Culture method for improving freeze-drying stress resistance and storage stress resistance of bifidobacterium and application of culture method

Abstract

The invention discloses a culture method for improving freeze-drying stress resistance and storage stress resistance of bifidobacterium and application of the culture method, and belongs to the technical field of microorganism culture. The method comprises the following steps: firstly, establishing a freeze-drying model of the pH dynamic regulation thalli for evaluating the influence of freeze-drying and storage activity of the thalli; based on the established freeze drying model of the pH dynamic regulation thalli, better culture conditions before freeze drying and better temperature during storage of the bifidobacterium are screened. According to the culture method for improving freeze-drying stress resistance and storage stress resistance of bifidobacteria and the application of the culture method, by regulating and controlling the active state of cultured thalli, the later-stage drying and storage stress resistance of strains is improved, and a foundation is provided for precise culture of probiotics and processing of active preparations.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial culture, and in particular to a culture method and application thereof for improving freeze-drying stress resistance and storage stress resistance of bifidobacteria. Background Art

[0002] Vacuum freeze-drying technology is a commonly used method for producing probiotic preparations. Since bacterial cells are extremely susceptible to damage or even death during the freeze-drying process, the survival rate, cell activity, cell membrane function and intracellular enzyme activity are reduced after freeze-drying. Therefore, the pH is adjusted during the fermentation process to reduce the degree of cell damage. Therefore, pH control is an important means and basic link in the production and processing of probiotics, aiming to improve the freeze-drying activity and storage resistance of bacteria through appropriate control.

[0003] Dynamic pH control technology has the characteristics of fast response and high precision. Compared with the process optimization of traditional vacuum freeze-drying and the optimization of freeze-drying protectants, it can greatly improve the freeze-drying activity and storage resistance of Bifidobacterium. Summary of the Invention

[0004] The purpose of the present invention is to provide a culture method and application thereof for improving the freeze-drying resistance and storage resistance of bifidobacteria, and to develop bifidobacteria with higher bacterial activity and storage resistance through dynamic pH control culture.

[0005] To achieve the above object, the present invention provides a method for cultivating bifidobacteria to improve freeze-drying resistance and storage resistance, comprising the following steps: S1. Cultivate bifidobacteria by dynamically controlling pH, freeze-dry bacteria at different pH values and growth stages, and establish a freeze-drying model for bacteria with dynamic pH control. S2. Analyze the bacterial cell damage in the pH dynamic control freeze-drying model of bacteria established in S1, select bacteria with the lowest bacterial cell damage, and record the culture pH value and growth period of the bacteria with the lowest bacterial cell damage; S3. Storing the freeze-drying model of the bacteria with dynamic pH control established in S1 at different temperatures, analyzing the stability of the freeze-drying model of the bacteria with dynamic pH control during storage, and selecting the storage temperature with the highest stability during storage; S4, the culture pH and growth period obtained by S2 are used as the optimal culture conditions before freeze-drying of bifidobacteria, and the storage temperature obtained by S3 is used as the optimal storage temperature of bifidobacteria; Cultivating bifidobacteria under optimal culture conditions can improve their freeze-drying resistance and storage resistance. Or the freeze-dried powder of bifidobacteria cultured under optimal culture conditions can improve the storage stress resistance of bifidobacteria at the optimal storage temperature.

[0006] Preferably, in S1, the dynamic pH regulation culture comprises: The activated third-generation seed liquid of Bifidobacterium was inoculated and anaerobically fermented at a constant temperature of 37.0℃. The fermentation was carried out naturally to a pH of 4.30-6.30. A 25% mass concentration NaOH solution was added for feeding and the pH was constantly controlled at different levels. The cultures were cultured to the logarithmic phase, stable phase, and decay phase respectively.

[0007] Preferably, in S2, the analysis of bacterial cell damage includes: cell membrane integrity, cell membrane fluidity, cell wall peptidoglycan content, extracellular β-galactosidase activity, extracellular Na + -K + -ATPase activity, bacterial fatty acid content, and peptidoglycan amino acid content.

[0008] Preferably, in S3, the analysis contents of the stability of the cells during storage at different temperatures include: cell viability, cell survival rate, cell recovery activity, extracellular β-galactosidase activity, extracellular Na + -K + -ATPase activity and sugar uptake capacity.

[0009] Preferably, in S4, the optimal culture conditions are: the culture pH is 4.30, and the culture period is the stable period.

[0010] Preferably, in S4, the optimal storage temperature is 4°C.

[0011] In another aspect, the present invention provides an application of the above-mentioned culture method in preparing freeze-dried bifidobacterium powder.

[0012] On the other hand, the present invention provides an application of the above-mentioned culture method in preparing a high-activity direct-injection starter of Bifidobacterium.

[0013] In another aspect, the present invention provides an application of the above-mentioned culture method in preparing a bifidobacterium probiotic solid beverage.

[0014] Therefore, the present invention provides a method for cultivating bifidobacteria to improve freeze-drying resistance and storage resistance and its application, which has the following beneficial effects: (1) The present invention establishes a freeze-drying model suitable for dynamically controlling the pH of bacteria, which is used to evaluate the impact of subsequent freeze-drying and storage of bacteria on activity.

[0015] (2) Based on the established pH dynamic regulation freeze-drying model of bacteria, by analyzing indicators such as cell membrane integrity and cell wall composition during the culture, drying and storage processes of bacteria, it was found that low pH culture can regulate the synthesis of unsaturated fatty acids such as oleic acid and palmitoleic acid, and the accumulation of amino acid metabolites such as L-cysteine and L-carnosine of peptidoglycan, thereby enhancing the stability of cell walls and membranes, thereby improving the resistance of bacteria to freeze-drying and storage environmental stresses, and increasing their quantity and activity.

[0016] (3) The present invention establishes a technical method for improving the strain's resistance to drying and storage stress by regulating the active state of cultured bacteria, providing a basis for the precise cultivation of probiotics and the processing of active preparations.

[0017] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The effect of different pH and different periods on the freeze-dried survival rate of Bifidobacterium breve B2798; in the figure, different letters indicate significant differences between the data ( P <0.05); Figure 2 The standard curve was determined by the β-galactosidase activity assay kit; Figure 3 β-galactosidase activity of Bifidobacterium breve B2798 before and after freeze-drying; In the figure, different letters indicate significant differences between the data ( P <0.05); Figure 4 Figure 2 is the relationship between β-galactosidase and freeze-dried survival rate, where A is the extracellular β-galactosidase content of Bifidobacterium breve B2798 after freeze-drying at different pH and different culture periods, B is the survival rate of Bifidobacterium breve B2798 after freeze-drying at pH 4.30, C is the survival rate of Bifidobacterium breve B2798 after freeze-drying at pH 5.30, and D is the survival rate of Bifidobacterium breve B2798 after freeze-drying at pH 6.30. Different letters indicate significant differences between the data ( P <0.05); Figure 5 For reference Na + -K + -Standard curve of ATPase activity assay kit; Figure 6 Na of Bifidobacterium breve B2798 before and after freeze-drying + -K + -ATPase activity; In the figure, capital letters indicate significant differences before and after lyophilization ( P <0.05, lowercase letters indicate significant differences between different periods ( P<0.05); Figure 7 The peptidoglycan content of Bifidobacterium breve B2798 at different pH and different periods; in the figure, different capital letters indicate significant differences before and after freeze-drying ( P <0.05), different lowercase letters indicate significant differences in different periods ( P <0.05); Figure 8 The changes of cell membrane integrity before and after freeze-drying at different pH and time periods; Figure 9 The fluidity of the cell membrane of Bifidobacterium breve B2798 at different pH and different periods; in the figure, different letters indicate significant differences between the data ( P <0.05), capital letters indicate significant differences before and after freeze-drying, and lowercase letters indicate significant differences at different periods; Figure 10 The ratio of UFA to SFA of Bifidobacterium breve B2798 before and after freeze-drying at pH 4.30, where A is before freeze-drying and B is after freeze-drying; Figure 11 The ratio of UFA to SFA of Bifidobacterium breve B2798 before and after freeze-drying at pH 5.30, where A is before freeze-drying and B is after freeze-drying; Figure 12 The ratio of UFA to SFA of Bifidobacterium breve B2798 before and after freeze-drying at pH 6.30, where A is before freeze-drying and B is after freeze-drying; Figure 13 The changes in unsaturated fatty acid content of Bifidobacterium breve B2798 cultured at different pH values before and after freeze-drying; in the figure, capital letters indicate significant differences before and after freeze-drying ( P <0.05), lowercase letters indicate significant differences at different pH values ( P <0.05); Figure 14 The changes of saturated fatty acid content of Bifidobacterium breve B2798 before and after freeze-drying; Figure 15 The changes of unsaturated fatty acid content of Bifidobacterium breve B2798 before and after freeze-drying; Figure 16 Correlation analysis between the main fatty acids of bacteria and freeze-dried activity; Figure 17 The changes of amino acid content of Bifidobacterium breve B2798 before and after freeze-drying; Figure 18 for Na + -K + - Correlation heat map between ATPase activity, GP value, cell membrane integrity, peptidoglycan content, β-galactosidase activity, and freeze-dried survival rate; Figure 19The inactivation kinetics of Bifidobacterium breve B2798 during storage; different lowercase letters indicate significant differences between different groups ( P <0.05); Figure 20 The changes in extracellular β-galactosidase activity of Bifidobacterium breve B2798 during storage, where A represents the storage temperature of 4°C and B represents the storage temperature of 30°C; in the figure, the same letters indicate no significant difference between the data ( P >0.05), different letters indicate significant differences between data ( P <0.05); Capital letters indicate significant differences among different storage times ( P <0.05, lowercase letters indicate significant differences among different pH groups ( P <0.05); Figure 21 The extracellular Na of Bifidobacterium breve B2798 during storage + -K + -ATPase activity changes, where A is the storage temperature of 4°C and B is the storage temperature of 30°C; in the figure, the same letters indicate that the difference between the data is not significant ( P >0.05), different letters indicate significant differences between data ( P <0.05); Capital letters indicate significant differences among different storage times ( P <0.05, lowercase letters indicate significant differences among different pH groups ( P <0.05); Figure 22 It is the sugar uptake capacity of Bifidobacterium breve B2798 during storage. DETAILED DESCRIPTION

[0019] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0020] The instruments, equipment, reagents and materials used in the examples were obtained from commercial sources.

[0021] 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.

[0022] The examples relate to the following culture media: 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.

[0023] Modified MRS solid medium: Add 12 g / L agar to the modified MRS liquid medium, adjust the pH to 6.20 ± 0.02, and sterilize at 121°C for 15 min.

[0024] 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.

[0025] Example 1 Preparation of freeze-dried Bifidobacterium breve B2798 cells: (1) Bacteria activation and proliferation culture The B2798 strain stored at -80°C was inoculated into a modified MRS medium and placed in a constant temperature incubator for anaerobic culture at 37.0°C for 24 h to restore bacterial viability. The culture was then subcultured twice using a pipette at a 2% (V / V) inoculation volume. The third-generation seed liquid was inoculated into a fermentation medium (2 L) using a graduated cylinder at a 2% (V / V) inoculation volume. The culture was placed in a bioreactor at a constant temperature and anaerobic culture at 37.0°C and naturally fermented to pH 4.30, pH 5.30, and pH 6.30. A 25% mass concentration NaOH solution was used for feeding and culture at different pH levels, and the culture was cultured to the logarithmic phase, stationary phase, and decay phase, respectively.

[0026] (2) Preparation of bacterial suspension After the culture is completed, the fermentation broth is centrifuged using a low-speed centrifuge (4°C, 4000 rpm, 15 min), the supernatant is discarded, and the bacterial sludge is washed three times with sterile PBS buffer. The bacterial sludge is collected and evenly mixed with the protective agent at a ratio of 1:1.2 to prepare a bacterial suspension for use.

[0027] (3) Preparation of bacterial powder The prepared bacterial suspension was divided into vials and placed in a freeze dryer for vacuum freeze drying.

[0028] Example 2 During the high-density culture of Bifidobacterium breve B2798, centrifugal samples were taken at different times for vacuum freeze-drying, and the changes in freeze-drying survival rate at different times were monitored. The results are as follows Figure 1 shown.

[0029] Depend on Figure 1 The results show that the freeze-dried survival rates of Bifidobacterium breve B2798 samples taken at different stages of the fermentation system at pH 4.30 were significantly higher than those taken at pH 5.30 and 6.30. Furthermore, the freeze-dried survival rates of cells sampled during the stationary phase were the highest across all fermentation systems at different pH values. The lower the pH value of the fermentation system, the higher the freeze-dried survival rate.

[0030] Example 3 The extracellular β-galactosidase activity of Bifidobacterium breve B2798 bacterial slurry and powder was determined according to the experimental process of the β-galactosidase activity assay kit, and three groups of biological parallel samples were set for data analysis. The standard curve was as follows: Figure 2 As shown in Figure 2, the β-galactosidase activity of Bifidobacterium breve B2798 before and after freeze-drying is as follows: Figure 3 shown.

[0031] Depend on Figure 3 As can be seen, extracellular β-galactosidase activity increased significantly after lyophilization compared to before lyophilization, indicating that lyophilization damages the bacterial cell membrane and releases intracellular β-galactosidase. The increase in extracellular β-galactosidase activity after lyophilization at pH 4.30 was smaller than that in the other two groups, indicating that low pH culturing better prevents cell membrane rupture during the vacuum freeze-drying process. The increase in extracellular β-galactosidase activity during the stationary phase was minimal compared to that during the logarithmic and decay phases, indicating that strains harvested during the stationary phase are more active and more resistant to low temperatures.

[0032] The relationship between β-galactosidase and freeze-drying survival rate is as follows Figure 4 As shown, during the logarithmic phase, the extracellular β-galactosidase activity of Bifidobacterium breve B2798 was low after vacuum freeze-drying, and the freeze-drying survival rate of the bacteria was at a moderate level. The freeze-drying survival rate of the bacteria at pH 4.30 was the highest (84.98%) compared to the other two pH conditions. This phenomenon indicates that the cell membrane of the bacteria cultured and freeze-dried at pH 4.30 is less damaged.

[0033] During the stable period, the freeze-dried survival rate of the bacteria and the activity of β-galactosidase changed significantly. During the stable period, the freeze-dried survival rate of the bacteria was significantly higher than that during the logarithmic period and the decay period ( P <0.05). The extracellular β-galactosidase activity of the bacteria was significantly lower than that in the logarithmic and decay phases ( P<0.05). This indicates that the bacterial cell membrane is less damaged and the cell activity is better during the stable period.

[0034] During the decay phase, the freeze-dried survival rate of Bifidobacterium breve B2798 decreased significantly, while the extracellular β-galactosidase activity increased significantly. At pH 4.30, the freeze-dried survival rate remained relatively high (77.73%) compared to the other two pH groups. The enzyme activity increased most significantly at pH 6.30, while the freeze-dried survival rate dropped to its lowest level (18.38%). This suggests that long-term exposure to alkaline conditions reduces the cells' ability to withstand freezing, significantly impacting their freeze-dried survival rate.

[0035] Example 4 The samples were pre-treated according to the experimental process of the enzyme activity assay kit, and the Na + -K + -ATPase activity was measured and three groups of biological parallel samples were set for data analysis. + -K + -ATPase kit method to draw the standard curve, such as Figure 5 As shown, Na of Bifidobacterium breve B2798 before and after freeze-drying + -K + -ATPase activity as Figure 6 shown.

[0036] The results showed that after vacuum freeze drying, Na + -K + The activity of the ATPase increased significantly, indicating that vacuum freeze-drying damaged the cell membrane. The increase in enzyme activity was minimal when the bacteria were cultured at pH 4.30. This may be because the low pH environment induces a stress response in the bacteria, enhancing cell membrane stability and thus improving the freeze-drying survival rate. The increase in extracellular enzyme activity was greatest when the bacteria were cultured at pH 6.30, indicating that the cell membrane was severely damaged during culture at pH 6.30.

[0037] Example 5 The bacterial peptidoglycan (PG) ELISA detection kit was used to process the samples according to the experimental procedures of the kit. Three parallel sets were set for each sample to determine the peptidoglycan content of Bifidobacterium breve B2798 before and after freeze-drying.

[0038] The peptidoglycan content of Bifidobacterium breve B2798 at different pH and different periods Figure 7 As shown in the figure, it can be seen that when the fermentation system is cultured at pH 4.30, the peptidoglycan content of Bifidobacterium breve before and after freeze-drying is significantly higher than that of other pH groups ( P <0.05). This indicates that acid stress can induce enhanced peptidoglycan synthesis in Bifidobacterium breve.

[0039] The peptidoglycan content of Bifidobacterium breve B2798 in the stable phase was significantly higher than that in the logarithmic and decay phases, indicating that the peptidoglycan content in the cell wall was highest when cultured at pH 4.30, at which time the bacteria had the strongest resistance to the outside world and the best cell protection effect.

[0040] Example 6 Changes in B2798 cell membrane integrity before and after lyophilization were assessed using flow cytometry. Double staining with PI and SYTO™ 9 was performed and the cells were analyzed based on the characteristics of the fluorescent dyes, categorizing them as live, damaged, and dead.

[0041] The bacterial mud and powder were diluted with PBS to prepare bacterial suspension, so that the number of bacteria in the bacterial suspension was controlled at 10 6 ~10 7 (CFU / mL). Add 980µL of bacterial suspension to each sterilized EP tube, add 10µL of 1mmol / L PI dye and 10µL of 1mmol / L SYTO™9 dye, shake for 1 minute to thoroughly mix the dye and bacterial suspension, incubate at room temperature in the dark for 15 minutes, and then analyze the cell membrane integrity before and after freeze-drying at different pH and time periods. Figure 8 shown.

[0042] Depend on Figure 8 It can be seen that when the fermentation system pH is 4.30, the proportion of undamaged cells at each stage before and after freeze-drying is higher than that of the other two groups, indicating that high-density culture at pH 4.30 is more conducive to maintaining the integrity of the bacterial cell membrane than the other two pH groups, and has a significant cell protection effect. When the fermentation system is cultured at pH 4.30, 5.30, and 6.30, the proportion of undamaged cells at each stage before and after freeze-drying showed the same trend, with the highest proportion of undamaged cells in the stable phase, followed by the logarithmic phase, and the lowest proportion of undamaged cells in the decay phase, indicating that vacuum freeze-drying during the stable phase is more effective in protecting cells.

[0043] Example 7 The vacuum freeze-dried bacterial powder and the bacterial mud before freeze-drying were rehydrated with PBS, shaken evenly, and then centrifuged and washed, and then resuspended in PBS solution. The OD value of the sample was diluted to about 0.1, and the sample was stained with 6-acryloyl-2-dimethylnaphthalene (Laurdan) fluorescent dye. The final concentration of the added sample was 50mM, vortexed and mixed, and incubated at 37°C in the dark for 30 minutes, and then immediately ice-bathed to terminate the reaction. The detection was performed using an enzyme-labeled instrument under the conditions of excitation wavelength of 360nm and emission dual wavelength (440nm / 490nm). The cell membrane fluidity of the sample was analyzed using generalized fluorescence polarization. After freeze-drying, the cell membrane fluidity (GP value) of Bifidobacterium breve B2798 was as follows: Figure 9shown.

[0044] As can be seen, when the fermentation system was cultured at a pH of 4.30, the GP values of Bifidobacterium breve B2798 were the lowest both before and after lyophilization, indicating that the cell membrane fluidity of the bacteria cultured at a pH of 4.30 was significantly higher than that of the other experimental groups. Comparison of cell membrane fluidity before and after lyophilization in the logarithmic, stationary, and decay phases revealed that the cell membrane fluidity in the stationary phase was the highest.

[0045] Example 8 The determination of bacterial fatty acids includes the following steps: (1) Sample preparation: Weigh the sample into a centrifuge tube, add methanol containing internal standard, vortex the sample extract at 2500 rpm for 10 min, ultrasonicate in a water bath at 4°C for 15 min, centrifuge at 4200 rpm for 5 min, accurately transfer 500 μL of supernatant to a glass tube, add sodium hydroxide methanol solution (NaOH-MeOH), concentrate to dryness with nitrogen blow, add sodium hydroxide methanol solution to the dry sample, react in a constant temperature box at 70°C for 10 min, remove the sample and cool it on ice, add dichloromethane and double distilled water, vortex at 2500 rpm for 5 min, centrifuge at 4°C and 4200 rpm for 5 min, accurately transfer 300 μL of supernatant to a glass tube, add hydrochloric acid methanol solution (HCl-MeOH), react in a constant temperature box at 70°C for 20 min, remove the sample and cool it on ice, add n-hexane, 300 μL of double distilled water, vortex at 2500 rpm for 5 min, centrifuge at 4°C and 4200 rpm for 5 min, remove the sample and cool it on ice, add n-hexane, 300 μL of double distilled water, vortex at 2500 rpm for 5 min, centrifuge at 4°C and 4200 rpm for 5 min, remove 200 μL of n-hexane layer solution for analysis.

[0046] (2) Determination of fatty acid content by GC-MS: Gas chromatography analysis conditions: chromatographic column DB-5MS (30 m × 0.25 mm × 0.25 µm); carrier gas: helium; flow rate: 1 mL / min; column temperature program: initial temperature 40 °C, hold for 2 min, then increase the temperature to 200 °C at a rate of 30 °C / min and hold for 1 min, then increase the temperature to 240 °C at a rate of 10 °C / min and hold for 1 min, and finally increase the temperature to 285 °C at a rate of 5 °C / min and hold for 3 min.

[0047] Prepare standard solutions of different concentrations and obtain the chromatographic peak intensity data corresponding to each concentration of standard. Draw standard curves for different substances with the external standard concentration (Concentration Ratio) as the horizontal axis and the external standard peak area (Area Ratio) as the vertical axis.

[0048] The integrated peak areas of all detected samples are substituted into the linear equation of the standard curve for calculation, and then further substituted into the calculation formula to finally obtain the content data of the substance in the actual sample.

[0049] The calculation formula is: ; Where, c is the concentration of fatty acids in the sample (μg / mL); v 1 is the volume of sample extract (μL); v 2 is the volume of collected supernatant (μL); v 3 is the reconstitution volume (μL); m is the sample mass (g).

[0050] A total of 20 fatty acid components were identified in Bifidobacterium breve B2798. The quantitative standard curve equations for each detected fatty acid are shown in Table 1 below: Table 1 Quantitative standard curve equations for each detected fatty acid ;

[0051] The 20 fatty acids identified can be divided into two categories based on the degree of carbon chain saturation: 12 are saturated fatty acids, including lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid, pearly acid, stearic acid, arachidic acid, behenic acid, caproic acid, caprylic acid, and capric acid; and 8 are unsaturated fatty acids, mainly including oleic acid, linoleic acid, trans-linoleic acid, arachidic acid, palmitoleic acid, α-linolenic acid, erucic acid, and cis-12-tridecenoic acid.

[0052] The structure and function of the cell membrane were evaluated by the ratio of saturated fatty acids (SFA) to unsaturated fatty acids (UFA). The ratio of UFA to SFA of Bifidobacterium breve B2798 before and after freeze-drying at pH 4.30, pH 5.30 and pH 6.30 was used to evaluate the structure and function of the cell membrane. Figure 10 、 11 , as shown in 12.

[0053] The results showed that the UFA ratio of Bifidobacterium breve B2798 in the pH 4.30 group before and after freeze-drying was higher than that of the other two groups, and the increase was the largest. During the freeze-drying process, a higher ratio of unsaturated fatty acids helps maintain good fluidity and structural toughness of the cell membrane, thereby better maintaining its integrity. The cis double bond configuration in UFA produces a steric hindrance effect, effectively inhibiting the regular arrangement of fatty acid molecules and avoiding the reduction of membrane fluidity due to the close stacking of molecules. The pH 4.30 group has the best protection effect on the bacteria during the freeze-drying process. The freeze-dried survival rate of the bacteria is also higher, which further shows that low pH culture can more effectively protect cells during the freeze-drying process.

[0054] Changes in unsaturated fatty acid content of Bifidobacterium breve B2798 before and after freeze-drying at different pH values Figure 13As shown, the UFA content of Bifidobacterium breve B2798 in the pH 4.30 group increased the most after freeze-drying, and the UFA content of the bacteria after freeze-drying was the highest.

[0055] The SFA of the cells before and after freeze-drying were quantitatively analyzed, such as Figure 14 As shown in the figure, stearic acid, palmitic acid, myristic acid, and arachidic acid showed the most significant differences before and after freeze-drying ( P <0.01). During the freeze-drying process, the stearic acid content of the bacteria in the pH 4.30, pH 5.30, and pH 6.30 groups increased by 4.60-fold, 5.49-fold, and 7.28-fold, respectively; the palmitic acid content increased by 4.35-fold, 5.47-fold, and 5.73-fold, respectively; the myristic acid content increased by 5.12-fold, 5.34-fold, and 16.45-fold, respectively; and the arachidic acid content increased by 1.62-fold, 1.64-fold, and 2.12-fold, respectively.

[0056] The results showed that the saturated fatty acid content of the three experimental groups increased significantly after vacuum freeze-drying. Among them, the saturated fatty acid content of the pH 4.30 group increased the least after freeze-drying, further indicating that the proportion of unsaturated fatty acids in the pH 4.30 group after freeze-drying was higher than that in the pH 5.30 group and the pH 6.30 group.

[0057] Quantitative analysis of UFA of bacteria before and after freeze-drying, such as Figure 15 As shown, oleic acid and palmitoleic acid showed the most significant differences ( P <0.01).

[0058] Oleic acid (C18:1), an unsaturated fatty acid with important physiological functions, has a unique curved carbon chain structure that significantly enhances cell membrane fluidity. This structural property not only helps maintain membrane integrity but also enhances its stability under environmental stress. During freeze-drying, oleic acid content in the pH 4.30, pH 5.30, and pH 6.30 groups increased by 14.17-fold, 3.55-fold, and 3.51-fold, respectively.

[0059] Palmitoleic acid is a monounsaturated fatty acid (MUFA) with a double bond at the seventh carbon atom, which makes it relatively stable. During the freeze-drying process, the palmitoleic acid content of bacteria in the pH 4.30, pH 5.30, and pH 6.30 groups increased by 5.91-fold, 3.59-fold, and 3.04-fold, respectively.

[0060] Results showed that the unsaturated fatty acid content of all three experimental groups increased significantly after vacuum freeze-drying. The pH 4.30 group exhibited the highest unsaturated fatty acid content after freeze-drying. Increased unsaturated fatty acid content can enhance cell membrane fluidity and flexibility, strengthening resistance to freezing during vacuum freeze-drying. Previous studies have shown that strains can improve their tolerance to acid stress by adjusting the fatty acid composition of their cell membranes under low pH conditions. The results of this study demonstrate that acidic culture conditions can significantly induce adaptive changes in the fatty acid composition of bacteria. The increased relative content of long-chain unsaturated fatty acids effectively blocks extracellular lactate penetration by enhancing membrane density and alleviating acid stress damage. Simultaneously, the increased proportion of unsaturated fatty acids, such as oleic acid (C18:1) and palmitoleic acid (C16:1), significantly improved membrane fluidity. These adjustments not only enhanced the strain's acid tolerance but also maintained membrane structural stability during freeze-drying, resulting in the optimal freeze-drying survival rate in the pH 4.30 culture group.

[0061] Correlation analysis between the main fatty acids of the bacteria and the freeze-dried activity Figure 16 The results showed that the relative contents of oleic acid and palmitoleic acid in vacuum freeze-dried Bifidobacterium breve B2798 cells were positively correlated with their freeze-drying activity, while the relative contents of palmitic acid, stearic acid, and myristic acid were negatively correlated with their freeze-drying activity. This suggests that oleic acid and palmitoleic acid can protect cells during the freeze-drying process. Increasing the content of unsaturated fatty acids effectively lowers the phase transition temperature, where the cell membrane transitions from a liquid crystal to a gel state. This property significantly enhances the cell's adaptability to low-temperature stress, thereby reducing damage to the cell structure during the freeze-drying process. This may be the reason why oleic acid and palmitoleic acid can enhance the freeze-drying activity of the strain.

[0062] Example 9 Extract the peptidoglycan from the bacteria and use gas chromatography-mass spectrometry (GC-MS) to quantitatively analyze the amino acid composition of the sample. The specific process includes the following: (1) Extraction of peptidoglycan: Collect the cells, centrifuge at 8000 rpm for 1 min, resuspend in distilled water, wash once by centrifugation, discard the supernatant, and resuspend the cells in 100 mL of distilled water; boil the resuspension for 10 min to prevent self-cleavage of peptidoglycan, then centrifuge at 11000 rpm for 8 min at 4°C to collect the cells. Resuspend the cells in preheated 5% (w / v) SDS buffer and boil for 25 min. Recollect the insoluble matter by centrifugation (11000 rpm, 20°C, 8 min), then resuspend the cells in preheated 4% (w / v) SDS buffer, boil for 15 min, centrifuge, and collect the cells. Wash the cells with preheated 60°C distilled water to remove SDS and centrifuge at least 6 times. Disrupt the cells by ultrasonication, with an interval of 5 seconds, a disruption of 5 seconds, a power of 400 W, 99 cycles per cycle, and at least 4 cycles until the supernatant becomes clear. The sample was centrifuged at 4000 rpm for 10 minutes, unbroken cells were removed, and the supernatant was retained. The broken cell walls were collected by centrifugation at 11000 rpm for 10 minutes and washed twice with water. The cell walls were resuspended in 20 mM PBS buffer (pH 7.6) and treated with DNase and RNase at a final concentration of 0.5 mg / mL, respectively, at 37°C for 3 hours. Trypsin was added at a final concentration of 0.5 mg / mL and treated at 37°C for 3 hours. Pronase was added at a final concentration of 2 mg / mL and treated at 37°C for 2 hours, followed by several washes. The cells were then treated with HF at 4°C for at least 24 hours. Teichoic acid was removed and the cells were washed several times with water.

[0063] (2) Determination of changes in peptidoglycan amino acid composition: Sample pretreatment: After thawing, add 20% acetonitrile / methanol aqueous solution; vortex mix and place in a -20°C refrigerator; centrifuge at 12000 rpm for 10 min at 4°C, and transfer the supernatant to a new centrifuge tube; centrifuge again, transfer the supernatant to a sample injection bottle, and temporarily store in a -20°C refrigerator for LC-MS / MS analysis.

[0064] Liquid phase conditions: Chromatographic column: ACQUITY BEH Amide column; Mobile phase: Phase A, ultrapure water (containing 2 mM ammonium acetate, 0.04% formic acid); Phase B, acetonitrile (containing 2 mM ammonium acetate, 0.04% formic acid); Gradient elution program: 0-1.2 min A / B: 10:90 (V / V), 9 min A / B: 40:60 (V / V), 10-11 min: 60:40 (V / V), 11.01-15 min: 10:90 (V / V); Flow rate: 0.4 mL / min; column temperature: 40°C; injection volume: 2 μL.

[0065] Prepare standard solutions of different concentrations and obtain the mass spectrometry peak intensity data corresponding to each concentration of standard. Draw standard curves for different substances with the external standard to internal standard concentration ratio (Concentration Ratio) as the horizontal axis and the external standard to internal standard peak area ratio (Area Ratio) as the vertical axis.

[0066] Substitute the integrated peak area ratio of all detected samples into the linear equation of the standard curve for calculation, and further substitute it into the following formula for calculation to finally obtain the content data of the substance in the actual sample.

[0067] Amino acid content in the sample: ; Where, c is the concentration of the sample amino acid in the sample (ng / mL); v is the volume of the solution used for extraction (μL).

[0068] Among the 94 amino acids detected, 54 amino acid components were identified in Bifidobacterium breve B2798. The quantitative standard curve equations of each detected amino acid are shown in Tables 2 and 3.

[0069] Table 2 Linear equations of the standard curve of amino acids detected in Bifidobacterium breve B2798 ; ;

[0070] Table 3 Linear equations of the standard curve of amino acids detected in Bifidobacterium breve B2798 ;

[0071] L-cysteine has a sulfhydryl group (-SH) with strong reducing properties, which can directly scavenge free radicals (such as hydroxyl radicals, superoxide anions, etc.), reducing oxidative stress during the freeze-drying process. In addition, L-cysteine is a precursor of glutathione (GSH), which can promote the synthesis of glutathione in cells and enhance the antioxidant capacity of cells. The amino acid content before and after freeze-drying was quantitatively analyzed, and the results were as follows: Figure 17 As shown, the differences in the contents of L-cysteine (Cys) and L-carnosine (L-Carnosine) before and after freeze-drying were the most significant ( P <0.01). The results showed that after vacuum freeze drying, the L-cysteine and L-carnosine contents of the three experimental groups decreased significantly. Among them, the pH 4.30 group showed the smallest decrease in L-cysteine and L-carnosine contents after freeze drying. The higher the peptidoglycan amino acid content, the stronger the resistance to freezing during vacuum freeze drying and the better the protection effect on the bacteria. Studies have found that exogenous addition of cysteine can significantly increase srtThe expression level of A gene is increased, and the anchoring efficiency of cell wall surface proteins is enhanced to maintain its structural integrity. Therefore, low pH culture is of great significance for the protection of bacteria during vacuum freeze-drying.

[0072] Example 10 Based on the above test indicators: Na + -K + -ATPase activity, GP value, cell membrane integrity, peptidoglycan content, β-galactosidase activity, freeze-dried survival rate, analyze the correlation between the indicators, the correlation heat map is as follows Figure 18 shown.

[0073] The results showed that GP value was negatively correlated with cell membrane fluidity. The freeze-dried survival rate was significantly positively correlated with cell membrane integrity, peptidoglycan content, and cell membrane fluidity ( P <0.01), and the activity of extracellular β-galactosidase, Na + -K + -ATPase activity was significantly negatively correlated. This indicates that when the cell membrane fluidity, integrity and peptidoglycan content are higher, the freeze-dried survival rate will also increase. In addition, the extracellular β-galactosidase activity and Na + -K + -The higher the ATPase activity, the lower the freeze-drying survival rate, which means that the more serious the cell membrane damage, the lower the freeze-drying survival rate.

[0074] In addition, correlation analysis also showed that peptidoglycan content was significantly positively correlated with cell membrane integrity ( P <0.01). This indicates that higher cell wall peptidoglycan content improves cell protection. Peptidoglycan indirectly protects cell membrane integrity by providing mechanical strength, maintaining osmotic pressure balance, and resisting external damage. Higher peptidoglycan content increases cell wall rigidity, reducing the risk of cell membrane rupture due to external pressure or osmotic changes. This is also a major reason for the high freeze-dried survival rate of strains.

[0075] Example 11 Freeze-dried powders of Bifidobacterium breve B2798 cultured at high densities at different pH conditions were evaluated for storage stability at both 4°C and 30°C. Samples were collected on days 0, 7, 14, and 28 of storage to assess the effects of storage at 4°C and 30°C on the viability of B. breve B2798 cells cultured at different pH levels.

[0076] (1) Changes in the inactivation kinetics of Bifidobacterium breve B2798 during storage Figure 19As shown in Figure 2, the inactivation kinetics showed that the survival of B. breve B2798 was temperature-dependent. Specifically, the survival rate of B. breve B2798 decreased as the storage temperature increased from 4°C to 30°C. According to the linear fit, K max The value (calculated according to the slope of the curve) is positively correlated with the bacterial survival rate, R 2 >0.95. The inactivation rate of cells cultured at pH 4.30 was the lowest during stable storage, likely because the low pH environment stimulates a stress response in bifidobacteria, enhancing their survival rate. The inactivation rate increased with increasing pH, suggesting that cells cultured at low pH offer more stable storage.

[0077] (2) After re-dissolving the freeze-dried bacterial powder, inoculate it into a modified MRS medium at a 2% inoculum volume. Measure the absorbance every hour using a microplate reader to calculate the specific growth rate. The maximum specific growth rates of Bifidobacterium breve B2798 at different temperatures are shown in Tables 4 and 5 below: Table 4 Changes in the maximum specific growth rate of Bifidobacterium breve B2798 during storage (h -1 ) (4℃) ;

[0078] Note: The same letters indicate no significant difference between the data ( P >0.05), different letters indicate significant differences between data ( P <0.05); Capital letters indicate significant differences between different pH values ( P <0.05), lowercase letters indicate significant differences among different storage times ( P <0.05).

[0079] As shown in Table 4, the specific growth rate of B2798 bacterial powder in different pH groups decreased during storage, and the downward trend became more significant as the storage temperature increased ( P <0.05).

[0080] Table 5 Changes in the maximum specific growth rate of Bifidobacterium breve B2798 during storage (h -1 ) (30℃) ;

[0081] Note: The same letters indicate no significant difference between the data ( P >0.05), different letters indicate significant differences between data ( P <0.05); Capital letters indicate significant differences between different pH values ( P <0.05), lowercase letters indicate significant differences among different storage times ( P <0.05).

[0082] The results showed that when stored at 4°C, the growth rate of the three groups of bacterial powder decreased more slowly than that at 30°C, which was consistent with the trend of the inactivation rate of Bifidobacterium breve. In addition, the bacterial powder in the pH 4.30 group had higher storage activity than the other two groups of bacterial powder, and the maximum growth rate after reconstitution was higher, which further proved that low pH culture was more conducive to the maintenance of bacterial powder activity. This may be because the low pH environment can reduce Na + -K + -A decrease in ATPase activity and a higher energy supply can support faster cell growth and division.

[0083] (3) By measuring the activity of β-galactosidase leaked out of the cell during storage, the effect of storage temperature on enzyme activity can be determined. The results are as follows: Figure 20 As shown. When stored at 4°C, the extracellular enzyme activities of Bifidobacterium breve B2798 powder in the pH 4.30 group, pH 5.30 group, and pH 6.30 group were the highest at 0.56, 1.34, and 2.12 IU / 10, respectively, during the 28-day storage period. 8 cell, and the extracellular enzyme activities of Bifidobacterium breve B2798 increased by 0.42, 1.00, and 1.39 IU / 10, respectively, compared with 0d 8 Compared with the three pH groups, the increase in extracellular β-galactosidase activity in the pH 4.30 group was the smallest.

[0084] When stored at 30℃, the extracellular enzyme activities of Bifidobacterium breve B2798 powder in pH 4.30 group, pH 5.30 group, and pH 6.30 group reached the highest values of 1.01, 1.37, and 2.78 IU / 10, respectively, during storage for 28 days. 8 cell, and the extracellular enzyme activities of Bifidobacterium breve B2798 increased by 0.87, 1.37, and 2.05 IU / 10, respectively, compared with 0d 8 Compared with the three pH groups, the increase in extracellular β-galactosidase activity in the pH 4.30 group was the smallest.

[0085] During storage, extracellular β-galactosidase activity showed an upward trend. As pH and temperature decreased, extracellular β-galactosidase activity also decreased. Furthermore, the pH 4.30 group had the highest specific growth rate and survival rate, indicating that the pH 4.30 group had greater storage stability than the other two groups and was better able to maintain cell membrane integrity. This is because low pH culture can alter the fatty acid composition of the bifidobacterial cell membrane, enhancing cell membrane stability and thus better maintaining cell storage stability.

[0086] (4) Take Na + -K + -ATPase activity was used as an indicator, and the Na+ -K + -ATPase activity, which is used to judge the effect of storage temperature on Na + -K + -ATPase activity. Figure 21 As shown in the figure, when stored at 4°C, the extracellular enzyme activities of Bifidobacterium breve B2798 powder in the pH 4.30 group, pH 5.30 group, and pH 6.30 group were the highest at 1.13, 2.44, and 5.01 IU / 10, respectively, during the 28-day storage period. 8 cell, and compared with 0d, the extracellular enzyme activities of Bifidobacterium breve B2798 increased by 0.68, 1.23, and 2.57 IU / 10 8 Compared with the three pH groups, the extracellular Na + -K + -ATPase activity increased the least. When stored at 30°C, the extracellular enzyme activity of Bifidobacterium breve B2798 powder in the pH 4.30, pH 5.30, and pH 6.30 groups reached the highest levels of 1.61, 4.55, and 7.56 IU / 10, respectively, during storage for 28 days. 8 cell, compared with 0d, the extracellular enzyme activity of Bifidobacterium breve B2798 increased by 1.16, 3.34, and 5.12 IU / 10 8 Compared with the three pH groups, the extracellular Na + -K + -ATPase activity increased the least. During storage, extracellular Na + -K + -ATPase activity showed an upward trend, and the extracellular Na + -K + -ATPase was the lowest, indicating that the bacterial powder stored in the pH 4.30 group was more stable than the other two groups.

[0087] (5) Determination of sugar uptake capacity of strains during storage The vacuum-freeze-dried bacterial powder was re-dissolved in PBS buffer, vortexed and mixed, centrifuged at 4000 × g, 4 °C for 5 min, washed twice, resuspended, and serially diluted with PBS to make the number of viable bacteria in the sample between 1 × 10 6 CFU / mL, 6-NDBG fluorescent dye was added to the sample at a final concentration of 5 μM, incubated at 37 °C in the dark for 30 min, and the fluorescence intensity of the sample was detected by flow cytometry.

[0088] The sugar uptake capacity of the strain during storage Figure 22 The changes in fluorescence intensity of sugar uptake ability of strains during storage at different temperatures are shown in Table 6 and Table 7 respectively: Table 6 Changes in fluorescence intensity of sugar uptake capacity of Bifidobacterium breve B2798 during storage (4°C) ;

[0089] Note: The same letters indicate no significant difference between the data ( P >0.05), different letters indicate significant differences between data ( P <0.05); Capital letters indicate significant differences between different pH values ( P <0.05), lowercase letters indicate significant differences among different storage times ( P <0.05).

[0090] according to Figure 22 As shown in Table 6, the sugar uptake capacity of B. breve B2798 after high-density fermentation and freeze-drying at different pH levels varied under different storage conditions. For cells cultured at pH 4.30 and stored at 4°C, the sugar uptake fluorescence intensity decreased by 14.25%, 33.43%, and 60.62% after 7, 14, and 28 days, respectively. For cells cultured at pH 5.30 and stored at 4°C, the sugar uptake fluorescence intensity decreased by 35.56%, 44.07%, and 65.91% after 7, 14, and 28 days, respectively. For cells cultured at pH 6.30 and stored at 4°C, the sugar uptake fluorescence intensity decreased by 44.41%, 57.27%, and 67.34% after 7, 14, and 28 days, respectively. The magnitude of the decrease in sugar uptake fluorescence intensity decreased with decreasing pH. Compared with the three pH groups, the sugar uptake fluorescence intensity of the pH 4.30 group decreased the least, indicating that the bacterial powder cultured under acidic conditions had a stronger sugar uptake ability during storage, which indirectly reflects that the activity of the bacterial powder cultured under acidic conditions can be better maintained.

[0091] Table 7 Changes in fluorescence intensity of sugar uptake capacity of Bifidobacterium breve B2798 during storage (30°C) ;

[0092] Note: The same letters indicate no significant difference between the data ( P >0.05), different letters indicate significant differences between data ( P <0.05); Capital letters indicate significant differences between different pH values ( P <0.05), lowercase letters indicate significant differences among different storage times ( P <0.05).

[0093] The sugar uptake fluorescence intensity of bacteria cultured at pH 4.30 and stored at 30°C decreased by 34.79%, 58.08%, and 71.58% after 7, 14, and 28 days, respectively. The sugar uptake fluorescence intensity of bacteria cultured at pH 5.30 and stored at 30°C decreased by 45.36%, 63.45%, and 74.36% after 7, 14, and 28 days, respectively. The sugar uptake fluorescence intensity of bacteria cultured at pH 6.30 and stored at 30°C decreased by 53.87%, 68.06%, and 80.23% after 7, 14, and 28 days, respectively.

[0094] In summary, the sugar uptake capacity of B. breve B2798 showed varying degrees of attenuation during storage in all experimental groups. The decline in sugar uptake capacity was greater when stored at 30°C than when stored at 4°C, indicating that bacterial activity is better maintained during low-temperature storage. After culture, fermentation, and freeze-dried at pH 4.30, the decline in sugar uptake capacity was significantly less than that observed at 4°C and 30°C compared to the other two pH groups. The slower inactivation rate during storage in cultures cultured at pH 4.30 is consistent with the trend in sugar uptake, further demonstrating that cultures cultured at low pH maintain better activity during storage and offer more stable storage.

[0095] 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 improving the freeze-drying resistance and storage resistance of bifidobacteria, characterized in that: The steps include: S1. Cultivate bifidobacteria by dynamically controlling pH, freeze-dry bacteria at different pH values and growth stages, and establish a freeze-drying model for bacteria with dynamic pH control. S2. Analyze the bacterial cell damage in the pH dynamic control freeze-drying model of bacteria established in S1, select bacteria with the lowest bacterial cell damage, and record the culture pH value and growth period of the bacteria with the lowest bacterial cell damage; S3. Storing the freeze-drying model of the bacteria with dynamic pH control established in S1 at different temperatures, analyzing the stability of the freeze-drying model of the bacteria with dynamic pH control during storage, and selecting the storage temperature with the highest stability during storage; S4, the culture pH and growth period obtained by S2 are used as the optimal culture conditions before freeze-drying of bifidobacteria, and the storage temperature obtained by S3 is used as the optimal storage temperature of bifidobacteria; Cultivating bifidobacteria under optimal culture conditions can improve their freeze-drying resistance and storage resistance. Or the freeze-dried powder of bifidobacteria cultured under optimal culture conditions can improve the storage stress resistance of bifidobacteria at the optimal storage temperature.

2. A method for cultivating bifidobacteria to improve freeze-drying resistance and storage resistance according to claim 1, characterized in that: In S1, the dynamic pH control culture includes: The activated third-generation seed liquid of Bifidobacterium was inoculated and anaerobically fermented at a constant temperature of 37.0℃. The fermentation was carried out naturally to a pH of 4.30-6.

30. A 25% mass concentration NaOH solution was added for feeding and the pH was constantly controlled at different levels. The cultures were cultured to the logarithmic phase, stable phase, and decay phase respectively.

3. The method for cultivating bifidobacteria to improve freeze-drying resistance and storage resistance according to claim 1, characterized in that: In S2, the analysis of bacterial cell damage includes: cell membrane integrity, cell membrane fluidity, cell wall peptidoglycan content, extracellular β-galactosidase activity, extracellular Na + -K + -ATPase activity, bacterial fatty acid content, and peptidoglycan amino acid content.

4. The method for cultivating bifidobacteria to improve freeze-drying resistance and storage resistance according to claim 1, characterized in that: In S3, the analysis of the stability of the cells during storage at different temperatures includes: cell viability, cell survival rate, cell recovery activity, extracellular β-galactosidase activity, extracellular Na + -K + -ATPase activity and sugar uptake capacity.

5. The method for cultivating bifidobacteria to improve freeze-drying resistance and storage resistance according to claim 1, characterized in that: In S4, the optimal culture conditions were: culture pH 4.30 and culture period was the stable period.

6. The method for cultivating bifidobacteria to improve freeze-drying resistance and storage resistance according to claim 1, characterized in that: In S4, the optimal storage temperature was 4°C.

7. Use of the culture method according to any one of claims 1 to 6 in preparing freeze-dried bifidobacterium powder.

8. Use of the culture method according to any one of claims 1 to 6 in preparing a high-activity direct-injection starter of Bifidobacterium.

9. Use of the culture method according to any one of claims 1 to 6 in preparing a bifidobacterium probiotic solid beverage.

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