High-density culture method of lactobacillus plantarum

Abstract

The invention discloses a high-density culture method of lactobacillus plantarum, and belongs to the technical field of microbial fermentation. The high-density culture method comprises the following steps: performing seed culture on lactobacillus plantarum, and inoculating the lactobacillus plantarum into a fermentation tank filled with a fermentation culture medium for fermentation culture; the fermentation culture medium comprises a carbon source, a nitrogen source and manganese sulfate; the mass concentration ratio of the carbon source to the nitrogen source to the manganese sulfate is (15-30): (75-90): (0.25-0.4). By optimizing the fermentation culture medium and culture conditions, the growth amount of the strain and the number of living cells are increased, and high-density culture of the lactobacillus plantarum is realized.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial fermentation, and particularly relates to a method for high-density cultivation of Lactobacillus plantarum. Background Art

[0002] Lactobacillus plantarum belongs to the genus Lactobacillus and mainly exists in fermented products such as cream, meat, and vegetables. Most strains are isolated from plant raw materials, so it is named Lactobacillus plantarum. As a probiotic group in the gastrointestinal tract, Lactobacillus plantarum has multiple functions such as maintaining the balance of the intestinal flora, enhancing the body's immunity, and promoting the absorption of nutrients, and is widely used in fields such as food fermentation and healthcare.

[0003] High-density cultivation refers to the application of certain cultivation techniques or devices to increase the fermentation density of bacteria. Specifically, it is to obtain a higher cell density with a lower cultivation volume and a shorter cultivation period, improve the fermentation speed and fermentation effect, and can reduce the usage amount of subsequent fermenting agents, control equipment investment, and reduce production costs when applied to production practice. At present, the raw material price of the commercial culture medium for Lactobacillus plantarum is high, and the traditional cultivation method has low efficiency, which can no longer meet the industrial requirements. Therefore, it is of great significance to seek an economical and efficient method for high-density cultivation of Lactobacillus plantarum. Summary of the Invention

[0004] In view of the above problems existing in the prior art, the applicant of the present invention provides a method for high-density cultivation of Lactobacillus plantarum. By optimizing the fermentation medium and cultivation conditions, the present invention improves the growth amount and viable cell number of the strain, and realizes the high-density cultivation of Lactobacillus plantarum.

[0005] The technical solution of the present invention is as follows:

[0006] A method for high-density cultivation of Lactobacillus plantarum, comprising the following steps: After the Lactobacillus plantarum is subjected to seed cultivation, it is inoculated into a fermenter filled with a fermentation medium for fermentation cultivation;

[0007] The fermentation medium comprises: a carbon source, a nitrogen source, and manganese sulfate; the mass concentration ratio of the carbon source, the nitrogen source, and manganese sulfate is (15-30):(75-90):(0.25-0.4).

[0008] Preferably, the carbon source comprises at least one of sucrose, glucose, lactose, and trehalose;

[0009] And / or, the addition amount of the carbon source in the fermentation medium is 15-30 g / L.

[0010] Preferably, the nitrogen source comprises at least one of corn steep liquor powder, yeast extract powder, soybean meal extract, soy peptone, and beef extract powder;

[0011] And / or, the addition amount of nitrogen source in the fermentation medium is 75-90 g / L.

[0012] Preferably, the addition amount of manganese sulfate in the fermentation medium is 0.25-0.4 g / L;

[0013] And / or, the fermentation medium further comprises 1-3 g / L of dipotassium hydrogen phosphate, 1-3 g / L of diammonium hydrogen citrate, 4-6 g / L of sodium acetate, 0.1-0.4 g / L of magnesium sulfate, 0.5-1.5 g / L of Tween, and the balance is water.

[0014] Preferably, the inoculation amount of the inoculation is 1-3% (v / v).

[0015] Preferably, the initial pH of the fermentation medium is 5.5-7.0.

[0016] Preferably, the fermentation culture includes static culture and / or dynamic culture;

[0017] During the dynamic culture, the pH is kept constant;

[0018] Preferably, the pH of the dynamic culture is pH 6.5-7.5;

[0019] And / or a neutralizing agent is added to the dynamic culture, and the neutralizing agent includes NaOH and HCl.

[0020] Preferably, the time of the dynamic culture is 20-28 h;

[0021] And / or, the temperature of the dynamic culture is 35-39 °C;

[0022] And / or, the dynamic culture is carried out in an exponential feeding manner; the exponential feeding starts when the dynamic culture reaches 10-14 h; the feeding substrate of the exponential feeding includes a carbon source.

[0023] Preferably, the parameter F1(0) of the exponential feeding is 0.012-0.022 L / h, μ = 0.3168-0.7188 h -1 ;

[0024] And / or, the feeding substrate of the exponential feeding is a 380-420 g / L trehalose solution.

[0025] The present invention also provides an application of Lactobacillus plantarum obtained by culturing by the above high-density culture method in the preparation of bacterial powder.

[0026] The beneficial technical effects of the present invention are as follows:

[0027] The present invention optimizes the culture medium and fermentation conditions during the fermentation of Lactobacillus plantarum, improving the cell growth amount and the number of viable cells. The cell growth amount reaches 1.96, and the number of viable cells reaches 1.50×10 12 CFU / mL, which is several orders of magnitude higher than the 2.49×10 8 CFU / mL of viable cells obtained by static culture in the unoptimized MRS liquid medium, achieving high-density culture of Lactobacillus plantarum. Description of the Drawings

[0028] Figure 1 It is a graph showing the optimization result of the number of hidden layers in the artificial neural network model in Example 3.

[0029] Figure 2 It is a graph showing the training process of the artificial neural network model in Example 3.

[0030] Figure 3 It is a topological structure diagram of the artificial neural network in Example 3.

[0031] Figure 4 It is a graph showing the establishment process of the artificial neural network model in Example 3.

[0032] Figure 5 It is a graph showing the training process of the artificial neural network model in Example 3.

[0033] Figure 6 It is a graph showing the optimization result of the genetic algorithm in Example 3.

[0034] Figure 7 It is the effect of the inoculum size of the strain on the growth of Lactobacillus plantarum ZJ-23 in Example 4.

[0035] Figure 8 It is the effect of the culture temperature on the growth of Lactobacillus plantarum ZJ-23 in Example 4.

[0036] Figure 9 It is the growth amount of Lactobacillus plantarum ZJ-23 at different fermentation broth pH values in Example 7.

[0037] Figure 10 It is the number of viable cells of Lactobacillus plantarum ZJ-23 at different fermentation broth pH values in Example 7.

[0038] Figure 11 It is the growth amount of Lactobacillus plantarum ZJ-23 under different neutralizing agents in Example 7.

[0039] Figure 12 It is the number of viable cells of Lactobacillus plantarum ZJ-23 under different neutralizing agents in Example 7.

[0040] Figure 13Growth characteristics of Lactobacillus plantarum ZJ-23 under exponential fed-batch culture in Example 7. Detailed implementation manners

[0041] The present invention will be specifically described below with reference to the accompanying drawings.

[0042] The first aspect of the present invention provides a method for high-density culture of Lactobacillus plantarum. The culture method includes: inoculating Lactobacillus plantarum after seed culture, and inoculating it into a fermenter filled with a fermentation medium for fermentation culture.

[0043] In some embodiments, the fermentation medium includes: a carbon source, a nitrogen source, and manganese sulfate; the mass concentration ratio of the carbon source, the nitrogen source, and manganese sulfate is (15-30):(75-90):(0.25-0.4), including but not limited to 15:75:0.25, 15:75:0.4, 15:90:0.25, 15:90:0.4, 30:75:0.25, 30:75:0.4, 30:90:0.25, 30:90:0.4.

[0044] In some embodiments, the carbon source includes at least one of sucrose, glucose, lactose, and trehalose.

[0045] In some embodiments, the addition amount of the carbon source in the fermentation medium is 15-30 g / L, including but not limited to 15 g / L, 20 g / L, 21.86 g / L, 25 g / L, 30 g / L.

[0046] In some embodiments, the nitrogen source in the fermentation medium includes at least one of corn steep liquor powder, yeast extract powder, soybean meal extract, soy peptone, and beef extract powder.

[0047] In some embodiments, the addition amount of the nitrogen source in the fermentation medium is 75-90 g / L, including but not limited to 75 g / L, 80 g / L, 83.64 g / L, 85 g / L, 90 g / L.

[0048] In some embodiments, the addition amount of manganese sulfate in the fermentation medium is 0.25-0.4 g / L, including but not limited to 0.25 g / L, 0.3 g / L, 0.324 g / L, 0.4 g / L.

[0049] In some embodiments, the fermentation medium further includes 1-3 g / L of dipotassium hydrogen phosphate, 1-3 g / L of diammonium hydrogen citrate, 4-6 g / L of sodium acetate, 0.1-0.4 g / L of magnesium sulfate, 0.5-1.5 g / L of Tween, and the balance is water.

[0050] In some embodiments, the inoculation amount of the inoculum is 1-3% (v / v), including but not limited to 1% (v / v), 1.5% (v / v), 2% (v / v), 2.5% (v / v), 3% (v / v).

[0051] In some embodiments, the initial pH of the fermentation medium is 5.5-7.0, including but not limited to 5.5, 5.8, 6.2, 6.5, 7.0.

[0052] In some embodiments, the fermentation culture includes static culture and / or dynamic culture.

[0053] In some embodiments, the pH is kept constant during the dynamic culture. Preferably, the pH of the dynamic culture is pH 6.5-7.5, including but not limited to 6.5, 7.0, 7.5.

[0054] In some embodiments, a neutralizing agent is further added to the dynamic culture. The neutralizing agent includes NaOH and HCl. In the neutralizing agent, the volume fraction of NaOH is 15-30%, including but not limited to 15%, 20%, 25%, 30%; the concentration of HCl is 1-3 mol / L, including but not limited to 1 mol / L, 1.5 mol / L, 2 mol / L, 2.5 mol / L, 3 mol / L. When the pH in the fermenter increases, an acidic neutralizing agent HCl needs to be fed into the fermenter. For example, when the pH in the fermenter decreases, a basic neutralizing agent NaOH needs to be fed into the fermenter.

[0055] In some embodiments, the time of the dynamic culture is 20-28 h, including but not limited to 20 h, 22 h, 24 h, 26 h, 28 h.

[0056] In some embodiments, the temperature of the dynamic culture is 35-39 °C, including but not limited to 35 °C, 36 °C, 37 °C, 38 °C, 39 °C.

[0057] In some embodiments, the dynamic culture is carried out in an exponential feeding manner. The exponential feeding starts at 10-14 h of the dynamic culture, including but not limited to starting at 10 h, 11 h, 12 h, 13 h, 14 h.

[0058] In some embodiments, the feeding substrate of the exponential feeding includes a carbon source, including but not limited to sucrose, glucose, lactose, trehalose.

[0059] In some embodiments, the parameter F1(0) of the exponential feeding is 0.012-0.022 L / h, including but not limited to 0.012 L / h, 0.014 L / h, 0.017 L / h, 0.020 L / h, 0.022 L / h; μ = 0.3168-0.3188 h-1 , including but not limited to 0.3168 h -1 、0.3172 h -1 、0.3176 h -1 、0.3178 h -1 、0.3182 h -1 、0.3184 h -1 、0.3188 h -1 。

[0060] In some embodiments, the feeding substrate for exponential feeding is a 380 - 420 g / L trehalose solution, including but not limited to 380 g / L, 390 g / L, 400 g / L, 410 g / L, 420 g / L.

[0061] The second aspect of the present invention provides the application of Lactiplantibacillus plantarum obtained by culturing using the above - mentioned high - density culture method in the preparation of bacterial powder.

[0062] In the present invention, Lactiplantibacillus plantarum ZJ - 23 is taxonomically named Lactiplantibacillus plantarum, and was deposited on April 21, 2025 at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, with the deposit number CGMCC No. 34276, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0063] In the present invention, the mentioned MRS liquid medium includes:

[0064] Nitrogen source: peptone 10.0 g / L, beef powder 8.0 g / L, yeast powder 4.0 g / L;

[0065] Carbon source: glucose 20.0 g / L;

[0066] Dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, Tween 80 1.0 g / L, pH 5.7.

[0067] That is, in the MRS liquid medium, the addition amount of the nitrogen source is 2.2% (w / v), and the addition amount of the carbon source is 2% (w / v).

[0068] Example 1

[0069] 1. Carbon source optimization

[0070] Fermentation media were obtained by replacing the carbon source in MRS liquid medium with 2% (w / v) sucrose, glucose, lactose, trehalose, and cane molasses, respectively. A single colony of Lactobacillus plantarum ZJ-23 was inoculated into 12 mL of sterilized MRS liquid medium and cultured at 37 °C for 24 h. This was repeated twice to obtain the seed liquid. The seed liquid was inoculated into the fermentation medium at an inoculation amount of 2% (v / v). The initial pH of the fermentation medium was 5.7, and OD was measured after static culture at 37 °C for 24 h 600 to investigate the effect of different carbon sources on the growth of Lactobacillus plantarum ZJ-23. Through the above experiments, it was found that there were significant differences in the growth of Lactobacillus plantarum with different carbon sources. Among them, trehalose had the most obvious growth-promoting effect on the bacteria; while cane molasses had the worst growth-promoting effect on the bacteria. Therefore, trehalose was selected as the optimal carbon source for the fermentation medium. The growth amount of Lactobacillus plantarum was the smallest when cane molasses was used as the carbon source of the medium. The reason is that the colloid contained in cane molasses belongs to polysaccharides and has a large viscosity and cannot be utilized by microorganisms, resulting in the smallest growth amount of Lactobacillus plantarum

[0071] After determining that the optimal carbon source was trehalose, different addition amounts of trehalose in the fermentation medium were set. Fermentation media were obtained by replacing 2% (w / v) of the carbon source in MRS liquid medium with 1% (w / v), 2% (w / v), 3% (w / v), 4% (w / v), and 5% (w / v) trehalose, respectively. A single colony of Lactobacillus plantarum ZJ-23 was inoculated into 12 mL of sterilized MRS liquid medium and cultured at 37 °C for 24 h. This was repeated twice to obtain the seed liquid. The seed liquid was inoculated into the fermentation medium at an inoculation amount of 2% (v / v). The initial pH of the fermentation medium was 5.7, and OD was measured after static culture at 37 °C for 24 h 600 to investigate the effect of different concentrations of trehalose on the growth of Lactobacillus plantarum ZJ-23 by measuring OD 600 The addition amount of trehalose at 2% (w / v) was the most suitable for the growth of Lactobacillus plantarum ZJ-23

[0072] 2. Nitrogen source optimization

[0073] The nitrogen source in MRS liquid medium was replaced with 2.2% (w / v) corn steep liquor powder, yeast extract powder, soybean meal extract, soy peptone, and beef extract, respectively. A single colony of Lactobacillus plantarum ZJ-23 was inoculated into 12 mL of sterilized MRS liquid medium and cultured at 37 °C for 24 h. This was repeated twice to obtain the seed liquid. The seed liquid was inoculated into the fermentation medium at an inoculation amount of 2% (v / v). The initial pH of the fermentation medium was 5.7, and OD was measured after static culture at 37 °C for 24 h 600, the effects of different nitrogen sources on the growth of ZJ-23 were investigated. Through the above experiments, it was found that there were certain differences in the growth of Lactobacillus plantarum with different nitrogen sources. Among them, beef extract had the most obvious growth-promoting effect on Lactobacillus plantarum; yeast extract, soybean meal extract, and corn steep liquor powder had basically similar effects on the growth of the bacteria. Therefore, beef extract was selected as the best nitrogen source.

[0074] After determining that the best nitrogen source was beef extract, different addition amounts of beef extract in the fermentation medium were set. Fermentation media were obtained by replacing the nitrogen source in MRS liquid medium with 1 (w / v), 3 (w / v), 5 (w / v), 7 (w / v), and 9% (w / v) beef extract, respectively. A single colony of Lactobacillus plantarum ZJ-23 was inoculated into 12 mL of sterilized MRS liquid medium and cultured at 37 °C for 24 h. This was repeated twice to obtain the seed liquid. The seed liquid was inoculated into the fermentation medium at an inoculation amount of 2% (v / v). The initial pH of the fermentation medium was 5.7, and after static culture at 37 °C for 24 h, OD was measured. 600 , by measuring OD 600 The effects of different concentrations of beef extract on the growth of ZJ-23 were investigated. Through the above experiments, it was found that as the addition amount of beef extract increased, the growth amount of the bacteria showed a trend of first increasing and then decreasing. At this time, the best addition amount of beef extract was 5% (w / v).

[0075] 3. Optimization of the addition amount of manganese sulfate

[0076] Manganese sulfate in MRS liquid medium was replaced with 0.03 (g / L), 0.05 (g / L), 0.1 (g / L), 0.2 (g / L), 0.3 (g / L), and 0.4 (g / L) manganese sulfate, respectively. A single colony of Lactobacillus plantarum ZJ-23 was inoculated into 12 mL of sterilized MRS liquid medium and cultured at 37 °C for 24 h. This was repeated twice to obtain the seed liquid. The seed liquid was inoculated into the fermentation medium at an inoculation amount of 2% (v / v). The initial pH of the fermentation medium was 5.7, and after static culture at 37 °C for 24 h, OD was measured. 600 , the effects of different addition amounts of manganese sulfate on the growth of Lactobacillus plantarum ZJ-23 were investigated. Through the above experiments, it was found that different addition amounts of manganese sulfate had certain effects on the growth of Lactobacillus plantarum ZJ-23. When the addition amount of manganese sulfate was 0.3 g / L, the growth amount of the bacteria OD 600 was the largest. Therefore, the best addition amount of manganese sulfate at this time was 0.3 g / L.

[0077] Example 2

[0078] 1. Optimization of the culture medium formula by response surface experiment

[0079] By analyzing the single-factor optimization experiment conducted in Example 1, it was found that when the contents of other substances in the MRS liquid medium remained unchanged, and the carbon source, nitrogen source, and manganese sulfate were replaced with trehalose 2% (w / v), beef extract powder 5% (w / v), and manganese sulfate 0.3 g / L respectively, the OD of the obtained fermentation medium 600 was relatively large. Therefore, this addition amount was selected as the benchmark for the response surface test. The variables and their levels in the central composite experiment are shown in Table 1.

[0080] Table 1 Variables and their levels in the central composite experiment

[0081]

[0082] Beef extract powder, trehalose, and manganese sulfate, which have a greater impact on the growth amount of the bacteria, were selected as the response factors, and OD 600 was used as the response value, and Design Expert software was used to optimize the medium. The corresponding levels of the variables were input into the Design-expert software, and 20 designs were obtained as shown in Table 2. Experiments were carried out. The composition of the medium during the experiment: compared with the MRS liquid medium, only the carbon source, nitrogen source, and manganese sulfate changed, and the other components remained unchanged; the culture conditions: the initial pH of the medium was 5.7, and it was statically cultured at 37 °C for 24 h to obtain the OD under the corresponding fermentation medium formula 600 .

[0083] Table 2 Central composite experimental design

[0084]

[0085]

[0086] 2. The relationship between the response predicted value and the independent variable influencing factors is as follows:

[0087] Using Design Expert software to analyze the data in Table 2, the following multiple regression equation was obtained:

[0088] Y = 0.9489 + 0.1065*A + 0.0885*B + 0.0212*C + 0.0727*AB - 0.0145*BC - 0.0244*A 2 - 0.1067*B 2 - 0.0433*C 2 ;

[0089] In the formula: Y is the OD of the bacterial strain 600The predicted response value, where A, B, and C are the actual values of beef extract powder, trehalose, and manganese sulfate respectively. The absolute value of the regression coefficient (such as 0.1065, 0.0885, -0.0145, etc.) represents the influence degree of the independent variable influencing factor on the predicted response value, and the positive or negative sign of the coefficient represents the increase or decrease of the response value caused by the change of the independent variable influencing factor.

[0090] 4. ANOVA and significance test of the response surface model

[0091] In the regression model, the significance of the independent variable influencing factor on the predicted response value is generally evaluated by the P value. To further evaluate the accuracy and reliability of the model, ANOVA is used to conduct a significance test on the above model, and the results are shown in Table 3. The P value of the model is less than 0.01, and the response surface F value is 5.16 (the F value is an important index to measure the influence of test factors on the response), indicating that this model is significant and has good credibility.

[0092] Table 3 ANOVA table

[0093]

[0094]

[0095] According to the ANOVA table, the F value of factor A is 14.53, the F value of factor B is 10.02, and the F value of factor C is 0.5753. The order of the influence of the three factors on the growth amount of the strain is A (beef extract powder) > B (trehalose) > C (manganese sulfate).

[0096] Example 3

[0097] 1. Optimization of the number of hidden layers

[0098] Taking the response surface experiment data in Example 2 as the training samples, an artificial neural network model is established using a neural network. First, the number of neurons in the hidden layer is set to 2 - 15 in sequence, and 14 neural network trainings are carried out. As the running curve of the neural network tends to be stable, the verification performance of the model gradually improves. The training process is as Figure 1 and Figure 2 shown. When the number of neurons in the hidden layer is set to 9 and the number of training epochs is 3, the training error reaches 0.0079197. At this time, the neural network model is stable, and the correlation coefficient is 0.97338, indicating that 97.3% of the model prediction data is consistent with the experimental data, and only 2.7% of the variance cannot be explained by the model. It shows that the model training accuracy is good under this parameter. Combining the above results, and corresponding to the three independent variable influencing factors and one predicted response value of the strain OD 600 , finally, a network topology structure with 3 input layer neurons, 9 hidden layer neurons, and 1 output layer neuron is established, as Figure 3 .

[0099] 2. Establishment of Artificial Neural Network Model

[0100] Use code to implement the construction and training process of the neural network model. As shown in Figure 4 and Figure 5 After 1247 iterations, the mean squared error reaches 0.0011649, and the training set R 2 reaches 0.99893. The mean squared error measures the average size of the error between the predicted value and the true value of the model. The smaller the value, the more accurate the prediction. R 2 is used to evaluate the goodness of fit of the model to the data. The closer it is to 1, the better the fitting effect of the model to the data. Therefore, the mean squared error is 0.0011649, and the training set R 2 being 0.99893 indicates good training accuracy.

[0101] 3. Optimization by Genetic Algorithm

[0102] Using the generated network model, apply the genetic algorithm toolbox optimtool to find the combination of culture medium formulas to increase biomass and maximize the biomass of Lactobacillus plantarum ZJ-23. After 70 iterations, the optimal combination is obtained. From Figure 6 it can be seen that considering the three influencing factors comprehensively, the genetic algorithm results show that when the addition amounts of nitrogen source (beef extract powder), carbon source (trehalose), and manganese sulfate are 8.364% (w / v), 2.186% (w / v), and 0.324 g / L respectively, the biomass of Lactobacillus plantarum is the largest, which is 1.443.

[0103] Use the culture medium optimized above as the fermentation medium to optimize the culture conditions. The fermentation medium includes: beef extract powder 83.64 g / L, trehalose 21.86 g / L, manganese sulfate 0.324 g / L, and the remaining components remain unchanged compared with the MRS liquid medium.

[0104] Example 4

[0105] 1. Influence of Inoculum Size on Strain Growth

[0106] The inoculum size determines the initial cell concentration. Under suitable inoculum size, the strain will quickly enter the logarithmic growth phase. Therefore, the initial inoculum size has an important impact on product formation and cell biomass. Inoculate a single colony of Lactobacillus plantarum ZJ-23 into 12 mL of sterilized MRS liquid medium and incubate at 37°C for 24 h. Repeat twice to obtain the seed solution. Inoculate the seed solution into the fermentation medium at inoculum sizes of 1 (v / v), 2 (v / v), 3 (v / v), 4 (v / v), and 6% (v / v) respectively. The initial pH of the fermentation medium is 5.7, and measure the OD after static culture at 37°C for 24 h 600, the effects of different inoculation amounts on the biomass of Lactobacillus plantarum ZJ-23 were investigated, and the measurement results are as Figure 7 shown. The fermentation medium includes: beef extract powder 83.64 g / L, trehalose 21.86 g / L, dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.324 g / L, Tween 80 1.0 g / L. According to Figure 7 it can be seen that the growth of the bacterial cells shows a trend of first increasing and then decreasing with the increase of the inoculum amount, and the growth amount reaches the maximum (OD 600 = 1.26) at an inoculum amount of 2% (v / v). When the inoculum amount is too high, the excessive number of cells in the later stage causes insufficient nutrients in the fermentation medium, resulting in no further increase in the biomass of the bacterial cells. Therefore, the optimal inoculum amount of the strain is determined to be 2% (v / v). When the inoculum amount reaches 4% or more, the OD value decreases significantly.

[0107] 2. Effects of culture temperature on the biomass of the strain

[0108] Temperature is a key factor affecting the growth and metabolism during the growth of Lactobacillus plantarum. Selecting an appropriate temperature for cultivation is beneficial for Lactobacillus plantarum to maintain a relatively fast growth rate. A single colony of Lactobacillus plantarum ZJ-23 was inoculated into 12 mL of sterilized MRS liquid medium and cultured at a constant temperature of 37 °C for 24 h. Repeating this twice obtained the seed liquid. The seed liquid was inoculated into the fermentation medium at an inoculum amount of 2% (v / v). The initial pH of the fermentation medium was 5.7. After static cultivation at different temperatures (20 °C, 25 °C, 30 °C, 37 °C, 43 °C) for 24 h, OD 600 was measured to investigate the effects of different culture temperatures on the biomass of Lactobacillus plantarum ZJ-23, and the measurement results are as Figure 8 shown. The fermentation medium includes: beef extract powder 83.64 g / L, trehalose 21.86 g / L, dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.324 g / L, Tween 80 1.0 g / L.

[0109] According to Figure 8 it can be seen that with the increase of temperature, the growth of the bacterial cells (OD 600 ) shows a trend of first increasing and then decreasing. When the culture temperature is 37 °C, the growth of the bacterial cells is the largest, and OD 600 = 1.29. This is because when the temperature exceeds the optimal growth temperature, the enzyme activity in the cells is inhibited and the types of metabolites change, resulting in a decrease in cell activity and even cell death, thus the biomass shows a downward trend. Therefore, the optimal culture temperature of the strain is determined to be 37 °C.

[0110] 3. Effects of the initial pH of the medium on the biomass of the bacterial cells

[0111] Inoculate a single colony of Lactobacillus plantarum ZJ-23 into 12 mL of sterilized MRS liquid medium, and incubate it at 37 °C for 24 h. Repeat this process twice to obtain the seed liquid. Then inoculate the seed liquid into fermentation media with different initial pH values (5.0, 5.5, 6.2, 6.5, 7.0) at an inoculation amount of 2% (v / v). After static incubation at 37 °C for 24 h, measure the OD 600 to investigate the effect of different initial pH values of the medium on the biomass of Lactobacillus plantarum ZJ-23. The fermentation medium includes: beef extract powder 83.64 g / L, trehalose 21.86 g / L, dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.324 g / L, Tween 80 1.0 g / L. Through the above experiments, it was found that the cell growth showed an upward trend first. After the pH reached 6.2, there was no significant change in the growth amount with the increase of pH. Considering the acid tolerance of Lactobacillus plantarum, the initial pH of the medium was selected as 6.5 for subsequent experiments.

[0112] Example 5

[0113] Verify the growth amount of Lactobacillus plantarum ZJ-23 under the fermentation medium formula and optimized conditions. Inoculate a single colony of Lactobacillus plantarum ZJ-23 into 12 mL of sterilized MRS liquid medium, and incubate it at 37 °C for 24 h. Repeat this process twice to obtain the seed liquid. Then inoculate the seed liquid into MRS liquid medium and fermentation medium respectively at an inoculation amount of 2% (v / v). The initial pH of the medium is 6.5. After static incubation at 37 °C for 24 h, measure the OD 600 . The MRS liquid medium includes: peptone 10.0 g / L, beef powder 8.0 g / L, yeast powder 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, Tween 80 1.0 g / L. The fermentation medium includes: beef extract powder 83.64 g / L, trehalose 21.86 g / L, dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.324 g / L, Tween 80 1.0 g / L.

[0114] Through the above experiments, it was found that the bacteria were in the logarithmic growth phase from 0 to 12 h, and the cell growth amount in the fermentation medium was greater after 10 h. After 24 h, there was a significant difference in the growth amount between the two media. The growth amount in the fermentation medium under the optimized conditions was 1.172 times that in the MRS liquid medium.

[0115] The viable cell counts of the strains in two kinds of media under optimized conditions were measured, and the results are shown in Table 4. It can be seen from the results that the viable cell count of Lactobacillus plantarum ZJ-23 in the fermentation medium was 2.66×10 9 CFU / mL, which was 9.68 times higher than the viable cell count (2.49×10 8 CFU / mL) in the liquid MRS medium.

[0116] Table 4 Growth status of Lactobacillus plantarum ZJ-23 before and after optimization

[0117] Number of viable cells (CFU / mL) MRS liquid medium <![CDATA[2.49×10 8 > Fermentation medium <![CDATA[2.66×10 9 > Multiplication factor 9.68

[0118] Example 6

[0119] A single colony of Lactobacillus plantarum ZJ-23 was inoculated into 12 mL of sterilized MRS liquid medium and cultured at a constant temperature of 37 °C for 24 h. This was repeated twice to obtain the seed liquid. The seed liquid was inoculated into a 5 L fermenter containing the fermentation medium at an inoculation amount of 2% (v / v). Under the conditions of a rotation speed of 80 rpm / min and aeration, fermentation was carried out at 37 °C for 24 h, and the growth amount of the bacteria, the sugar content, and the lactic acid content in the fermentation broth were measured. Through the above experiments, it was found that as the fermentation proceeded, the OD 600 of Lactobacillus plantarum ZJ-23 gradually increased. During this process, the lactic acid content in the fermentation broth gradually increased, while the trehalose content in the fermentation broth gradually decreased and reached the lowest amount at 15 h. At this time, the cell OD 600 also gradually leveled off, indicating that the decrease in sugar content restricted the growth of the strain. Therefore, fed-batch culture was considered. After 12 h, the cell OD 600 had already started to grow slowly, so the feeding could start at 12 - 15 h of fermentation.

[0120] Example 7

[0121] During the fermentation process, affected by the accumulation of acidic substances in the metabolites and the insufficient supply of initial nutrients, the growth amount of the strain will be restricted. The fermenter has the advantages of adjusting the culture pH by feeding and supplementing nutrients to carry out continuous fermentation to increase the biomass. Therefore, the growth conditions of the strain in the 5 L fermenter were further optimized.

[0122] 1. Optimization of pH in constant pH fermentation

[0123] Inoculate a single colony of Lactobacillus plantarum ZJ-23 into 12 mL of sterilized MRS liquid medium and incubate it at 37 °C for 24 h. Repeat this process twice to obtain the seed liquid. Inoculate the seed liquid into a 5 L fermenter containing the fermentation medium at an inoculation amount of 2% (v / v). Under the conditions of a rotation speed of 80 rpm / min and aeration, monitor the pH in real time through a pH electrode and control the fermentation broth to conduct a preliminary fermentation at a constant pH of 6.0, 6.5, 7.0, and 7.5 at 37 °C for 24 h. To maintain a constant pH fermentation environment in the fermenter, a neutralizing agent needs to be added to the fermenter. For example, when the pH in the fermenter increases, an acidic neutralizing agent needs to be added to the fermenter, and when the pH in the fermenter decreases, a basic neutralizing agent needs to be added to the fermenter. The neutralizing agent in the fermenter is a basic neutralizing agent, NaOH solution (volume fraction of 25%), and an acidic neutralizing agent, 2 mol / L HCl. After fermentation for 24 h, measure the biomass and viable cell count of the strain. The results are as Figure 9 and Figure 10 shown. Under the condition of pH = 7.0, the final OD 600 and viable cell count of Lactobacillus plantarum ZJ-23 are the highest, reaching 1.80 and 4.44×10 10 CFU / g respectively. Therefore, the fermentation is carried out under the condition of a constant pH of 7.0.

[0124] 2. Optimization of the types of neutralizing agents for constant pH fermentation

[0125] There are differences in the buffering capacity of different types of neutralizing agents, and the salt ions therein will also affect the growth of microorganisms and the production of products. Inoculate a single colony of Lactobacillus plantarum ZJ-23 into 12 mL of sterilized MRS liquid medium and incubate it at 37 °C for 24 h. Repeat this process twice to obtain the seed liquid. Inoculate the seed liquid into a 5 L fermenter containing the fermentation medium at an inoculation amount of 2% (v / v). Under the conditions of a rotation speed of 80 rpm / min and aeration, ferment at 37 °C for 24 h. Select combinations of NaOH, Na2CO3, NH3·H2O with a volume fraction of 25% and 2 mol / L HCl as neutralizing agents respectively. After fermentation at a constant pH of 7.0 for 24 h, measure the biomass to determine the optimal neutralizing agent. The results are as Figure 11 and Figure 12 shown. When the neutralizing agent is a combination of NaOH with a volume fraction of 25% and 2 mol / L HCl, the final OD 600 and viable cell count of the strain are the highest, reaching 1.80 and 4.44×10 10 CFU / g respectively. Therefore, for subsequent fermentation, NaOH with a volume fraction of 25% and 2 mol / L HCl are selected as neutralizing agents.

[0126] 3. Study on the exponential feeding and growth characteristics of Lactobacillus plantarum ZJ-23 in a 5 L fermenter

[0127] Exponential feeding is based on the law of bacterial growth curve, which helps the bacteria grow steadily and evenly and increase product yield, avoiding the waste of raw materials. It is an effective strategy to improve production efficiency. A single colony of Lactobacillus plantarum ZJ-23 was inoculated into 12mL of sterilized MRS liquid culture medium, and cultured at 37℃ for 24h. The seed liquid was repeated twice to obtain the seed liquid. The seed liquid was inoculated into a 5L fermentation tank containing fermentation medium at an inoculum amount of 2% (v / v), and fermented at 37℃ for 24h under ventilation conditions at a speed of 80rpm / min. Exponential feeding was performed after 12h of fermentation. The exponential feeding parameters were F1(0)=0.017L / h, μ=0.3178h -1 The feed material is 400g / L trehalose solution. The cell growth status during the fermentation process is measured every 3h, and samples are retained for subsequent index determination. In exponential feeding, the calculation formula for the feed rate F1(t) is: F1(t) = F1(0)e μt , where F1(0) is the initial feed rate, μ is the specific growth rate of the bacteria, and t is time.

[0128] The results are as follows Figure 13 As shown in the figure, the experimental results show that the cell growth rate gradually increases with the fermentation process, gradually stabilizes after 15 hours, and the maximum cell growth rate is 1.96 at 19 hours. 600 The number of viable cells continued to increase gradually, reaching 1.50×10 12 CFU / mL, compared with 2.49×10 8 CFU / mL, fermentation medium 2.66×10 9 The CFU / mL increased by 4 and 3 orders of magnitude, respectively.

[0129] The results of the determination of the fermentation products showed that the lactic acid in the supernatant showed a gradual increase trend, indicating that Lactobacillus plantarum ZJ-23 gradually produced acid during the exponential feeding fermentation process, and there was still an upward trend after 24 hours. For the trehalose content, it began to gradually decrease at 6 hours, and the feeding was started at 12 hours. The sugar content in the fermentation broth was at a low level from 12 to 18 hours, indicating that most of the carbon source of the feeding was used by the bacteria at this stage, and the bacteria no longer grew after 21 hours. The possible reason is that when the feeding amount is greater than the amount of sugar required by lactic acid bacteria, the increase in osmotic pressure will inhibit the growth of the bacteria, so the number of live bacteria no longer grows rapidly.

[0130] The above is only a preferred embodiment of the present invention, and the present invention is not limited to the above embodiments. It is understood that other improvements and changes directly derived or associated by those skilled in the art without departing from the spirit and concept of the present invention should be considered to be included in the protection scope of the present invention.

Claims

1. A high-density cultivation method of Lactobacillus plantarum, characterized in that, It includes the following steps: After the Lactobacillus plantarum is subjected to seed culture, it is inoculated into a fermenter filled with a fermentation medium for fermentation culture; The fermentation medium includes: a carbon source, a nitrogen source, and manganese sulfate; The mass concentration ratio of the carbon source, the nitrogen source, and manganese sulfate is (15-30):(75-90):(0.25-0.4).

2. The high-density culture method of Lactobacillus plantarum according to claim 1, wherein The carbon source includes at least one of sucrose, glucose, lactose, and trehalose; And / or, the addition amount of the carbon source in the fermentation medium is 15-30 g / L.

3. The high-density culture method of Lactobacillus plantarum according to claim 1, characterized in that The nitrogen source includes at least one of corn steep liquor dry powder, yeast extract powder, soybean meal extract, soy peptone, and beef extract powder; And / or, the addition amount of the nitrogen source in the fermentation medium is 75-90 g / L.

4. The high-density cultivation method of Lactobacillus plantarum according to claim 1, wherein, The addition amount of manganese sulfate in the fermentation medium is 0.25-0.4 g / L; And / or, the fermentation medium further includes 1-3 g / L of dipotassium hydrogen phosphate, 1-3 g / L of diammonium hydrogen citrate, 4-6 g / L of sodium acetate, 0.1-0.4 g / L of magnesium sulfate, 0.5-1.5 g / L of Tween, and the balance is water.

5. The high-density culture method of Lactobacillus plantarum according to claim 1, characterized in that The inoculation amount of the inoculation is 1-3% (v / v).

6. The high-density culture method of Lactobacillus plantarum according to claim 1, characterized in that, The initial pH of the fermentation medium is 5.5-7.

0.

7. The high-density cultivation method of Lactobacillus plantarum according to claim 1, characterized in that The fermentation culture includes static culture and / or dynamic culture; During the dynamic culture process, the pH is kept constant; Preferably, the pH of the dynamic culture is pH 6.5-7.5; And / or a neutralizing agent is further added to the dynamic culture, and the neutralizing agent includes NaOH and HCl.

8. The high-density culture method of Lactobacillus plantarum according to claim 7, characterized in that, The time of the dynamic culture is 20-28 h; And / or, the temperature of the dynamic culture is 35-39 °C; And / or, the dynamic culture is carried out in an exponential feeding manner; the exponential feeding starts at 10-14 h of the dynamic culture; the feeding substrate of the exponential feeding includes a carbon source.

9. The high-density cultivation method of Lactobacillus plantarum according to claim 1 or 8, characterized in that, The parameter F1(0) of the exponential feeding is 0.012 - 0.022 L / h, and μ = 0.3168 - 0.7188 h -1 ; And / or, the feeding substrate of the exponential feeding is a 380-420 g / L trehalose solution.

10. Use of the Lactobacillus plantarum obtained by the high-density culture method according to any one of claims 1-9 in the preparation of bacterial powder.

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