A method for improving survival rate of probiotic bacteria based on cell membrane regulation

Abstract

The application discloses a method for improving survival rate of probiotics based on cell membrane regulation, and the method comprises the following steps: adding lipid components during the culture of probiotics and / or adding the lipid components as a protective agent after the culture of probiotics; the lipid components added during the culture of probiotics comprise cytidine diphosphate diacylglycerol and / or stearic acid; and the lipid components added as a protective agent after the culture of probiotics comprise cardiolipin and / or oleic acid. The method breaks through the research scope of traditional optimization of freeze-drying process (such as protective agent and parameter), and through the establishment of lipidomics means and a screening method of anti-resistance related lipid molecules, key lipid molecules directly related to resistance are screened out; through the dual mode of supplementing key lipid components and precursor substances, the strain resistance is improved from the metabolic level, and the freeze-drying resistance and survival rate of the strain are significantly improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biotechnology, and particularly relates to a method for improving the survival rate of probiotics based on cell membrane regulation. BACKGROUND

[0002] Drying treatment is one of the important means for the production and storage of probiotics. In order to reduce transportation costs and storage space, most probiotic products used in industrial production are commercialized in the form of freeze-drying. However, during the freeze-drying treatment and storage process, probiotics are often affected by various stress factors, leading to cell damage, inactivation and reduced activity. Therefore, studying the tolerance and mechanism of strain cells during the drying treatment and storage process is of great significance for improving production efficiency and quality, and ensuring product stability.

[0003] The cell membrane is the main barrier between the cell and the external environment. When the external temperature and pressure change dramatically, the cell membrane is the first to perceive the external environmental pressure and is the first target substrate to be damaged, playing a key role in protecting cells from external pressure stress and damage. Whether the mortality rate of strain cells after drying is closely related to the cell membrane composition, and whether the differences in cell membrane composition of different strains will cause differences in their resistance to drying, still lack systematic research.

[0004] Therefore, in-depth study of the tolerance and mechanism of strain cells during the drying treatment and storage process can not only provide scientific basis for the production and application of probiotics, but also develop new drying treatment and storage technology, and improve the quality and market competitiveness of lactic acid bacteria products.

[0005] Currently, the general technology development for probiotic powder is mostly based on the optimization of auxiliary materials and process general parameters in the fermentation and drying stages of the strain. This has a very limited improvement on the high activity powder technology. The biggest defect is that it cannot be targeted and cannot fundamentally solve the problems of low survival rate and poor stability of the strain. SUMMARY

[0006] To solve the above technical problems, the present application provides a method for improving the survival rate of probiotics based on cell membrane regulation. By adding lipid components during the cultivation of probiotics and / or adding them as protective agents after the cultivation of probiotics, the survival rate of freeze-drying is significantly improved, and the stability is also significantly improved.

[0007] The present application provides a method for improving the survival of probiotics based on cell membrane regulation, wherein the method comprises:

[0008] adding lipid components during the cultivation of probiotics and / or adding them as protective agents after the cultivation of probiotics;

[0009] The lipid component added during the culture of the probiotic bacteria includes cytidine diphosphate diacylglycerol (CDP-DAG) and / or stearic acid (C18:0);

[0010] The lipid component added as a protective agent after the culture includes cardiolipin (CL) and / or oleic acid (C18:1).

[0011] In the above method, preferably, the time of adding the lipid component during the culture of the probiotic bacteria is 0-4 hours and / or 6-14 hours. The present application sets the time when the culture is started with the inoculation of the probiotic bacteria as 0 hour, and the lipid component can be added at the initial stage of the culture for 0-4 hours or at the logarithmic phase of the culture for 6-14 hours, or added at both time periods.

[0012] In the above method, preferably, the molar concentration of the cytidine diphosphate diacylglycerol is 0.1-10 μM, more preferably 0.4-10 μM, further preferably 0.4-5 μM, further preferably 0.5-2 μM, further preferably 0.5-1 μM, based on the mixture obtained after the addition of the lipid component during the culture of the probiotic bacteria.

[0013] In the above method, preferably, the molar concentration of the stearic acid is 5-200 nM, more preferably 20-100 nM, further preferably 60-100 nM, further preferably 80 nM, based on the mixture obtained after the addition of the lipid component during the culture of the probiotic bacteria.

[0014] In the above method, preferably, the concentration of the cardiolipin is 0.1-5% w / w, more preferably 1-2% w / w, based on the mixture obtained after the addition of the lipid component as a protective agent after the culture.

[0015] In the above method, preferably, the concentration of the oleic acid is 0.2-10% w / w, more preferably 0.3-2% w / w, based on the mixture obtained after the addition of the lipid component as a protective agent after the culture.

[0016] In the above method, preferably, the genus of the probiotic bacteria includes one or a combination of two or more of the genus of Lactobacillus, Lactococcus, Lactosus, Lactosus, Lactosus, Lactosus, Bifidobacterium, Streptococcus, and Lactococcus.

[0017] In the above method, preferably, the probiotic bacteria include one or a combination of two or more of Paracasei, Lactobacillus acidophilus, Lactobacillus crispatus, and Lactobacillus gasseri.

[0018] In the above method, preferably, the method for improving the survival of the probiotic bacteria based on the regulation of the cell membrane further comprises:

[0019] The lipid component is added during the culture of probiotics and / or after the culture of probiotics as a protective agent, and the obtained suspension containing probiotics is subjected to freezing treatment.

[0020] In the above method, preferably, the freezing treatment comprises pre-freezing treatment and freeze-drying treatment.

[0021] In the above method, preferably, the pre-freezing treatment is performed at a temperature of -80 to -45°C for 4-12 hours; more preferably, the pre-freezing treatment is performed at a temperature of -80 to -50°C for 6-10 hours.

[0022] In the above method, preferably, the freeze-drying treatment is performed at a temperature of -80 to -40°C, a vacuum degree of ≤20 Pa, and a freeze-drying time of 20-40 h. More preferably, the freeze-drying treatment is performed at a temperature of -60 to -45°C, a vacuum degree of 8-10 Pa, and a freeze-drying time of 20-40 h.

[0023] In some embodiments of the present application, the method for improving the survival rate of probiotics based on cell membrane regulation comprises:

[0024] The lipid component is added after the culture of probiotics as a protective agent, and the lipid component added after the culture as a protective agent comprises cardiolipin and / or oleic acid;

[0025] The addition of the lipid component after the culture of probiotics as a protective agent comprises the following steps:

[0026] Strain activation: the activation of the probiotic strain is performed with MRS liquid medium.

[0027] Preparation of slurry: the activated probiotic strain is cultured to the end of the logarithmic growth phase, and the obtained bacterial suspension is subjected to centrifugal treatment to obtain slurry;

[0028] Preparation of emulsion: after washing the slurry, a bacterial suspension is obtained by adding a buffer solution; the bacterial suspension is uniformly mixed with a cardiolipin solution and / or an oleic acid solution to obtain an emulsion (i.e., a mixture obtained after the addition of the lipid component as a protective agent after the culture).

[0029] Pre-freezing of emulsion: the emulsion is pre-frozen at a temperature of -80 to -45°C for 4-12 hours.

[0030] Freeze-drying: the pre-frozen emulsion is subjected to vacuum freeze-drying at a temperature of -80 to -40°C and a vacuum degree of ≤20 Pa.

[0031] According to the embodiments of the present application, the buffer solution is a PBS buffer solution, and the volume of the buffer solution is 70 ml.

[0032] According to the embodiment of the present application, in the method for improving the survival rate of probiotics based on the regulation of cell membrane, the concentration of the oleic acid solution is 0.1-10M; in the emulsion, the amount of the oleic acid solution added is 0.1-10ml.

[0033] According to the embodiment of the present application, in the method for improving the survival rate of probiotics based on the regulation of cell membrane, the concentration of the cardiolipin solution is 0.25-50mM; in the emulsion, the amount of the cardiolipin solution added is 0.1-10ml.

[0034] According to the embodiment of the present application, in the method for improving the survival rate of probiotics based on the regulation of cell membrane, the step of activating the bacterial strain is performed at least three times.

[0035] In some specific embodiments of the present application, the method for improving the survival rate of probiotics based on the regulation of cell membrane comprises:

[0036] adding a lipid component during the culture of probiotics; the lipid component comprises cytidine diphosphate diacylglycerol (CDP-DAG) and / or stearic acid (C18:0);

[0037] the step of adding the lipid component during the culture of probiotics comprises:

[0038] activating the bacterial strain, activating the probiotic bacterial strain with MRS liquid medium;

[0039] preparing the bacterial slurry, inoculating the activated probiotic bacterial strain into the culture medium for culture, adding the lipid component including stearic acid and / or cytidine diphosphate diacylglycerol at 0-4 hours or 6-14 hours of the culture period, or at both time periods, to obtain a mixed solution (i.e. the mixed solution obtained after adding the lipid component during the culture of probiotics), and culturing to the end of the logarithmic growth phase, and then centrifuging the cultured probiotic suspension to obtain the bacterial slurry;

[0040] preparing the emulsion: after washing the bacterial slurry, adding a buffer to obtain a bacterial suspension; adding or not adding cardiolipin, oleic acid or a compound of the two substances, and adding or not adding other protective carriers to obtain the emulsion for standby;

[0041] pre-freezing the emulsion, pre-freezing the emulsion at a temperature of-80℃ to-50℃ for 4-12 hours.

[0042] freeze-drying, vacuum freeze-drying the pre-frozen bacterial suspension at a vacuum degree of ≤10Pa and a temperature of-80℃ to-40℃.

[0043] According to an embodiment of the present application, the culture medium is MRS culture medium, and the volume of the culture medium is 200 mL.

[0044] According to an embodiment of the present application, the buffer is PBS buffer, and the volume of the buffer is 70 mL.

[0045] According to an embodiment of the present application, in the method for improving the survival rate of probiotics based on cell membrane regulation, the concentration of the stearic acid solution is 0.01-10 mM; and in the mixed solution, the amount of the stearic acid solution added is 1-100 μL.

[0046] According to an embodiment of the present application, in the method for improving the survival rate of probiotics based on cell membrane regulation, the concentration of the cytidine diphosphate diacylglycerol solution is 1-100 mM; and in the mixed solution, the amount of the cytidine diphosphate diacylglycerol solution added is 1-100 μL.

[0047] According to an embodiment of the present application, in the method for improving the survival rate of probiotics based on cell membrane regulation, the step of activating the bacterial strain is performed at least three times.

[0048] The present application discloses a method for improving the survival rate of probiotics based on cell membrane regulation. The method breaks through the research scope of traditional optimization of freeze-drying process (such as protective agent and parameter), selects key lipid molecules directly related to resistance through lipidomics means and the establishment of screening method of resistance-related lipid molecules, and significantly improves the freeze-drying ability and survival rate of the strain through the dual mode of supplementing key lipid components and their precursor substances, i.e. improving the strain resistance from the metabolic level.

[0049] The present application finds that glycerophospholipid metabolism is the core regulation pathway through KEGG pathway analysis; based on the metabolic pathway of membrane lipids, the key lipid molecules cardiolipin and C18:1 are selected as protective agents or their precursors C18:0 and CDP-DAG are used for strain metabolic intervention and protective agents, which can significantly improve the survival rate of probiotics after freeze-drying. The method for improving the survival rate of probiotics based on cell membrane regulation provided by the present application can improve the processing and storage stability of the strain by developing key technologies from the changes of strain cell membrane structure components and cell membrane composition regulation. BRIEF DESCRIPTION OF DRAWINGS

[0050] Figure 1 The lipid composition of the bacterial membrane of K56 in different growth periods (early growth and logarithmic phase) is different (a-c).

[0051] Figure 2 The lipid composition of the bacterial membrane of K56 in different growth periods (early growth and stationary phase) is different (d-f).

[0052] Figure 3 The results of the difference in the lipid composition of the bacterial membrane of K56 in different growth periods (log phase and stationary phase).

[0053] Figure 4 The results of the survival rate of the freeze-dried viable cell count of P. frexicanum K56 with the addition of C18:1 precursor C18:0.

[0054] Figure 5 The metabolic pathway of C18:1 and precursor C18:0.

[0055] Figure 6 The results of the survival rate of the freeze-dried viable cell count of P. frexicanum K56 obtained under freeze-drying conditions with the addition of cardiolipin precursor CDP-DAG.

[0056] Figure 7 The metabolic pathway of cardiolipin (CL) and its precursor CDP-DAG.

[0057] Figure 8 The results of the significant improvement in membrane integrity after the addition of C18:0 (stearic acid) and CDP-DAG (cytidine diphosphate diacylglycerol) at the initial stage of culture (0 h).

[0058] Figure 9 The infrared spectrum of K56, K56+C18:0, and K56+CDP-DAG, and the change in the peak intensity of the bacterial membrane of K56 after the addition of exogenous substances.

[0059] Figure 10 The results of the effect of the addition of C18:1 as a protective agent on the survival rate of K56 strain after freeze-drying.

[0060] Figure 11 The results of the area occupied by each lipid molecule per nm2 and the distance between the head groups of phospholipid molecules in the molecular dynamics simulation. 2

[0061] Figure 12 The results of the verification of the interaction mechanism of C18:1 with the bacterial membrane in the molecular dynamics simulation.

[0062] Figure 13 The results of the verification of the interaction mechanism of CL with the bacterial membrane in the molecular dynamics simulation.

[0063] Figure 14 The detailed observation results of the process of cardiolipin entering the bacterial membrane in the molecular dynamics simulation. DETAILED DESCRIPTION

[0064] ​In order to have a clearer understanding of the technical features, objectives and beneficial effects of the present application, the technical solutions of the present application are described in detail as follows, but cannot be understood as limiting the implementable scope of the present application.

[0065] Hereinafter, the embodiments of the present application are described. It should be noted that sometimes, “pre-freeze freeze-drying” or “freeze-drying” is simply referred to as “freeze-drying”, “culture or fermentation life cycle” is simply referred to as “life cycle”, “pre-mixing treatment of protective carrier and slurry” is simply referred to as “emulsification”, and “protective carrier” is simply referred to as “protective agent”.

[0066] In a specific embodiment of the present application, the method for improving the survival of probiotics based on cell membrane regulation provided by the present application utilizes key cell membrane lipid components C18:1 and cardiolipin and their precursor substances, and is realized by directly adding protective agents or life cycles. The method comprises the following steps: strain activation, expansion culture, slurry preparation, emulsion preparation, pre-freezing, vacuum drying, etc.

[0067] In the step of strain activation, MRS liquid medium is used to activate the probiotic strain. The step of strain activation is performed at least three times or more.

[0068] In the step of strain expansion culture, any one of C18:1 and cardiolipin or a combination of precursor substances thereof is supplemented at 0-4 h in the early stage of culture and 6-14 h in the logarithmic phase of culture, respectively.

[0069] In the step of slurry preparation, the activated probiotic strain is cultured for a certain time, and the cultured probiotic suspension is subjected to centrifugal treatment to obtain slurry for standby use.

[0070] In the step of emulsion preparation, any one of C18:1 and cardiolipin or a combination thereof is supplemented in the prepared slurry.

[0071] In the step of freeze-drying, the slurry or emulsion is pre-frozen in a vacuum freeze-drying machine, the pre-freezing temperature is -40-80℃, and the vacuum degree is less than 20 Pa.

[0072] In the preliminary study of the present application, the phospholipid composition of the bacterial membrane of K56 strain in different growth cycles (early growth, logarithmic phase, stationary phase) was analyzed and compared, and the results are shown in Figure 1 a-c of Figure 2 d-f of Figure 3As shown in g-i, after comparing the K56 growth in the initial stage, logarithmic phase and stationary phase, it was found that the phospholipid composition of the biofilm was significantly different in different growth stages of the strain. The composition characteristics of the biofilm in the initial growth stage mainly revolve around phospholipid precursors. In this stage, there are more phospholipid precursors with high reactivity in the biofilm, which play a key role in the construction of the initial architecture of the biofilm. When the strain reaches the logarithmic growth phase, the length and saturation of the fatty acid chain contained in the phospholipid show characteristics suitable for the vigorous metabolism of the strain. The longer and moderately saturated fatty acid chain gives the biofilm good fluidity, which is conducive to the exchange of substances inside and outside the cell, meeting the needs of nutrient uptake and metabolic product discharge in the process of high-speed proliferation of the strain.

[0073] The cell membrane composition changes greatly and is in the remodeling stage (0-4 h) in the initial growth stage and the logarithmic phase (6-14 h), so the present application sets the life cycle time of the lipid composition during the culture of probiotics as 0-4 h in the initial culture period and / or 6-14 h in the logarithmic phase of culture.

[0074] Example 1

[0075] The present embodiment provides a method for improving the freeze-drying survival rate of probiotics (Paracasei K56) based on cell membrane regulation, which specifically comprises the following steps:

[0076] After activating the Paracasei K56 strain with MRS liquid medium, 200 mL of MRS broth was added with an inoculation amount of 2% (v / v), and the culture was statically cultured at 37°C during the culture. Four groups of experiments were set up:

[0077] Oleic acid precursor C18:0 was added at 0 hours of culture, cardiolipin precursor CDP-DAG was added at 0 hours of culture, oleic acid precursor C18:0 was added at 8 hours of culture, and cardiolipin precursor CDP-DAG was added at 8 hours of culture.

[0078] After 20 h of culture at 37°C, the culture was centrifuged to harvest the cell precipitate, washed twice and resuspended with 70 mL of PBS buffer to obtain a bacterial suspension.

[0079] The bacterial suspension was pre-frozen, the pre-freezing temperature was -80°C, and the pre-freezing time was 6 hours.

[0080] The bacterial suspension after pre-freezing treatment was freeze-dried, and the freeze-drying conditions were: cold trap temperature -60°C, vacuum degree 10 Pa, and freeze-drying time about 24 hours.

[0081] It should be noted that C18:0 or CDP-DAG is added to the culture medium at 0 or 8 hours, respectively, to participate in the process of bacterial membrane formation, and the exogenous addition of C18:0 and CDP-DAG reduces the direct damage of the drying process to the cells of lactic acid bacteria. In the method of the above embodiment, the concentration of the C18:0 solution is 0.4 mM, and the addition amount is 10, 20, 40 μL, respectively; the concentration of the CDP-DAG solution is 10 mM, and the addition amount is 10, 20, 40 μL, respectively.

[0082] Example 2

[0083] The present embodiment provides a method for improving the survival rate of freeze-dried probiotics (Paracasei K56) based on cell membrane regulation, which specifically comprises the following steps:

[0084] C18:1 is configured into a solution for standby, and the solution concentration is 1 mol / L, and the addition amount is 1, 2, 4 ml.

[0085] Using MRS liquid medium, Paracasei strain is inoculated into 200 mL MRS broth at an inoculation amount of 2% (v / v) and cultured at 37 ℃ for 20 h. Then the culture is centrifuged to harvest the cell precipitate, washed twice and resuspended with 70 mL PBS buffer, and different volumes of oleic acid are added.

[0086] The bacterial suspension is pre-frozen at a pre-freezing temperature of -45 ℃ for 10 hours.

[0087] Freeze-drying conditions: cold trap temperature -45 ℃, vacuum degree 8 Pa, freeze-drying time about 36 hours.

[0088] It should be noted that C18:1 is added to the bacterial slurry as a protective carrier to participate in the repair process of damaged bacterial membranes, and the exogenous addition of C18:1 reduces the direct damage of the drying process to the cells of lactic acid bacteria.

[0089] Test Example 1

[0090] The freeze-dried bacterial powder obtained by the method described in Example 1 is evaluated, and the change in survival rate before and after freeze-drying is tested, as follows:

[0091] (1) Anti-freeze-drying ability determination

[0092] After freeze-drying, 1 g of freeze-dried bacterial powder K56-F is dissolved in 10 mL of sterilized 0.85% (v / v) NaCl solution, and then serially diluted. The bacterial solution diluted to the final concentration is inoculated on MRS agar plates, and cultured at 37 ℃ for 72 h. The plate bacterial colony number is between 30~300 CFU. Three parallel plates are set for each dilution gradient, and the survival rate is calculated:

[0093] Survival rate (%) = viable cell count after freeze-drying (CFU / g) / viable cell count before freeze-drying (CFU / g) x 100%.

[0094] Figure 4 The results of the survival rate of freeze-dried viable cell count of P. freudenreichii subsp. casei K56 with the addition of C18:1 precursor C18:0. From Figure 4 It can be seen that the C18:0 experimental group has a higher overall freeze-drying survival rate than the control group after supplementing C18:0 at the beginning of the culture 0 h. Among the three doses supplemented, the addition of 40 μL of K56 has the most significant survival rate improvement effect of 43.99% (about 1.47 times), which may be because the K56 cell membrane may cause membrane remodeling by taking up C18:0 during the formation stage Figure 5 for the metabolic pathway of C18:1 and its precursor C18:0), thereby improving membrane stress resistance.

[0095] Figure 6 The results of the survival rate of freeze-dried viable cell count of P. freudenreichii subsp. casei K56 under freeze-drying conditions with the addition of cardiolipin precursor CDP-DAG. From Figure 6 It can be seen that the CDP-DAG experimental group, from the 10 μL dose to 40 μL, the freeze-drying survival rate of K56 strain shows a trend of first increasing and then decreasing, and the addition of the highest dose of 40 μL leads to a decrease in strain stress resistance. Cardiolipin itself has a low content in the cell membrane, so if the content is too high, it may cause negative feedback regulation of the cell membrane Figure 7 for the metabolic pathway of cardiolipin and its precursor CDP-DAG), resulting in poor membrane stress resistance. For 20 μL of CDP-DAG added at 0 h, the survival rate of K56 after freeze-drying increased by the largest margin, from 31.58% to 46.24% (about 1.46 times), indicating that CDP-DAG phospholipid molecules may improve the freeze-drying resistance of the cell membrane by synthesizing cardiolipin in the cell membrane.

[0096] (2) Lactate dehydrogenase (LDH) activity determination

[0097] Weigh 0.016 g of freeze-dried bacterial powder, dissolve it in 1 mL of sterilized PBS filtered through a 0.22 filter membrane, centrifuge (10000 g, 4 ℃, 10 min) and collect the supernatant. Enzyme activity is determined according to the steps of the LDH determination kit. One unit of enzyme activity is defined as the production of 1 μmol of pyruvic acid in the reaction system. Enzyme determination is performed in triplicate.

[0098]

[0099] During freeze-drying, the stress on lactic acid bacteria causes cell membrane rupture, leading to the leakage of intracellular enzymes. However, this does not affect the activity of lactases (such as lactate dehydrogenase and ATPase). Therefore, measuring extracellular enzyme activity can indirectly reflect the degree of cell membrane damage. Figure 8 As shown in a1 and a2, after the exogenous addition of C18:0 (a1) and CDP-DAG (a2) respectively, the K56 bacterial membrane leaked into the extracellular LDH enzyme activity results after freeze-drying. The results showed that the enzyme activity in the experimental group was significantly lower than that in the control group, indicating that the membrane integrity was significantly improved.

[0100] (3) Na + -K + -ATPase activity assay

[0101] Weigh approximately 0.02 g of the lyophilized bacterial powder, dissolve it in 1 mL of sterile PBS filtered through a 0.22 aqueous membrane, centrifuge (10000 g, 4 ℃, 10 min), and collect the supernatant. Enzyme activity is determined by ultra-micro Na... + -K + The enzyme assay kit was used to perform the enzymatic reaction and phosphorus determination reaction sequentially. Finally, the absorbance of the sample was measured at 636 nm. The enzyme assay was performed in triplicate.

[0102] Similarly, Na + -K + -ATPase is a protein in the cell membrane bilayer, acting as both a carrier and an enzyme. It catalyzes the hydrolysis of ATP to provide energy, maintains the membrane potential across the cell membrane, regulates cell osmotic pressure, and provides the driving force for nutrient absorption. This is achieved by measuring Na+. + -K + -ATPase activity can also indirectly reflect the degree of cell membrane damage. For example Figure 8 As shown in b1 and b2, by adding 20 μL and 40 μL of C18:0 (b1) and CDP-DAG (b2) to K56 medium, Na + -K + - The significant decrease in ATPase activity indicates that membrane integrity has been effectively improved.

[0103] like Figure 8 The integrity of K56 cells and bacterial biofilms supplemented with C18:0 and CDP-DAG was measured using bacterial live / dead fluorescence staining. The results showed that, under the SYTO9 staining channel, the green fluorescence intensity of K56 (24.03±2.69%) was relatively lower than that of K56-C18:0 (40.36±5.77%) and K56-CDP-DAG (38.51±4.62%), indicating a relatively lower number of live cells in K56. Under the PI staining channel, K56 exhibited the highest proportion of red fluorescence intensity.

[0104] Therefore, based on the above-mentioned lactate dehydrogenase (LDH) activity, Na + -K + -ATPase activity and the results of bacterial live / dead fluorescent staining, the membrane integrity of K56 can be significantly improved after supplementing C18:0 and CDP-DAG, which indirectly increases the number of viable cells of K56 after freeze-drying.

[0105] (4) Infrared spectrum

[0106] An appropriate amount of freeze-dried K56, K56+C18:0 and K56+CDP-DAG powders were taken and placed in a marble mortar. According to the mass ratio of sample to KBr powder of about 1:100, they were thoroughly ground and mixed until the sample and KBr powder formed a fine and uniform mixture. An appropriate amount of the ground mixture was carefully placed in the mold of a tablet press under a certain pressure (usually 8 kgf / cm 2 ) for 1 minute to obtain a transparent thin film sample. The Fourier infrared spectrometer was set with the corresponding scanning parameters, the scanning wave number range was 4000-400 cm -1 , the resolution was 4 cm -1 , and the scanning number was 64 times to obtain the infrared spectrum.

[0107] As Figure 9 The infrared spectrum shows the infrared absorption characteristics of K56, K56+C18:0 and K56+CDP-DAG and the change in the peak intensity of K56 membrane after supplementing exogenous substances. At about 3295 cm -1 , there is an absorption peak in K56, K56+C18:0 and K56+CDP-DAG, which may be related to the stretching vibration of hydroxyl (―OH), indicating that K56 may contain compounds or groups containing hydroxyl. At 1371 cm -1 ~1505 cm -1 , the absorption peak may be related to the skeletal vibration of aromatic ring or certain specific carbon-hydrogen bending vibration. In addition, compared with K56, the absorption peak at about 1666 cm -1 vibrates weakly after adding C18:0 and CDP-DAG, and the peak intensity is slightly different, which may be due to the substitution reaction of the stretching vibration peak of carbonyl (C=O) with the functional groups in C18:0 and CDP-DAG to generate ester groups, ketone groups, etc., which has a significant impact on the membrane phospholipid structure. There are also vibration peaks at other wave number regions, such as 1247 cm -1 , which may be related to the stretching vibration of carbon-oxygen (C―O) or other related vibration modes, further reflecting the changes in the membrane phospholipid structure after supplementing C18:0 and CDP-DAG.

[0108] Test Example 2

[0109] The freeze-dried bacterial powder obtained by the method described in Example 2 was evaluated, and various properties before and after freeze-drying were tested, as follows:

[0110] Anti-freeze-drying capacity determination

[0111] After freeze-drying, 1 g of freeze-dried bacterial powder K56-F was dissolved in 10 mL of sterilized 0.85% (v / v) NaCl solution, and then serially diluted. The bacterial solution diluted to the final concentration was inoculated on MRS agar plates, and incubated at 37 ℃ for 72 h. The plate bacterial colony count was between 30 and 300 CFU. Three parallel plates were set for each dilution gradient, and the survival rate was calculated:

[0112] Survival rate (%) = number of viable bacteria after freeze-drying (CFU / g) / number of viable bacteria before freeze-drying (CFU / g) x 100%.

[0113] As Figure 10 The results of the effect of exogenous addition of C18:1 as a protective agent on the anti-freeze-drying survival rate of K56 strain showed that the survival rate of P. parnui K56 increased from 31.82% to 43.95% (P<0.05), 44.8% (P<0.05) and 47.08% (P<0.01) respectively before freeze-drying by adding different volumes of oleic acid (1 M), with a maximum increase of 15.25%. The main mechanisms of action of oleic acid as a freeze-drying protective agent include: (1) its surface active properties can form a protective film around the molecules, stabilizing protein and cell membrane structure; (2) the hydrophobicity of oleic acid helps to reduce protein aggregation and denaturation; (3) it can interact with membrane lipids to maintain the integrity of the biological membrane. These properties enable oleic acid to exhibit good protective effects under freeze and dry stress conditions. Therefore, based on the above results, it can be concluded that exogenous addition of oleic acid protective agent can also greatly protect the bacterial membrane during the freeze-drying process.

[0114] In addition to the above traditional freeze-drying survival rate detection, the present application also explores the interaction mechanism of key lipid molecules C18:1, CL and bacterial membrane.

[0115] Molecular dynamics (MD) simulation has become an important tool for studying the structure and function of biological membranes and the effects of external molecules on membranes. In recent years, MD simulation has made significant progress in exploring the effects of key lipid molecules on the structure and function of bacterial membranes (bacterial cell membranes). The Martini force field is a widely used force field in coarse-grained MD, designed specifically for simulating large biological macromolecular systems, especially biological membranes, proteins, nucleic acids, etc. Its coarse-grained nature allows it to simulate large systems over long time scales, simplifying complex molecular systems by reducing the number of particles (usually 4 atoms are represented as 1 particle), which greatly reduces the number of particles and computational load of the system, providing an important tool for understanding cell membranes and membrane-related processes. Therefore, based on the Martini force field, the present invention simulates the cell membrane of Lactobacillus paracasei.

[0116] As shown in Figure 11 , Figure 12 , Figure 13 shows the effect of C18:1 and CL intervention on the area per lipid molecule / nm 2 and the distance between the head groups of phospholipid molecules. The total simulation time for molecular dynamics simulation is 300 ns, with temperature changes set to gradually decrease from -20 ℃ to -60 ℃. After the temperature changes to -60 ℃, the system is balanced, and the data results at the 200 ns simulation time are output as shown in Figures 11-13 From Figures 11-13 , it can be seen that when the temperature changes from -20 ℃ to -60 ℃, the area per lipid molecule (Area per lipid) of the membrane significantly decreases after adding C18:1 and CL in the membrane. This indicates that the arrangement of phospholipid molecules becomes more loose when the content of C18:1 and CL in the membrane increases, which helps the bacterial membrane resist low temperature environment.

[0117] As shown in Table 1, the change in the distance between the head groups of C18:1 and CL phospholipid molecules during the molecular dynamics simulation of the bacterial membrane low-temperature freezing process. From Table 1, it can be seen that the maximum distance between the head groups of phospholipids does not change significantly, indicating that the membrane thickness remains essentially unchanged.

[0118] Table 1

[0119]

[0120] As shown in Figure 14The detailed observation and analysis results of the process of cardiolipin entering the bacterial membrane by molecular dynamics simulation are shown, aiming at deeply understanding the interaction mechanism between cardiolipin and the bacterial membrane. At the initial moment (0 ns), the cardiolipin molecules (represented by white balls) are mainly distributed in the water environment (pink part) around the bacterial membrane composed of DPPC (dark gray) and POPG (green). With the passage of time, by 200 ns, part of the cardiolipin molecules begin to contact the surface of the bacterial membrane and gradually embed in the outer layer of the bacterial membrane. By 400 ns, more cardiolipin molecules enter the bacterial membrane, and the distribution range in the bacterial membrane is expanded, showing that the process of cardiolipin entering the bacterial membrane has a certain continuity and gradualness. By 600 ns, the distribution of cardiolipin molecules in the bacterial membrane is further diffused, and begins to penetrate into the interior of the bacterial membrane. By 800 ns, the distribution of cardiolipin molecules in the bacterial membrane is more uniform, and the depth is further increased, indicating that the process of cardiolipin entering the bacterial membrane is still continuing. Finally, by 1000 ns, the cardiolipin molecules in the bacterial membrane reach a relatively stable distribution state, and interact with the bacterial membrane lipid molecules. Therefore, it is speculated that cardiolipin may interact with specific binding sites on the surface of the bacterial membrane, gradually insert into the bacterial membrane, and diffuse within the membrane. This process may be influenced by various factors, such as the concentration of cardiolipin molecules, the lipid composition of the bacterial membrane, and environmental conditions, etc.

[0121] From the above test results, it can be seen that the method for improving the survival rate of probiotics based on cell membrane regulation of the application utilizes key lipid molecules directly related to resistance, and significantly improves the strain's resistance to freeze-drying and its survival rate through the dual mode of supplementing key lipid components and their precursor substances, i.e., improving strain resistance from the metabolic level, while also significantly improving the stability in the later stage.

Claims

1. A method for improving the survival rate of probiotic bacteria after freeze-drying based on cell membrane regulation, characterized in that, The method comprises: adding a lipid component during the culture of probiotics; the lipid component added during the culture of probiotics is cytidine diphosphate diacylglycerol and / or stearic acid; the molar concentration of cytidine diphosphate diacylglycerol is 0.5-1 μM, calculated based on the mixture obtained after adding the lipid component during the culture of probiotics; the molar concentration of stearic acid is 60-100 nM, calculated based on the mixture obtained after adding the lipid component during the culture of probiotics; the time for adding the lipid component during the culture of probiotics is 0-4 hours and / or 6-14 hours; the probiotics are Lactobacillus paracasei K56.

2. The method of claim 1, wherein, The method further comprises: freezing the suspension containing probiotics obtained after adding the lipid component during the culture of probiotics.

3. The method of claim 2, wherein, The freezing treatment comprises pre-freezing treatment and freeze-drying treatment; the temperature of the pre-freezing treatment is -80 to -45℃, and the time is 4-12 hours; the conditions of the freeze-drying treatment are: the temperature is -80 to -40℃, the vacuum degree is ≤20 Pa, and the freeze-drying time is 20-40 h.

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