 48 h, indicating a relatively long stationary phase, which is suitable for industrial production. The 20th hour was also determined to be the optimal measurement time for subsequent culture medium optimization.

[0047] 2. Optimization of culture medium formulation for Lactobacillus paracasei

[0048] 2.1 Single-factor optimization of culture medium: Screening for optimal carbon source

[0049] The carbon source (glucose) in MRS medium was replaced by different carbon sources, namely 20 g / L glucose, sucrose, maltose, fructose, lactose, galactose, and soluble starch, while keeping other components unchanged. The inoculum size was 2% (v / v), the initial pH was 6.5, and the culture was carried out at 37°C for 20 h. The bacterial density was calculated after incubation. The optimal carbon source culture component was screened using the OD600nm value as the evaluation index. Figure 2 The results showed that glucose, lactose, and galactose significantly increased the absorbance of *Lactobacillus paracasei*, with lactose showing the highest absorbance. Sucrose, soluble starch, and other polysaccharides had no significant promoting effect on *Lactobacillus paracasei*, indicating that this bacterium utilizes lactose most efficiently, making lactose the optimal carbon source.

[0050] 2.2 Single-factor optimization of culture medium: Screening for the optimal nitrogen source

[0051] The nitrogen source in MRS medium (peptone + beef extract + yeast extract) was replaced by different nitrogen sources, including 20 g / L yeast extract, peptone, beef extract, soybean peptone, and trypsin, with a peptone:yeast extract ratio of 2:1. Other components remained constant. The inoculum size was 2% (v / v), the initial pH was 6.5, and the culture was carried out at 37℃ for 20 h. The bacterial density was calculated after incubation. The OD600nm value was used as the evaluation index to investigate the effect of different nitrogen sources on the growth of *Lactobacillus paracasei*, and to screen the optimal nitrogen source culture components. The results are as follows: Figure 3 As shown, yeast extract of the same concentration has the best promoting effect on the growth of Lactobacillus paracasei. Considering the simplification of production formula and cost, yeast extract is selected as the best nitrogen source.

[0052] 2.3 Single-factor optimization of culture medium: screening for optimal trace elements

[0053] The trace elements (manganese sulfate monohydrate and magnesium sulfate heptahydrate) in the MRS were replaced with 0.2 g / L of (manganese sulfate monohydrate, magnesium sulfate heptahydrate, zinc sulfate heptahydrate, and ferric sulfate, manganese sulfate monohydrate: magnesium sulfate heptahydrate = 1:1), keeping other components unchanged. The inoculum size was 2% (v / v), the initial pH was 6.5, and the bacterial density was calculated after culturing at 37℃ for 20 h. Figure 4 Using OD600nm as the evaluation index, the effects of different trace elements on the growth of *Lactobacillus paracasei* were investigated, and the optimal trace element culture composition was screened. The experimental results showed that, compared with MRS basal medium, manganese sulfate monohydrate and manganese sulfate monohydrate: magnesium sulfate heptahydrate = 1:1 had a better promoting effect on the bacterial density of *Lactobacillus paracasei*, while magnesium sulfate heptahydrate, zinc sulfate heptahydrate, and ferric sulfate had a poor promoting effect on *Lactobacillus paracasei*. Furthermore, based on research showing that manganese ions have a significant promoting effect on the growth of *Lactobacillus paracasei*, manganese sulfate monohydrate was selected as the sole trace element.

[0054] 2.4 Single-factor optimization of culture medium: screening for optimal buffer salt

[0055] The buffer salts in the MRS medium were replaced with different buffer salts at a ratio of 9 g / L anhydrous sodium acetate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, diammonium citrate, potassium dihydrogen phosphate, and anhydrous sodium acetate: diammonium hydrogen phosphate: dipotassium hydrogen phosphate = 1:1:1 (3 g / L each), while keeping other components unchanged. The inoculum size was 2% (v / v), the initial pH was 6.5, and the bacterial density was calculated after culturing at 37℃ for 20 h. Figure 5 The optimal buffer salt culture component was selected using the OD600nm value as the evaluation index. Figure 5 It was found that 9 g / L anhydrous sodium acetate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, and potassium dihydrogen phosphate all promoted the OD value of *Lactobacillus paracasei*, and showed significant differences compared to the basal MRS. However, diammonium citrate did not significantly promote the bacterial OD value. Furthermore, the combined buffer salt ratio (anhydrous sodium acetate: diammonium citrate: dipotassium hydrogen phosphate = 1:1:1) showed the best promoting effect, and showed no significant difference compared to the basal MRS. Therefore, the 9 g / L combined buffer salt ratio (anhydrous sodium acetate: diammonium citrate: dipotassium hydrogen phosphate = 1:1:1) can be selected as the optimal buffer salt.

[0056] 2.5 Optimal Carbon Source: Single-Factor Optimization of Lactose Addition Amount

[0057] Based on the previous experiment that lactose was the optimal carbon source, the effect of different levels of lactose addition on the growth of the bacterial strain was investigated. Lactose concentration was optimized according to a gradient of 10, 20, 30, 40, and 50 g / L. Keeping other components constant, the inoculum size was 2% (v / v), the initial pH was 6.5, and the bacterial density was calculated after culturing at 37℃ for 20 h. Figure 6 Using OD600nm as the evaluation index, the effect of different lactose concentrations on the growth of *Lactobacillus paracasei* was investigated to screen for the optimal lactose concentration range. Figure 6 As shown, the density of *Lactobacillus paracasei* first increases and then decreases with increasing lactose concentration, reaching its highest level at a lactose concentration of 40 g / L. Therefore, the optimal range for lactose addition is considered to be 30-50 g / L.

[0058] 2.6 Optimal Nitrogen Source: Single-Factor Optimization of Yeast Extract Addition Amount

[0059] Previous experiments have shown that yeast extract is the optimal nitrogen source. Based on this, the effect of different levels of yeast extract addition on strain growth was investigated. Yeast extract concentrations of 10, 20, 30, 40, 50, 60, and 70 g / L were optimized, while other components remained constant. The inoculum size was 2% (v / v), the initial pH was 6.5, and the bacterial density was calculated after culturing at 37℃ for 20 h. Figure 7Using OD600nm as the evaluation index, the optimal yeast extract concentration range was screened. The optimal addition range was determined for subsequent response surface methodology analysis. Figure 7 It can be seen that as the concentration of yeast extract increases, the bacterial density of *Lactobacillus paracasei* first increases and then decreases, reaching its highest concentration at 60 g / L. Therefore, the optimal addition range for yeast extract is considered to be 50-70 g / L.

[0060] 2.7 Optimal Micronutrient: Single-Factor Optimization of Manganese Sulfate Addition Amount

[0061] Previous experiments have shown that the optimal trace element is manganese sulfate monohydrate. Based on this, the effect of different levels of manganese sulfate monohydrate addition on bacterial growth was investigated. Manganese sulfate monohydrate concentrations of 0.05, 0.1, 0.2, 0.3, and 0.4 g / L were optimized, while other components remained constant. The inoculum size was 2% (v / v), the initial pH was 6.5, and the bacterial density was calculated after culturing at 37℃ for 20 h. Figure 8 Using OD600nm as the evaluation index, the effect of different concentrations of manganese sulfate monohydrate on the growth of *Lactobacillus paracasei* was investigated, and the optimal concentration range of manganese sulfate monohydrate was screened. The results showed that the OD value of *Lactobacillus paracasei* first increased and then decreased with increasing manganese sulfate monohydrate concentration. The bacterial density was highest at a manganese sulfate monohydrate concentration of 0.1 g / L. Therefore, the optimal addition range of manganese sulfate monohydrate is considered to be 0.05-0.3 g / L.

[0062] 2.8 Optimal buffer salt: anhydrous sodium acetate: diammonium citrate: dipotassium hydrogen phosphate = 1:1:1 addition amount optimized by single factor

[0063] Previous experiments showed that the optimal buffer salt ratio was anhydrous sodium acetate: diammonium citrate: dipotassium hydrogen phosphate = 1:1:1. Based on this, the effect of different buffer salt concentrations on bacterial growth was investigated. The buffer salt concentrations were optimized according to anhydrous sodium acetate: diammonium citrate: dipotassium hydrogen phosphate = 1:1:1 (2, 3, 4, 5, and 6 g / L of each buffer salt), keeping other components constant. The inoculum size was 2% (v / v), the initial pH was 6.5, and the bacterial density was calculated after culturing at 37℃ for 20 h. Figure 9 Using OD600nm as the evaluation index, the optimal buffer salt concentration range was screened. The results showed that as the buffer salt concentration increased, the bacterial density of *Lactobacillus paracasei* fluctuated. Although the concentration was highest at 12 g / L, there was no significant difference between the different data sets, and it was not considered as a factor for subsequent response surface methodology.

[0064] 3. Optimizing culture medium formulation using response surface methodology

[0065] Based on the Box-Benhnken central composite design principle and the previous single-factor experimental results, the culture temperature was fixed at 37℃, the inoculum size was 2% (v / v), and the initial pH of the culture medium was 6.5. Lactose, yeast extract, and manganese sulfate (i.e., manganese sulfate monohydrate) were selected as the three independent variables (Table 1). The culture medium of Lactobacillus paracasei was optimized using response surface methodology, resulting in 17 experimental schemes. The experimental design is shown in Table 2 below, and the regression model variance analysis is shown in Table 3.

[0066] Table 1. Factors and levels for optimizing the response surface methodology of Lactobacillus paracasei culture medium.

[0067]

[0068] Table 2. Optimized response surface methodology and results for Lactobacillus paracasei culture medium

[0069]

[0070] Table 3. Analysis of Variance for Regression Models

[0071]

[0072] The experimental data were fitted using Design Expert 13, and the resulting quadratic multinomial regression equation is: OD600 = 1.5588 + -0.010625 * A + 0.002125 * B + -0.00025 * C + -0.00025 * AB + 0.003 * AC + 0.0005 * BC + -0.035775 * A^2 + -0.025775 * B^2 + -0.014025 *C^2. In the model, the F-value is an important indicator of the degree of influence of each variable on the response variable. When the significance test probability P < 0.05, it indicates that the variable has a significant impact on the response value and has mathematical statistical significance.

[0073] As shown in Tables 2 and 3, the regression model is highly significant (P < 0.01), and the lack-of-fit term is not significant (P > 0.05). The regression coefficients R0 of the model are... 2 The R² value is 0.9914, and the R²adj value is 0.9803, indicating that the model can explain 98.03% of the data, demonstrating a high model fit and a small difference between the experimental data and the predicted values. Therefore, this model is suitable for the analysis and prediction of the optimization of Lactobacillus paracasei culture medium. A, A 2 B 2 and C 2 The effect on OD600 was extremely significant (P<0.01). The order of influence was: lactose > yeast extract > manganese sulfate.

[0074] Analysis of the response surface methodology results yielded response surface plots and contour plots illustrating the effects of interactions between various factors on the OD value of *Lactobacillus paracasei*, as shown below. Figures 10-12 The influence of various factors on the OD value of *Lactobacillus paracasei* and the strength of interactions among these factors can be visually analyzed graphically. The steepness of the response surface is positively correlated with the significance of each factor's influence on the OD value of *Lactobacillus paracasei*, while the shape of the contour lines and the density of axial changes are related to the strength of interactions among the factors. The response surface results are parabolic, indicating the existence of a maximum point in the regression model. The contour lines for manganese sulfate and lactose, and manganese sulfate and yeast extract are elliptical, indicating significant interactions, while the contour lines for lactose and yeast extract are more rounded, indicating weaker interactions. These results correspond to the analysis of variance results.

[0075] Based on the regression model analysis, the optimized culture medium parameters for *Lactobacillus paracasei* were determined to be lactose 38.50 g / L, yeast extract 60.42 g / L, and manganese sulfate 0.147 g / L. Under these conditions, the predicted OD600 value was 1.560. Based on operability, the optimal process was adjusted to lactose 40 g / L, yeast extract 60 g / L, and manganese sulfate 0.15 g / L. Actual experiments were conducted under these conditions, and the measured OD value for *Lactobacillus paracasei* was 1.565, which is largely consistent with the model prediction.

[0076] In summary, the optimal modified MRS liquid culture medium is: lactose 40 g / L, yeast extract 60 g / L, manganese sulfate monohydrate 0.15 g / L, Tween-80 1 g / L, anhydrous sodium acetate 3 g / L, diammonium hydrogen citrate 3 g / L, and dipotassium hydrogen phosphate 3 g / L. If using solid culture medium, 2% agar (m / v) should be added, the pH adjusted to 6.5, and sterilized at 121℃ for 15 min.

[0077] 4. Optimization of cultivation conditions

[0078] 4.1 Optimal Dosage and Concentration Optimization

[0079] During cultivation, the initial inoculum size, pH, and temperature all significantly influence the growth and physiological activity of the microbial strain. Using the optimal culture medium formulation, activated fermentation seed culture was added to the optimal MRS modified liquid medium at concentrations of 1%, 2%, 3%, 4%, 5%, 6%, and 7% (v / v), keeping other components constant. The initial pH was 6.5. After culturing at 37°C for 20 h, the bacterial density was calculated. Figure 13 Using OD600nm as the evaluation index, the optimal concentration of seed culture was screened. The results showed that as the amount of seed culture added increased, the bacterial density of *Lactobacillus paracasei* fluctuated, with the highest bacterial density at 5% (v / v).

[0080] 4.2 Optimal pH Optimization of Culture Medium

[0081] Based on the optimal modified MRS liquid medium, under different pH conditions (5.5, 6, 6.5, 7, 7.5), while keeping other culture conditions constant, the seed culture addition was 2% (v / v), and the bacterial density was calculated after culturing at 37℃ for 20 h. Figure 14 Using OD600nm as the evaluation index, the effect of different pH values ​​on the growth of *Lactobacillus paracasei* was investigated to screen for the optimal pH. The results showed that as the pH of the culture medium increased, the OD value of *Lactobacillus paracasei* exhibited a trend of first decreasing, then increasing, and then decreasing again. The highest OD value was observed at pH=7, thus determining the optimal pH.

[0082] 4.3 Optimal Temperature Optimization

[0083] Based on the optimal modified MRS liquid medium, under different temperatures (34, 35.5, 37, 38.5, 40℃), while keeping other culture conditions constant, the seed culture addition was 2% (v / v), and the bacterial density was calculated after 20 h of culture. Figure 15 Using OD600nm as the evaluation index, the effect of different temperatures on the growth of *Lactobacillus paracasei* was investigated to screen for the optimal culture temperature. The results showed that the bacterial density of *Lactobacillus paracasei* fluctuated with increasing culture temperature, reaching its highest value at 38.5℃. Therefore, the optimal culture temperature was determined to be 38.5℃.

[0084] In summary, the optimal culture conditions are: seed culture inoculum volume of 5% (v / v), pH=7, and temperature of 38.5℃.

[0085] 5. The bacterial density before and after optimization was measured and compared.

[0086] The bacterial culture, preserved in glycerol at -80℃, was streaked onto MRS solid medium and incubated upside down in a 37℃ incubator for 48 hours. Single colonies were then picked and transferred to MRS liquid medium at 37℃ and 200 rpm. -1 The fermentation seed culture was obtained by shaking and culturing for 11 hours and activating twice. The seed culture was inoculated into MRS liquid medium at an inoculum of 2% (v / v) and cultured at 37℃ for 20 hours. Simultaneously, fermentation was carried out under the experimentally determined optimal MRS modified liquid medium formula and optimal culture conditions. The optical density of the bacteria at 600 nm was measured using a spectrophotometer under both the MRS liquid medium and the optimal MRS modified liquid medium, and the viable cell count was determined using the dilution plating method. The experimental results showed that *Lactobacillus paracasei* cultured in MRS liquid medium for 20 hours had an OD600 of 1.396, and the viable cell count obtained by dilution plating was 1.6 × 10⁻⁶. 9CFU / mL; OD600 was 1.603 in the optimal modified MRS liquid medium, and the viable count after dilution and plating was 4.6 × 10⁻⁶. 9 The CFU / mL concentration is 2.88 times that of MRS liquid culture medium.

[0087] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for the rejuvenation and high-density culture of *Lactobacillus paracasei*, characterized in that, Includes the following steps: S1. Strain activation: Select a single colony of Lactobacillus paracasei and place it in MRS liquid medium for activation to obtain fermentation seed liquid; S2. Rejuvenation and High-Density Culture of Strains: The fermentation seed liquid was inoculated into MRS modified liquid medium at an inoculation rate of 1-7% v / v and fermented to obtain rejuvenated and high-density Lactobacillus paracasei culture.

2. The method for revitalizing and high-density culturing *Lactobacillus paracasei* according to claim 1, characterized in that: In step S1, the activation is performed twice.

3. The method for revitalizing and high-density culturing *Lactobacillus paracasei* according to claim 2, characterized in that: The conditions for the two activations are: 34–39°C, shaking culture at 180–220 rpm for 10–12 h, and activation twice.

4. The method for revitalizing and high-density culturing *Lactobacillus paracasei* according to claim 3, characterized in that: The conditions for the two activations were: 37±1℃, 200 rpm shaking culture for 11±1h, and activation was performed twice.

5. The method for revitalizing and high-density culturing *Lactobacillus paracasei* according to any one of claims 1 to 4, characterized in that: In step S2, the inoculation amount is 2-7% v / v; further, it is 3-7% v / v; and even further, it is 5% v / v.

6. The method for revitalizing and high-density culturing *Lactobacillus paracasei* according to any one of claims 1 to 4, characterized in that: In step S2, the formula of the MRS modified liquid culture medium is as follows: lactose 30-50 g / L, yeast extract 50-70 g / L, manganese sulfate monohydrate 0.05-0.3 g / L, Tween-80 1 g / L, anhydrous sodium acetate 2-6 g / L, diammonium hydrogen citrate 2-6 g / L, dipotassium hydrogen phosphate 2-6 g / L, pH 6.5-7.

5.

7. The method for revitalizing and high-density culturing *Lactobacillus paracasei* according to claim 6, characterized in that: The formula for the modified MRS liquid culture medium is as follows: lactose 40 g / L, yeast extract 60 g / L, manganese sulfate monohydrate 0.15 g / L, Tween-80 1 g / L, anhydrous sodium acetate 2–6 g / L, diammonium hydrogen citrate 2–6 g / L, dipotassium hydrogen phosphate 2–6 g / L, pH 7.

0.

8. The method for revitalizing and high-density culturing *Lactobacillus paracasei* according to claim 6 or 7, characterized in that: Anhydrous sodium acetate: diammonium hydrogen citrate: dipotassium hydrogen phosphate = 1:1:1, mass ratio.

9. The method for revitalizing and high-density culturing *Lactobacillus paracasei* according to any one of claims 1 to 4, characterized in that: In step S2, the fermentation culture temperature is 34–38.5°C; more specifically, it is 38.5°C.

10. The method for revitalizing and high-density culturing *Lactobacillus paracasei* according to any one of claims 1 to 4, characterized in that: In step S2, the fermentation culture time is 20-48 hours; more specifically, it is 20 hours.

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