Bifidobacterium splitting activity detection method based on pH regulation and application thereof

Abstract

The application discloses a bifidobacterium splitting activity detection method based on pH regulation and application thereof, and belongs to the technical field of microbial fermentation. The detection method comprises high-density fermentation of bifidobacterium with fluorescent labeling under pH regulation and splitting cycle detection through flow cytometry, and the pH regulation range is 4.30-6.30. The bifidobacterium splitting activity detection method based on pH regulation and application thereof can quickly and accurately quantitatively and qualitatively detect the real-time fermentation state of bifidobacterium. The bifidobacterium splitting activity detection method based on pH regulation can be used for quantitative evaluation of cell splitting in a complex fermentation system, and realizes bifidobacterium fermentation optimization and precise fermentation control.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial fermentation, and in particular to a method for detecting bifidobacterium division activity based on pH regulation and application thereof. Background Art

[0002] pH control is an important control method and basic link in the fermentation production of probiotics. It aims to regulate the division of bacteria by regulating the pH of the fermentation system, and jointly form an environment conducive to the rapid division and proliferation of bacteria, thereby achieving precise fermentation and improving the processing activity of probiotic preparations.

[0003] As an important probiotic, bifidobacterium faces the problems of low biomass and easy attenuation of activity in industrial production. In the existing technology, although pH control is widely used in probiotic fermentation, the research on the molecular mechanism of the bifidobacterium division cycle is still insufficient. Traditional pH control methods mainly rely on empirical adjustments and lack precise control of division gene expression and division dynamics. In addition, conventional detection methods (such as plate counting method) are inefficient and cannot reflect the division status of bacteria in real time. In contrast, flow cytometry has the advantages of high throughput, high sensitivity and high accuracy, and can detect 10 6 -10 7 The system can detect and analyze individual cells and accurately quantify the number of bacterial populations, which is suitable for analyzing the physiological status of Bifidobacterium. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for detecting the division activity of bifidobacteria based on pH regulation and its application. By optimizing the pH range of the fermentation system and combining it with flow cytometry, the effect of pH on the division cycle of bifidobacteria is revealed, and the number of viable bacteria and the division efficiency in bifidobacterium fermentation production are significantly improved.

[0005] To achieve the above objectives, on the one hand, the present invention provides a method for detecting the division activity of bifidobacteria based on pH regulation, comprising: using fluorescently labeled bifidobacteria as seed liquid, subjecting the seed liquid to high-density fermentation under pH regulation, and detecting the division cycle of bifidobacteria by flow cytometry, wherein the pH regulation range is 4.30-6.30.

[0006] Preferably, the fluorescent labeling method for Bifidobacterium comprises: staining Bifidobacterium with a cell proliferation fluorescent probe CFDA-SE at a final concentration of 900 μmol / L.

[0007] Preferably, the pH control includes: inoculating the seed liquid into the bioreactor at a 2.0% inoculation rate for high-density fermentation, adjusting the initial fermentation pH to 6.50±0.02 using a 25% mass fraction NaOH solution, naturally fermenting to the pH control range, and constantly controlling the pH by adding a 25% mass fraction NaOH solution.

[0008] Preferably, after the high-density fermentation is completed, the bacterial cells are collected and washed and then resuspended to the original volume, and the bacterial concentration is adjusted to 10 6 -10 7 CFU / mL was tested by flow cytometry;

[0009] The flow cytometry was set to excitation wavelength Ex = 494 nm, emission wavelength Em = 521 nm, and 1 × 10 4 The bifidobacterial division cycle was analyzed by fitting the deconvolution peaks based on the events data points.

[0010] On the other hand, the present invention provides an application of the above-mentioned bifidobacterium division activity detection method based on pH regulation in bifidobacterium fermentation, which is used to improve the number of viable bacteria and division efficiency in bifidobacterium fermentation production, and the pH value with the shortest bifidobacterium division cycle and the largest number of bacterial division generations detected by flow cytometry within the pH control range is screened as the optimal pH value of the bifidobacterium fermentation system.

[0011] On the other hand, the present invention provides an application of the above-mentioned method for detecting bifidobacterium division activity based on pH regulation in the preparation of a high-activity direct-injection starter of bifidobacteria.

[0012] On the other hand, the present invention provides an application of the above-mentioned method for detecting bifidobacterium division activity based on pH regulation in the preparation of a probiotic solid beverage containing bifidobacteria.

[0013] On the other hand, the present invention provides a molecular marker for regulating the division activity of bifidobacteria, the molecular marker comprising fts Z. fts X、 fts L. fts W. fts Q. fts I. sep F. whi A, molecular markers are obtained by combining the above-mentioned pH-controlled bifidobacterium division activity detection method with the division cycle, cell growth phenotype and expression characteristics of division-related genes of bifidobacteria within the pH control range.

[0014] Preferably, the suitable fermentation pH is fts X、 fts W and fts Q expression is upregulated, and the stability of cell wall and membrane structure increases; when the fermentation pH is lower than the critical value, fts Z. fts X、 fts L. fts W. fts Q. fts I. sepF. whi A expression is downregulated, inhibiting the division process and prolonging the division cycle.

[0015] Therefore, the present invention provides a method for detecting bifidobacterium division activity based on pH regulation and its application, which has the following beneficial effects:

[0016] (1) The present invention established a real-time detection technology for the division cycle of Bifidobacterium breve based on CFDA-SE fluorescence labeling. By optimizing the fluorescent dye concentration and constant pH gradient experiments, the optimal final concentration of CFDA-SE was determined to be 900 μmol / L and the pH gradient was determined to be (4.30, 5.30, and 6.30).

[0017] (2) Through dynamic pH control, the optimal pH of 5.30 was screened out. Compared with the culture of bifidobacteria at constant pH 6.30, the number of divisions at pH 5.30 increased by 40.00%, the number of viable bacteria increased significantly by 2.48 times, and the average division cycle was shortened by 19.65%. At pH 4.30, the number of bacterial divisions decreased by 20.00%, the number of viable bacteria decreased significantly by 76.47%, and the membrane damage rate increased from 28.54% to 89.20% from the early logarithmic stage to the stable stage. The bacterial division was inhibited, and there was no significant change in the bacterial size during the culture process.

[0018] (3) The molecular mechanism by which pH affects fission activity by regulating fission-related gene expression, membrane integrity, and key enzyme activity was revealed. Appropriate fermentation pH can promote fts X、 fts W and fts The expression of Q-division-related genes maintains the stability of the bacterial wall membrane structure; when the pH is lower than a certain threshold, fts Z. fts I. fts The expression levels of eight division-related genes, including L, decreased, the division process was inhibited, and the division cycle was prolonged, which ultimately led to a decrease in the division activity and proliferation number of bacterial cells, providing a theoretical basis for the design of precise fermentation processes.

[0019] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The effect of dye concentration on the cell density of Bifidobacterium breve B2798;

[0021] Figure 2 The effect of dye concentration on the acid production capacity of Bifidobacterium breve B2798;

[0022] Figure 3 The effect of dye concentration on the number of viable bacteria of Bifidobacterium breve B2798 at different growth stages. Different lowercase letters indicate significant differences within the data group.P <0.05;

[0023] Figure 4 The fluorescence intensity changes of Bifidobacterium breve B2798 labeled with CFDA-SE dye during storage;

[0024] Figure 5 Figure 2 shows the effect of fermentation system pH on the growth of Bifidobacterium breve B2798, where A is the change of bacterial density under different fermentation system pH conditions, B is the change of viable bacterial count under different fermentation system pH conditions, and C is the maximum specific growth rate under different fermentation system pH conditions;

[0025] Figure 6 The effect of pH on F6PPK activity during the culture of Bifidobacterium breve B2798; A is the F6PPK standard curve; B is the F6PPK activity graph converted to bacterial cell number; different lowercase letters indicate significant differences within the same culture period; different uppercase letters indicate significant differences between groups at different culture periods ( P <0.05);

[0026] Figure 7 Effect of pH on the culture of Bifidobacterium breve B2798 + -ATPase activity; different lowercase letters indicate significant differences within the same culture period; different uppercase letters indicate significant differences between groups at different culture periods ( P <0.05);

[0027] Figure 8 The effect of pH on the cell area of ​​Bifidobacterium breve B2798 at different culture periods. In the figure, S represents the average cell area, in μm 2 ; n represents the sample size; R 2 represents the fitted regression coefficient;

[0028] Figure 9 Effect of pH on the cell membrane integrity of Bifidobacterium breve B2798 at different culture periods

[0029] Figure 10 The deconvolution peak fitting diagram of Bifidobacterium breve B2798 under different fermentation system pH conditions

[0030] Figure 11 The results of agarose gel electrophoresis are shown;

[0031] Figure 12 For absolute quantification of division-related genes;

[0032] Figure 13 is the relationship between bacterial size and division gene expression; different lowercase letters in the figure indicate significant differences in bacterial area at different stages ( P <0.05);

[0033] Figure 14 Figure 2 is the relationship between membrane integrity and gene expression, where A is the membrane damage ratio at different culture periods under different pH conditions; B is the gene expression calorimeter for each culture reagent under different pH conditions. DETAILED DESCRIPTION

[0034] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0035] The instruments, equipment, reagents and materials used in the examples were obtained from commercial sources.

[0036] The embodiments relate to the microorganism Bifidobacterium breve ( Bifidobacterium breve ) B2798 is a publicly available strain, currently deposited in the China General Microbiological Culture Collection Center, with the deposit number CGMCC No. 22765, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0037] The examples relate to the following culture media:

[0038] Modified MRS liquid medium: peptone (animal source) 10.0 g, beef extract powder 8.0 g, yeast extract powder 4.0 g, glucose 20.0 g, Tween-80 1.0 mL, dipotassium hydrogen phosphate 2.0 g, sodium acetate 5.0 g, triammine citrate 2.0 g, manganese sulfate 0.05 g, magnesium sulfate 0.2 g, L-cysteine ​​hydrochloride 0.5 g, distilled water 1 L, adjust the pH to 6.20 ± 0.02, and sterilize at 121°C for 15 min.

[0039] Modified MRS solid medium: agar 1.2 g, peptone (animal source) 10.0 g, beef extract powder 8.0 g, yeast extract powder 4.0 g, glucose 20.0 g, Tween-80 1.0 mL, dipotassium hydrogen phosphate 2.0 g, sodium acetate 5.0 g, triamcinolone citrate 2.0 g, manganese sulfate 0.05 g, magnesium sulfate 0.2 g, L-cysteine ​​hydrochloride 0.5 g, distilled water 1 L, adjust pH to 6.20 ± 0.02, and sterilize at 121°C for 15 min.

[0040] The present invention relates to a method for regulating pH to enhance bifidobacterium fission activity, which is a rapid detection method based on flow cytometry. The method is not only applicable to Bifidobacterium breve B2798, but also to species such as Bifidobacterium animalis lactis subsp. Probio-M8, Bifidobacterium animalis lactis subsp. V9, Bifidobacterium animalis lactis subsp. BB-69, Bifidobacterium animalis lactis subsp. BX-246, and Bifidobacterium longum infantis subsp. B8762. Bifidobacterium breve B2798 and its application are described in detail below through specific examples.

[0041] Example 1

[0042] The cell proliferation fluorescent probe (CFDA-SE) can penetrate the cell membrane and enter the cell through passive diffusion. Once inside the cell, under the catalysis of endogenous esterases, CFDA-SE is converted into a cation, fluorescein succinimidyl ester (CFSE). This product can covalently bind to intracellular proteins, thereby achieving long-term retention. After labeling cells with this dye, its uniform and stable fluorescent signal is evenly transmitted to daughter cells during cell division. The decay of fluorescence intensity is directly proportional to the number of cell divisions, allowing flow cytometry to detect cells that have divided eight or more times.

[0043] The fluorescent dye staining method of Bifidobacterium breve B2798 comprises the following steps:

[0044] S1. Seed liquid pretreatment: After activating and subculture Bifidobacterium breve B2798 to the third generation, take 30.0 mL of the third generation culture medium and place it in a 50 mL light-proof centrifuge tube on ice. Centrifuge (5000 rpm, 5 min, 4°C), discard the supernatant, and retain the bacteria.

[0045] S2. Wash and resuspend: Resuspend the retained bacteria in 4.0°C sterile phosphate buffered saline (PBS) solution to the original volume, and then perform a second centrifugation (5000 rpm, 5 min, 4°C) to thoroughly wash away impurities such as the culture medium. After centrifugation, discard the supernatant and resuspend the culture medium in PBS solution to the original volume.

[0046] S3. Dye infection: Thaw the CFDA-SE solution stored at -20.0°C at room temperature, add the optimized dye concentration to the bacterial suspension, incubate in a water bath at 37.0°C for 15 min, centrifuge (5000 rpm, 5 min, 4°C), discard the supernatant and retain the bacteria.

[0047] S4. Wash and resuspend: Place the centrifuged cells on ice, add culture medium to resuspend to the original volume, and then centrifuge (5000 rpm, 5 min, 4°C). Discard the supernatant and add culture medium to resuspend to the original volume to wash away a small amount of free dye.

[0048] S4. Preparation of seed solution: The bacterial suspension after resuspending the culture medium was placed in a water bath at 37°C for 5.0 min to obtain the seed solution.

[0049] S5. Inoculation: Inoculate the seed solution at a rate of 2.0% (v / v) into the bioreactor for high-density fermentation in the dark.

[0050] (1) Optimization of fluorescent dye concentration:

[0051] During the dye infection process in S3, the CFDA-SE solution was a solution obtained by dissolving the CFDA-SE probe powder using dimethyl sulfoxide (DMSO), and the final concentrations of the added CFDA-SE solution were 0, 100 μmol / L, 200 μmol / L, 300 μmol / L, 900 μmol / L, 1500 μmol / L and 2100 μmol / L, respectively. It was then transferred to the parallel bioreactor with an inoculation volume of 2.0% (v / v), and the fermentation temperature was regulated at 37°C and the initial pH was 6.50. When the pH reached 5.90, a 25% mass concentration of NAOH solution was added for constant control, and the fermentation was carried out under anaerobic culture conditions. Sampling was performed regularly to detect the growth of the bacteria during the fermentation process, and the optimal final concentration of the dye was determined based on the test data. The effect of dye concentration on the bacterial density of Bifidobacterium breve B2798 is shown in Figure 2. Figure 1 As shown, the maximum specific growth rate after staining with different concentrations of dye ( ±S, n=3) as shown in Table 1:

[0052] Table 1 Maximum specific growth rate after staining with different concentrations of dyes ( ±S, n=3)

[0053] ;

[0054] Note: Different lowercase letters indicate significant differences between different groups of data in the same column ( P <0.05).

[0055] Depend on Figure 1 It can be seen that under different concentrations of dye infection, there are differences in the growth of the labeled group and the unlabeled group, but the trends of the two are consistent. The lag phase is about 3 hours, and the logarithmic phase is between 3 and 9 hours. When the final concentration of the dye is less than or equal to 900 μmol / L, the bacterial density is slightly lower than that of the unlabeled group, but there is no significant difference ( P >0.05).

[0056] As shown in Table 1, when the final dye concentration was greater than 900 μmol / L, the maximum specific growth rate decreased. When the dye concentration reached 1500 μmol / L, the maximum specific growth rate was (0.881 ± 0.011) h -1 , which was significantly lower than that of the unlabeled group (0.960±0.010) h -1 In summary, higher concentrations of dyes can affect bacterial growth. The bacterial density and maximum specific growth rate show a downward trend with increasing dye concentrations. When the final concentration is greater than 900 μmol / L, the bacterial density and maximum specific growth rate begin to be significantly lower than those of the unlabeled group ( P <0.05).

[0057] The labeling rate of CFDA-SE on Bifidobacterium breve B2798 bacteria was detected by flow cytometry. It was found that the labeling rate of the bacteria at each concentration of the dye reached more than 99.0%. However, the bacterial labeling rate of the 300 μmol / L dye concentration test group decreased to 86.7% when the culture reached the stable period, indicating that the culture under this concentration condition would result in insufficient concentration, resulting in a large deviation in the division number measurement.

[0058] (2) Effect of dye labeling on the acid production capacity of bacteria: bacteria without dye labeling were set as the control group, and bacteria infected with different dye concentrations were set as the experimental group. Samples were taken at regular intervals during the fermentation process, and the acid production of bacteria during the fermentation process was measured using an M-online biochemical analyzer to evaluate the degree of influence of fluorescent dye infection on the metabolic capacity of bacteria.

[0059] Effect of dye concentration on acid production capacity of Bifidobacterium breve B2798 Figure 2 The effect of dye concentration on the lactic acid change rate of Bifidobacterium breve B2798 is shown in Table 2:

[0060] Table 2 Effect of dye concentration on the lactic acid change rate of Bifidobacterium breve B2798

[0061] ;

[0062] Note: Different lowercase letters in the table indicate significant differences between different groups of data in the same column ( P <0.05).

[0063] Depend on Figure 2 As shown in Table 2, after infection with different dye concentrations, the acid production trends of the experimental group and the control group were consistent. As the dye concentration increased, the acid production of the bacteria was inhibited. When the final dye concentration was 900 μmol / L, the accumulated lactic acid amount in the stable period was 10.21 g / L, and the lactic acid change rate was (1.789 ± 0.026) g / L·h -1 When the final concentration of the dye was ≤900 μmol / L, the cumulative amount of lactic acid in each test group at the end of fermentation was not significantly different from that of the unlabeled group (10.32 g / L). P >0.05); when the final dye concentration was >900 μmol / L, the acid production rate and lactate accumulation were significantly lower than those in the unlabeled group ( P The effects of different dye concentrations on the acid production capacity of the bacteria were comprehensively evaluated. After dye labeling with a final concentration of 900 μmol / L, the accumulated acid and the acid production rate of the bacteria were slightly lower than those in the unlabeled group, but there was no significant difference. This indicates that lower dye concentration labeling has no significant inhibitory effect on the acid production capacity of Bifidobacterium breve B2798.

[0064] (3) Fluorescent dye toxicity test: Bacteria treated with different concentrations of dye were used as the experimental group, and unstained bacteria were used as the control group. During the high-density culture process, samples were collected at regular intervals, and the number of viable bacteria in the culture medium at each time point was determined by the plate count method. The plate culture was poured using solid modified MRS medium (containing 0.5 g / L L-cysteine ​​hydrochloride) and inverted anaerobically cultured at 37°C for 72 hours. Three biological replicates were set for each sample, and the unit of viable bacteria count was CFU / mL. Finally, the cytotoxicity of the dye to Bifidobacterium breve B2798 bacteria was evaluated by statistically comparing the number of viable bacteria between the groups.

[0065] The results are as follows Figure 3 As shown by Figure 3 It can be seen that there is no significant difference in the number of viable bacteria in each experimental group during the lag period after inoculation ( P >0.05); at the mid-logarithmic stage, the number of viable bacteria in the experimental group with a final dye concentration of ≤900 μmol / L was lower than that in the unlabeled group, but there was no significant difference ( P >0.05), when the final concentration of the dye was >900 μmol / L, the number of viable bacteria began to decrease. When the final concentration of the dye was 1500 μmol / L, the number of viable bacteria was (1.97±0.17)×10 9 The CFU / mL was significantly reduced by 35.62% compared with the unlabeled group (P<0.05). At the stable stage, the number of viable bacteria was (8.72±0.41)×10-1 at the final concentration of the dye of 1500μmol / L. 9 CFU / mL, significantly decreased by 13.41% compared with the unlabeled group ( P <0.05). When the final concentration of the dye was 900 μmol / L, the viable bacterial counts in the mid-logarithmic phase and the stationary phase were (2.57±0.31)×10 9 CFU / mL, (9.63±0.49)×10 9 CFU / mL. Although slightly lower than the unlabeled group, there was no significant difference ( P >0.05), indicating that when the final concentration of the dye is 900 μmol / L, it has little effect on bacterial proliferation and metabolism, and has no obvious inhibitory effect on the bacterial culture process.

[0066] (4) Fluorescent dye labeling stability test: The cultured bacterial solution was inoculated into sterile PBS buffer to simulate an environment where the bacteria do not divide or die. Under this condition, the stability of CFDA-SE labeling (the final concentration of the dye is 900 μmol / L) was evaluated. The labeled group was used as the experimental group, and the unlabeled group was used as the control group. The stable bacteria were stored in the dark for 12.0 hours under the above simulated environment. First, the viable bacteria were counted to evaluate the effect of the simulated environment on the activity of the bacteria during storage. Then 1.0 mL of the stored sample was taken into a 1.5 mL light-proof EP tube and tested on the machine. Flow cytometry was used, and the excitation wavelength was set to E x=494nm, emission wavelength E m = 521 nm, collection (1 × 10 4 ) event data points were processed and analyzed using FlowJo 10.9.0 software to assess changes in fluorescence intensity during storage. The effect of CFDA-SE dye labeling on the viable count of Bifidobacterium breve B2798 during storage is shown in Table 3:

[0067] Table 3 Effect of CFDA-SE dye labeling on the viable count of Bifidobacterium breve B2798 during storage

[0068] ;

[0069] Note: The same lowercase letters in the table indicate that the differences between different groups in the same column of data are not significant ( P >0.05).

[0070] As shown in Table 3, after storage for 12 h, the number of viable bacteria fluctuated, but there was no significant difference in the number of viable bacteria ( P >0.05). This indicates that the system has no significant effect on bacterial activity and can meet the requirements of dye stability test.

[0071] Fluorescence intensity changes of CFDA-SE dye-labeled Bifidobacterium breve B2798 during storage Figure 4 As shown in the figure, the left red area is the peak graph of the unlabeled bacteria, and the green area is the peak graph of the labeled bacteria. If the peak graph is offset, it means that the bacteria are continuing to divide. In this experimental result, the average fluorescence intensity of the labeled bacteria at 0h is 5.78×10 4 The average fluorescence intensity at 12 h was 5.54×10 4 , the fluorescence intensity decreased slightly, but the shift peak did not shift significantly with the increase of storage time.

[0072] In summary, the experiment concluded that CFDA-SE dye with a final concentration of 900 μmol / L had good stability after labeling Bifidobacterium breve B2798 and met the experimental requirements, so CFDA-SE dye with a final concentration of 900 μmol / L was finally selected for the study.

[0073] Example 2

[0074] Effect of pH on bacterial growth

[0075] (1) During the high-density fermentation of Bifidobacterium breve B2798, samples were taken at regular intervals and the number of viable bacteria was determined by plate count. The fermentation broth samples were diluted with sterile saline until the appropriate concentration was reached (the original resuspended sample was loaded at the beginning of the fermentation, and the sample was diluted to 10 in the middle and late stages). -1 -10 -2, the dilution multiple in the experiment depends on the specific sample concentration). Use a pipette to draw 1 mL of bacterial suspension and inject it into a sterile culture dish. Then, use the pouring method to pour an appropriate amount of solid modified MRS culture medium into the culture dish. After shaking and solidifying, place it in an inverted culture environment at 37.0℃ for 72.0 hours. Set up three replicates for each sample, and the unit of viable bacteria count is CFU / mL.

[0076] (2) During the high-density fermentation process, samples of Bifidobacterium breve B2798 were taken at regular intervals and diluted to an appropriate concentration (0.20≤A=600nm≤0.80). 200 μL of fermentation liquid was aspirated using a pipette to determine its absorbance (A=600nm). Three replicates were set for each sample, and the measured value was multiplied by the dilution factor to obtain the change in the biomass of Bifidobacterium breve B2798 during the fermentation process.

[0077] (3) Based on the growth curve drawn from the bacterial density (A = 600 nm), the maximum specific growth rate of Bifidobacterium breve B2798 under different fermentation system pH conditions was calculated using the SLogistic2 equation in Origin. The calculation formula is:

[0078] ;

[0079] in, For cultivation time, y is the bacterial density, y 0 is the initial bacterial density, is the maximum bacterial density, is the maximum specific growth rate.

[0080] like Figure 5 As shown, A is a graph showing changes in bacterial density under different fermentation system pH conditions; B is a graph showing changes in viable bacterial count under different fermentation system pH conditions; and C is a graph showing the maximum specific growth rate under different fermentation system pH conditions.

[0081] Figure 5 The results showed that with the decrease of pH value in the fermentation system, the cell density, viable count and maximum specific growth rate of Bifidobacterium breve B2798 showed a trend of first increasing and then decreasing; there was no significant difference in the viable count during the lag phase among the experimental groups ( P >0.05), the difference in viable bacterial count increased significantly in the logarithmic phase. At the end of the logarithmic phase, the viable bacterial count in the fermentation system pH 5.30 test group was (1.39±0.04)×10 10 CFU / mL, significantly higher than those in other experimental groups ( P <0.05); the maximum specific growth rate of the fermentation system pH 5.30 test group was (0.94±0.01)h -1 , which was significantly greater than that of the other experimental groups ( P<0.05), there was no significant difference in the maximum specific growth rate between the pH 4.30 and pH 4.80 experimental groups and between the pH 6.30 and pH 6.80 experimental groups ( P >0.05). Therefore, the fermentation system pH values ​​of 4.30, 5.30, and 6.30 were selected as experimental groups for subsequent experiments.

[0082] (4) During the high-density fermentation process, samples were taken regularly from Bifidobacterium breve B2798, and the bacterial precipitate was collected by centrifugation. The precipitate was then washed with sterile PBS solution, resuspended to the original volume with sterile triple-distilled water, and then subjected to ultrasonic disruption in an ice bath. The disruption conditions were: 100W, ultrasonic for 3 seconds, then stop for 6 seconds, and cycle for 600 seconds. The supernatant was then collected as the bacterial cell content by centrifugation at 10,000 rpm and 4°C for 5 minutes. After disruption, the intracellular F6PPK activity of Bifidobacterium breve B2798 was detected according to the instructions of the F6PPK enzyme activity detection kit. The enzyme activity unit was IU. Three groups of biological parallel samples were set for data analysis.

[0083] Effect of pH on F6PPK activity during the culture of Bifidobacterium breve B2798 Figure 6 As shown, Figure 6 The F6PPK activity of each experimental group showed the same trend during the culture process. There was no significant difference in enzyme activity at the beginning of the logarithmic growth phase. The enzyme activity gradually increased during the logarithmic growth phase and reached its highest level at the end of the logarithmic growth phase. When the stable phase was reached, the enzyme activity decreased and the bacterial metabolic activity decreased. F6PPK activity increased with decreasing pH. At the end of the logarithmic growth phase, the enzyme activity was (24.86±0.66) IU / 10 4 cells, compared with pH 6.30 enzyme activity (10.76±0.55) IU / 10 4 cells significantly increased by 131.04%, and the enzyme activity in the pH 4.30 test group was (3.15±0.36) IU / 10 4 cells, significantly downregulated by 87.33% compared with pH 5.30 ( P <0.05). This is because when the pH is lower than the pKa of the organic acid, the organic acid produced by the bacteria exists in the form of undissociated molecules, diffuses into the cell and releases H + It destroys the acid balance inside and outside the cell, causing damage to the cell and affecting bacterial proliferation and metabolism.

[0084] (5) Same as the test method in (4) above, after crushing, refer to H + -ATPase enzyme activity detection kit instructions for detecting intracellular H + -ATPase activity, enzyme activity unit is U, set up three groups of biological parallel samples for data analysis, the results are as follows Figure 7 shown.

[0085] Depend on Figure 7 It can be seen that when the pH of the fermentation system decreases, the activity of the bacteria gradually increases. When the pH is 4.30, the H + -ATPase activity has been maintained at a high level since the mid-log phase (5.89~6.15U / 10 4 cells), and there was no significant difference in activity between the mid-logarithmic phase and the late-logarithmic phase ( P >0.05), to the stable period H + -ATPase activity began to decrease significantly ( P <0.05). Under pH 6.30 and pH 5.30 conditions, the H + -ATPase activity showed a gradual upward trend, with the initial activity lower than that of the pH 4.30 test group and close to the activity under pH 4.30 conditions at the end of the logarithmic phase ( P >0.05), this phenomenon is related to the activation of bacterial H + -ATPase enzyme activity, in the stable period, the bacterial metabolic capacity decreases, H + -ATPase enzyme activity was significantly reduced.

[0086] Different pH values ​​will affect the efficiency of bacterial ATP and intermediate product production, and the synthesis of substances required for division will also be affected, resulting in the inhibition of bacterial division and proliferation. + -When the ATPase enzyme activity is higher, a large amount of ATP will be consumed. Under low pH conditions, the activity of bacterial F6PPK decreases, bacterial metabolic activity is inhibited, and ATP production decreases, resulting in a decrease in the energy supply for bacterial division, which inhibits bacterial division and proliferation.

[0087] (6) During the fermentation process, samples were taken regularly, 1 mL of fermentation liquid was aspirated, and the bacterial sludge was collected by centrifugation. The precipitate was washed with sterile PBS solution and resuspended to the original volume. The bacterial suspension was applied to a glass slide using an inoculation loop. After staining and fixing with crystal violet solution, the suspension was detected and photographed using an optical microscope BX-53. The area of ​​the bacteria was identified and marked using Image View software. Origin2021 software was used to analyze and fit the distribution of bacterial area at each stage under different pH conditions. The effect of different fermentation system pH on the bacterial size of Bifidobacterium breve B2798 during high-density culture was analyzed, and the relationship between bacterial division and proliferation and bacterial size was quantified.

[0088] The results are as follows Figure 8 As shown in Figure 2, the average bacterial area of ​​Bifidobacterium breve B2798 at each culture period under different pH conditions showed a normal distribution through Gaussian function fitting. At the beginning of the logarithm, the bacterial area of ​​the three experimental groups was maintained at 0.87~0.91μm2 There was no significant difference between the two groups; at pH 4.30, the bacterial area was maintained at 0.83~0.96μm 2 There was no significant change in the bacterial area during each culture period ( P >0.05); at pH 5.30, the bacterial area was maintained at 0.91~1.65μm 2 The bacterial area at the middle logarithmic stage increased significantly by 81.32% compared with the early logarithmic stage ( P <0.05), and at the end of the logarithmic phase, the bacterial area began to decrease, decreasing by 17.58% compared with the middle logarithmic phase, but still significantly higher than the early logarithmic phase ( P <0.05); at pH 6.30, the bacterial area was maintained at 0.87~1.37μm 2 The bacterial area at the mid-logarithmic stage increased significantly by 57.47% compared with the early logarithmic stage ( P <0.05), and at the end of the logarithmic phase, the bacterial area began to decrease, down 13.24% from the middle logarithmic phase, but still significantly higher than the early logarithmic phase ( P <0.05).

[0089] Combine Figure 5 The results showed that the pH 5.30 experimental group significantly promoted the growth of Bifidobacterium breve B2798 compared with the pH 6.30 and pH 4.30 experimental groups, indicating that a suitable acidic environment helps enhance bacterial expansion before cell division, resulting in a greater increase in bacterial area. Low pH affects the activity of enzymes involved in peptidoglycan synthesis (such as transpeptidase FtsI), hindering the synthesis of cell wall components and thus inhibiting bacterial expansion. Low pH also changes the degree of peptidoglycan cross-linking, increasing cell wall rigidity and preventing normal bacterial growth. Furthermore, low pH affects the expression and localization of division proteins (such as FtsZ), further inhibiting normal bacterial expansion. Therefore, at pH 4.30, the average bacterial area of ​​Bifidobacterium breve B2798 did not change significantly during cultivation.

[0090] (7) After centrifugation of the sample to be tested, the cells were collected (1000×g, 5 min, 4°C), the culture medium was removed, and the cells were resuspended in PBS buffer to a concentration of approximately 1×10 6 CFU / mL, using PI and Syto TM 9 Double staining combined with flow cytometry to assess cell membrane integrity. Figure 9 shown.

[0091] PI staining of the pH 4.30 experimental group at the early logarithmic stage showed a membrane damage rate of 28.54%, which was high. It is speculated that the low pH inhibits protein function, affecting transmembrane transport and membrane stability, resulting in a high cell membrane damage rate. At the mid-logarithmic stage, PI staining showed that the membrane damage rate accounted for 31.36%. Although some bacteria were still proliferating, the overall growth rate was significantly lower than that of the pH 5.30 and pH 6.30 experimental groups ( Figure 5 Middle C, P <0.05), H + -ATPase activity is at a high level ( Figure 7 ), but the degree of membrane damage continued to increase, presumably because the proton gradient of the bacteria was difficult to maintain under acid stress, resulting in impaired energy metabolism and ultimately an increase in the degree of membrane damage; at the end of the logarithmic phase, due to continuous acid stress, a large number of bacteria suffered membrane damage, and the membrane damage rate increased to 69.47%, and most bacteria entered the VBNC state; during the stable phase, membrane damage was most serious, with a high cell mortality rate. PI staining showed that the membrane damage rate reached 92.20%, a large number of bacteria died due to membrane damage, and some bacteria were in the VBNC state.

[0092] In the pH 5.30 experimental group, PI staining results showed that the proportion of membrane-damaged bacteria was 10.83% at the early logarithmic stage, which was at a low level. At this time, the bacteria began to proliferate and the cell membrane integrity was good. In the middle logarithmic stage, PI staining results showed that the proportion of membrane-intact bacteria was the highest (90.10%) and the proportion of damaged bacteria was the lowest (6.36%). At this time, the proton pump activity was high ( Figure 7 ), the cell proliferation rate is the fastest ( Figure 5 Middle C), presumably due to the optimal state of membrane phospholipids and proteins at this stage, enhancing membrane stability. At the end of the logarithmic phase, due to active metabolism, some cells began to age, and membrane damage increased. PI staining revealed an increase in the percentage of cells with damaged membranes (15.84%). The degree of membrane damage increased slightly at this stage, but was still lower than that in the pH 6.30 and pH 4.30 experimental groups. At the stationary phase, due to nutrient depletion and weakened metabolism, some cells entered the VBNC state, resulting in a significant increase in membrane damage (54.10%), but still lower than in the other experimental groups.

[0093] The membrane integrity of the pH 6.30 test group was higher (16.21%) at the beginning of the logarithmic period, close to that of the pH 5.30 test group, but the proton pump activity was lower than that of the pH 5.30 test group ( Figure 7 ), the proton gradient was established slowly; in the mid-logarithmic phase, the bacteria began to proliferate rapidly, and the membrane damage rate was 17.63%, slightly lower than that of the pH 5.30 test group. At this time, the metabolic capacity of the bacteria in the pH 6.30 test group ( Figure 6 ), proton pump activity ( Figure 7 ) and bacterial growth rate ( Figure 5The test group below pH 5.30 may cause the synthesis of cell membrane phospholipids to be inhibited, the transmembrane potential to be reduced, and the membrane stability to be affected; at the late logarithmic phase, due to the gradual depletion of nutrients, the cells enter a decline state, and the membrane damage significantly increases P <0.05), and PI staining showed that the ratio of membrane-damaged cells was 39.10%; at the stationary phase, the lack of nutrients caused a large number of cells to enter the VBNC state, and part of the cells died, and PI staining showed that the membrane damage ratio (56.75%) significantly increased P <0.05).

[0094] In summary, the membrane damage of the pH 4.30 test group was the most serious throughout the fermentation process, and the cell viability was the lowest, and a large number of cells died at the stationary phase, indicating that acid stress caused significant damage to the cell membrane of B. breve B2798. In contrast, the membrane damage of the pH 5.30 test group was lower, and the cell survival rate was higher, which was beneficial to the growth and metabolism of the cells. The pH 6.30 test group maintained good membrane integrity at the logarithmic growth phase, but at the late logarithmic phase and the stationary phase, the membrane damage was significantly higher than that of the pH 5.30 test group P <0.05), and a high proportion of cells entered the VBNC state, which may be due to the weak regulation of membrane fatty acid metabolism under the condition of pH 6.30, resulting in a decrease in membrane stability.

[0095] Example Three

[0096] Effect of pH on the cell division of B. breve B2798.

[0097] According to the staining method in Example One, the optimal CFDA-SE concentration selected in Example One was used to label the B. breve B2798 cells, and the CFDA-SE fluorescently labeled cell suspension was used as the seed liquid. The volume ratio of the seed liquid to the fermentation medium was 2% (v / v), and three parallel tanks were set for each pH test group. A 25% NaOH solution was used to uniformly adjust the initial pH to 6.50, and natural fermentation was carried out until the pH reached 6.30, 5.30, and 4.30. Then, a 25% NaOH solution was added to control the pH, and the fermentation temperature was set to 37°C. Nitrogen was supplied for high-density fermentation.

[0098] Division cycle detection:

[0099] During the high-density fermentation process, samples were taken at regular intervals, placed on ice, and then centrifuged. The supernatant was discarded, and the sample was resuspended to the original volume with sterile PBS solution. The cell concentration was adjusted to 10 6 ~10 7CFU / mL, after vortex mixing, use a pipette to transfer 1.0mL to a 1.5mL light-proof EP tube for detection. Flow cytometry was used to detect the bacterial division cycle. The flow cytometer was set to excitation wavelength Ex = 494nm and emission wavelength Em = 521nm for detection. 1×10 4 ) events data and fit the deconvoluted peaks.

[0100] Split cycle detection:

[0101] The viable cell count method in Example 2 was used to detect the viable cell count obtained during the fermentation process, and the division number was calculated using the following formula:

[0102] ;

[0103] in, N t : number of viable bacteria at time t, unit: CFU / mL; N 0 : Initial viable bacterial count, unit: CFU / mL.

[0104] like Figure 10 As shown, the black curve represents the unfitted fluorescence distribution histogram. After fitting, green peaks are used to identify different generations. The accuracy of the fitted curve is determined by the root mean square error (RMSE). For cell proliferation fitting, an RMSE ≤ 0.5 is considered valid. The RMSE values ​​of the deconvolution peak fitting under all three pH conditions were less than 0.5, indicating a valid fit. Post-incubation testing revealed fluorescence shifts in all experimental groups, indicating bacterial proliferation.

[0105] The division number and division cycle of Bifidobacterium breve B2798 under different fermentation system pH conditions are shown in Table 4 below:

[0106] Table 4 The division number and division cycle of Bifidobacterium breve B2798 under different fermentation system pH conditions

[0107] ;

[0108] Note: Different lowercase letters in the table indicate significant differences between different groups of data in the same column ( P <0.05).

[0109] Quantitative analysis of each generation population, combined with the fitted deconvolution peak, revealed that the decrease in the "Generation 0" population and the increase in the populations of other generations indicate that the bacterial division process is ongoing, and the population sizes of each generation also exhibit dynamic changes. According to the data in Table 4, at a pH of 6.30, the bacterial divisions were 5.24 ± 0.04, with a Flowjo fit of 5; at a pH of 5.30, the bacterial divisions were 6.87 ± 0.11, with a Flowjo fit of 7; and at a pH of 4.30, the bacterial divisions were 3.23 ± 0.03, with a Flowjo fit of 3. The number of divisions calculated from the viable cell count is essentially consistent with that obtained by Flowjo fitting, indicating a good fit. While viable cell counts require approximately (3.0 ± 0.5) days to obtain results, the detection method in this example can measure the number of divisions in real time, enabling precise control of the fermentation process.

[0110] Example 4

[0111] The absolute quantification method was used to accurately quantify the expression levels of division-related genes during the fermentation process.

[0112] (1) Annotation of key genes for splitting:

[0113] The genome sequence of Bifidobacterium breve B2798 was obtained from the iLABab (https: / / www.imhpc.com / iLABdb) lactic acid bacteria genome database. The genome of Bifidobacterium breve B2798 was annotated using Prokka software and the Rapid Annotation using Subsystem Technology (RAST) server (http: / / rast.nmpdr.org / rast.cgi). The Prokka-generated annotation file was retrieved to extract gene IDs and functional descriptions, and genes related to bacterial division were screened.

[0114] The key genes for division of Bifidobacterium breve B2798 are shown in Table 5:

[0115] Table 5 Key genes for division of Bifidobacterium breve B2798

[0116] ;

[0117] As shown in Table 5, a total of 8 functional genes related to divisome formation, membrane synthesis and separation, and division timing control were obtained, among which: ftsThe SepF family genes are a group of genes that play a core role in the bacterial division process. They encode a variety of key division proteins and participate in the assembly of cell division bodies, cell wall synthesis and final division. SepF protein directly affects the division of bacteria by enhancing the polymerization of FtsZ protein, stabilizing the Z ring and promoting the assembly of other division proteins. Fts Localization and activity of family proteins at the cleavage site; WhiA protein acts as a transcriptional regulator by regulating Fts The expression of family proteins affects the timing of bacterial division and the development process; the synergistic effect of SepF and WhiA ensures the stability of the Z ring and the timely expression of Fts family proteins, ensuring the smooth progress of cell division and development.

[0118] (2) Based on the complete genome information of Bifidobacterium breve B2798, specific primers were designed for the key division genes retrieved through annotation. The results are shown in Table 6.

[0119] Table 6 Specific primer information for key genes involved in cleavage

[0120] ;

[0121] (3) Extraction of bacterial RNA.

[0122] S1. Pipette 1.0 mL of sample into a 1.5 mL EP tube, centrifuge (4°C, 12,000 rpm, 5 min), and discard the supernatant.

[0123] S2. Add an appropriate amount of TE buffer containing lysozyme to the EP tube and resuspend. Incubate at room temperature for 6 minutes to break the cell wall.

[0124] S3. Add 1.0 mL of Trizol to the EP tube treated above, shake and mix thoroughly, and let it stand at room temperature for 10 minutes.

[0125] S4. Add 200 μL of chloroform to each EP tube after the above treatment, shake and mix, let it stand for 10 minutes, and then centrifuge (4°C, 12000 rpm, 15 minutes).

[0126] S5. Take 400 μL of supernatant, add 600 μL of pre-cooled isopropanol, shake to mix, let stand at -20℃ for 1 hour, and then centrifuge (4℃, 12000 rpm, 15 min).

[0127] S6. Add 1 mL of pre-cooled 75% alcohol for washing, then centrifuge (4°C, 12,000 rpm, 10 min), discard the supernatant, and air dry naturally (about 5 min).

[0128] S7. Add 30 μL of Nuclease-free to the EP tube after the above treatment and dissolve it.

[0129] (4) Reverse transcription.

[0130] S1, take 1.5 mL EP tube, take 1 μL Totl RNA, 2 μL Primer Mix, 0.2 μL 10 mM RT, 4 μL 2.5 mM dNTP, and finally add double distilled water to 13 μL.

[0131] S2, after mixing the above solution, water bath at 70°C for 10 min, and then ice bath for 2 min.

[0132] S3, after the above treatment, add 4 μL 5x First-Strand Buffer, 0.1 μL 0.1 M DTT, 1 μL 200 U / μL HiFiScript M-MLV to the EP tube.

[0133] S4, continue reverse transcription program: 42°C, 50 min; 85°C, 5 min.

[0134] (5) Standard curve establishment

[0135] S1, PCR product ligation system: add the materials in Table 7 to 0.2 mL EP tube at one time, mix immediately, and connect in PCR instrument for 2-3 h (16°C).

[0136] Table 7 PCR product ligation system

[0137] ;

[0138] S2, heat shock of competent cells: take the competent cells stored in -80°C refrigerator and place on ice until completely dissolved; take 5 μL ligation solution and add to 50 μL competent cells, ice bath for 30 min; then add 1 mL SOC liquid medium, incubate in 37°C constant temperature shaker at 250 rpm for 50 min; then pour sterile LB solid medium containing ampicillin into a sterile petri dish, add 60 μ-gal, evenly smear with sterile coating rod; finally centrifuge the activated bacterial solution (5000 rpm, 10 min, 4°C), use a pipette to remove 800 μL supernatant and discard, add 20 μL IPTG to each tube, resuspend, and evenly smear the bacterial suspension on the solidified LB solid medium using the coating method, and incubate in a 37°C constant temperature incubator overnight.

[0139] S3, prepare standard curve samples: first, sequence the constructed plasmid to ensure accuracy, then use a ultramicro visible light ultraviolet spectrophotometer to measure the OD value of the plasmid, as shown in Table 8, and convert it to copy number (copies / μL) by the following formula. 260nm

[0140] ​ ;

[0141] N A is Avogadro's constant, 6.023 x 10 23 ; C is concentration, ng / μL; molecular weight: target gene size vector size.

[0142] Table 8 Plasmid OD 260nm value

[0143] ;

[0144] S4, standard curve construction: 10-fold gradient dilution of the prepared plasmid, 10~10 5 dilution was used to prepare the standard curve.

[0145] (6) RNA quality inspection, as shown in the figure, M represents DL2000 DNA Marker; A is pH 6.30; B is pH 5.30; C is pH 4.30; CK: negative control. The results show that the amplification bands of each experimental group are clear and continuous, indicating that the specific primers have good specificity and no non-specific amplification, which can be used for subsequent quantitative analysis. Figure 11

[0146] (7) Reaction system configuration: take 2 mL EP tube, add 10 μL 2 x UltraSYBR Mixture (2 x), 0.25 μL 5 μM Primer F and Primer R, 2 μL cDNA, and use sterile enzyme-free double distilled water to make up to 20 μL.

[0147] (8) qPCR procedure: the cycle stage includes denaturation, annealing and extension. The cycle number is 35 times.

[0148] S1, initial denaturation: 95℃, 5min;

[0149] S2, denaturation: 95℃, 10s;

[0150] S3, annealing: 60℃, 30s;

[0151] S4, extension: 72℃, 30s;

[0152] S5, fluorescence signal collection: at the end of each cycle, collect fluorescence signal at the extension stage.

[0153] (9) Result analysis:

[0154] ① Split gene absolute quantitative verification:

[0155] The absolute quantitative standard curve is shown in Table 9:

[0156] ​Table 9 Standard curve

[0157] ;

[0158] As can be seen from Table 9, the standard curve constructed in this example is of high quality and shows a good linear relationship (R 2 ≥0.99), indicating a high correlation between Ct values ​​and the logarithmic copy number of the standard, ensuring accurate quantification. Furthermore, the slopes of the standard curves were close to -0.30, demonstrating that the qPCR reaction conditions were properly optimized and the amplification efficiency was near ideal. Furthermore, the standard dilution gradient covered the entire sample Ct value range, ensuring reliable copy number estimation for unknown samples. Overall, the standard curve was well constructed, ensuring accurate quantification of the target gene.

[0159] Absolute quantification of division-related genes Figure 12 As shown, fts Z-1, 2, 3, 4, and 5 represent the gene expression levels of the experimental groups corresponding to the early logarithmic phase, mid-logarithmic phase, late logarithmic phase, stable phase, and decay phase; fts X-1, 2, 3, 4, 5, fts L-1, 2, 3, 4, 5, fts I-1, 2, 3, 4, 5, fts W-1, 2, 3, 4, 5, fts Q-1, 2, 3, 4, 5, sep F-1, 2, 3, 4, 5, whi A-1, 2, 3, 4, 5 are the same as above.

[0160] The results showed that the expression patterns of division-related genes were significantly different under different pH conditions and growth periods. Combined with the division cycle detection results under different pH conditions, it was found that the overall expression of division-related genes was the highest under pH 5.30, the division activity was the strongest, and the number of bacterial divisions was significantly higher than that of other experimental groups ( P <0.05); pH 6.30 is a neutral environment, and the expression of division genes is suppressed, resulting in fewer divisions than the pH 5.30 test group. Under pH 4.30 conditions, due to long-term acid stress, gene expression at all stages was significantly suppressed ( P <0.05), with poor division activity and the lowest division number. After the end of the logarithmic phase, the expression of division-related genes approached the detection limit, the bacteria no longer grew, and the population distribution detected by flow cytometry was fixed at the last generation population.

[0161] ③Effect of gene expression on bacterial size:

[0162] The relationship between bacterial size and division gene expression is as follows Figure 13 As shown, fts Z-1, 2, and 3 represent the expression levels at the early, middle, and late logarithmic stages, respectively;fts L-1, 2, 3, ftsI-1, 2, 3, sep F-1, 2, 3, whi The results showed that the changes in the cell size of Bifidobacterium breve B2798 under different pH conditions were closely related to the expression levels of key genes for division.

[0163] In the early logarithmic phase, cells are about to enter a rapid proliferation phase, and the expression levels of division-related genes increase. fts Z. sep F. whi A high expression ( P <0.05), indicating that this environment promotes the formation of the Z ring and the stability of the division complex, which is beneficial to the expansion of pre-mitotic cells. fts I and fts L also maintains high expression, promoting cell wall synthesis; under pH 6.30 fts Z and sep The expression levels of F were (1.15±0.10)×10 6 copies and (5.49±0.49)×10 5 The pH value of the experimental group was slightly lower than 5.30, which may be due to the fact that the alkaline environment affected the assembly efficiency of the Z ring, resulting in lower cell division activity; the pH 4.30 experimental group fts The expression levels of Z and its regulatory genes were significantly lower than those in the pH 5.30 and pH 6.30 groups ( P <0.05), indicating that acid stress inhibited the polymerization of the Z ring, leading to the obstruction of the fission complex formation.

[0164] At the mid-logarithmic stage, the cell proliferation rate reached its peak, and the bacterial size of the pH 5.30 and pH 6.30 experimental groups was the largest, and the pH 5.30 group was significantly larger than the pH 6.30 group ( P <0.05), reflecting its stronger splitting activity. Quantitative analysis showed that the pH 5.30 group fts Z. fts I. fts L. sep F. whi A expression levels reached peak values, especially fts Z (4.74 ± 0.09) × 10 6 copies, fts I (7.84 ± 1.16) × 10 6 copies, fts L (3.43 ± 0.73) × 10 6 copies, and significantly increased by 1.76-fold, 1.31-fold, and 1.51-fold compared with the pH 6.30 group (P <0.05). fts I and fts The results of high gene expression of L indicated that pH 5.30 may provide the best catalytic environment for peptidoglycan synthesis, promote the rapid synthesis of cell wall, and thus promote bacterial division; although the division-related genes in the pH 6.30 group were still highly expressed, the overall expression level was lower than that in the pH 5.30 group, especially fts L (2.67 ± 0.20) × 10 6 copies, significantly decreased by 22.75% compared with the pH 5.30 test group ( P <0.05), indicating that the stability of bacterial division bodies and peptidoglycan synthesis were inhibited under this environment, and the division efficiency decreased; pH 4.30 group fts Z (1.12 ± 0.21) × 10 6 copies, fts I (3.53 ± 0.32) × 10 6 copies, sep F (1.12 ± 0.10) × 10 6 The number of copies was only 10%~50% of that in the pH 5.30 group, indicating that the acid stress environment significantly inhibited Z ring assembly and cell wall synthesis, resulting in the inability of cells to grow and expand normally before division and the small size of the bacteria.

[0165] At the end of the logarithmic phase, the cell growth rate decreased, the expression of division-related genes decreased, and the bacterial size decreased. fts Z (2.41 ± 0.32) × 10 6 copies and sep F (5.63 ± 0.35) × 10 5 copies decreased by 49.16% and 60.71% respectively compared with the mid-logarithmic period ( P <0.05), the bacterial size was significantly reduced ( P <0.05); pH 6.30 group fts I and fts Z decreased by 81.30% and 76.73% compared with the mid-logarithmic period ( P <0.05), the cell wall synthesis and schizont formation abilities were weakened, resulting in a decrease in bacterial size ( P <0.05); in the pH 4.30 group, the division genes maintained a low level, division stagnated, and the bacterial size decreased but did not change significantly ( P >0.05).

[0166] ④Effects of gene expression on bacterial membrane integrity:

[0167] The relationship between membrane integrity and gene expression Figure 14As shown, fts X-1, 2, and 3 represent the expression levels at the early, middle, and late logarithmic stages, respectively; fts W-1, 2, 3, fts Q-1, 2, and 3 are the same as above.

[0168] The results showed that in the early logarithmic period, the metabolism of Bifidobacterium breve B2798 cells gradually became active, but the division frequency was low and the membrane integrity remained at a high level. Flow cytometry showed that under pH 6.30 and pH 5.30 conditions, the membrane integrity remained above 80%, and fts X、 fts W. fts The expression level of Q gene is low, indicating that the cells are still in the stage of environmental adaptation and have not yet entered the rapid division stage. Under pH 4.30 conditions, the bacteria have not yet been subjected to strong acid stress, but the proportion of cells with intact membranes is only 71.46%. fts X、 fts W. fts Q expression was significantly lower than that at pH 5.30 and pH 6.30 ( P <0.05), indicating that low pH may have inhibited the activation of the division gene.

[0169] In the mid-logarithmic phase, the bacteria enter the rapid division phase, and the membrane integrity and division gene expression change. The membrane integrity of the pH 6.30 group remained at 82.39%, and fts X、 fts W. fts The expression of Q gene increased significantly by 4.01-fold, 3.67-fold and 2.45-fold in this stage (P<0.01), which was consistent with the increase of cell division activity. The membrane integrity of the pH 5.30 group was higher (93.64%), and fts X、 fts W. fts The expression of Q was also higher than that in the pH 6.30 group and significantly higher than that in the pH 4.30 group ( P <0.05), indicating that the neutral to slightly acidic environment can maintain good fission activity and membrane stability. In contrast, the membrane integrity of the pH 4.30 group continued to decline to 68.64%, fts X、 fts W. fts The expression level of Q was significantly lower than that of other groups (P<0.01), which may be because the low pH affected the role of the FtsX-WQ complex in peptidoglycan synthesis and transmembrane signal transduction, resulting in decreased division efficiency and an increase in the proportion of membrane-damaged cells.

[0170] At the end of the logarithmic phase, bacterial proliferation slowed down and the cell number tended to be stable. The membrane integrity of the pH 5.30 group decreased slightly (83.20%), but fts X、 ftsW. fts Q still maintained a high expression, supporting cell proliferation. The membrane integrity of the pH 6.30 group further decreased to 59.91%, and gene expression decreased simultaneously ( P <0.05), indicating that the long-term alkaline environment leads to the damage of membrane protein function, increased cell membrane permeability, and the membrane damage rate increases significantly at the end of the logarithmic phase ( P <0.05). The membrane integrity of the pH 4.30 group decreased most significantly (30.51%). fts X、 fts W. fts The expression of Q gene dropped to the lowest level, indicating that the accumulation of acid stress caused serious damage to the cell membrane, while the transcription of division genes was inhibited and the proliferation ability of the bacteria was seriously affected.

[0171] Therefore, the present invention provides a method for detecting bifidobacterium division activity based on pH regulation and its application, which significantly improves the bacterial division activity and the number of viable bacteria through real-time detection technology of bifidobacterium division cycle and pH dynamic regulation strategy, providing key technical support for the industrial high-density cultivation of bifidobacteria.

[0172] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for detecting bifidobacterium division activity based on pH regulation, characterized in that: include: Fluorescently labeled bifidobacteria were used as seed liquid, and the seed liquid was fermented at high density under pH control. The division cycle of bifidobacteria was detected by flow cytometry. The pH control range was 4.30-6.

30. The fluorescent labeling method of bifidobacteria comprises: applying a cell proliferation fluorescent probe CFDA-SE with a final concentration of 900 μmol / L to stain bifidobacteria; After high-density fermentation, the bacterial cells were collected and washed and resuspended to the original volume. The bacterial concentration was adjusted to 10 6 -10 7 CFU / mL was tested by flow cytometry; The flow cytometry was set to excitation wavelength Ex = 494 nm, emission wavelength Em = 521 nm, and 1 × 10 4 The bifidobacterial division cycle was analyzed by fitting the deconvolution peaks based on the events data points.

2. The method for detecting bifidobacterium division activity based on pH regulation according to claim 1, characterized in that: The pH control includes: inoculating the seed liquid into the bioreactor at an inoculum size of 2.0% for high-density fermentation, using a 25% mass fraction NaOH solution to adjust the initial fermentation pH to 6.50±0.02, naturally fermenting to the pH control range, and constantly controlling the pH by adding a 25% mass fraction NaOH solution.

3. An application of the method for detecting bifidobacterium fission activity based on pH regulation in bifidobacterium fermentation according to claim 1 or 2, characterized in that: The pH value with the shortest bifidobacterium division cycle and the largest number of bacterial division generations detected by flow cytometry within the pH control range is the optimal pH value for the bifidobacterium fermentation system. Cultivating bifidobacteria at the optimal pH value can increase the number of viable bacteria and division efficiency in bifidobacterium fermentation production.

4. Use of the method for detecting bifidobacterium division activity based on pH regulation as claimed in claim 1 or 2 in preparing a high-activity direct-injection starter of bifidobacteria.

5. Use of the method for detecting bifidobacterium division activity based on pH regulation according to claim 1 or 2 in the preparation of a probiotic solid beverage containing bifidobacteria.

6. A molecular marker for regulating the division activity of bifidobacteria, characterized by: Molecular markers include The molecular marker is obtained by combining the bifidobacterium division activity detection method based on pH regulation according to claim 1 or 2 with the division cycle, cell growth phenotype and expression characteristic analysis of division-related genes of bifidobacteria within the pH regulation range.

7. A molecular marker for regulating the division activity of bifidobacteria according to claim 6, characterized in that: Suitable fermentation pH and The expression is upregulated, and the stability of the cell wall and membrane structure is increased; when the fermentation pH is lower than the critical value, Down-regulation of expression inhibits the division process and prolongs the division cycle.

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