Method for improving survival rate of probiotics based on cell membrane regulation and control

Abstract

The invention discloses a method for improving the survival rate of probiotics based on cell membrane regulation and control. The method comprises the following steps: adding a lipid component in a probiotic culture process and / or adding the lipid component as a protective agent after probiotic culture, the lipid component added in the probiotic culture process comprises cytidine diphosphate diacylglycerol and / or stearic acid; the lipid component added as the protective agent after culture comprises cardiolipin and / or oleic acid. The method breaks through the research category of a traditional optimized freeze drying process (such as a protective agent and parameters), and key lipid molecules directly related to resistance are screened out through a lipidomics means and establishment of a screening method of stress resistance related lipid molecules; through a dual mode of supplementing key lipid components and precursor substances thereof, the tolerance of the strain is improved from the metabolic level, and the freeze-drying resistance and the survival rate of the strain are remarkably improved.

Description

Technical Field

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

[0002] Drying treatment is one of the important means for the production and storage of probiotics. 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, resulting in problems such as 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 its production efficiency and quality, as well as ensuring product stability.

[0003] The cell membrane is the main barrier separating the bacterial cell from the external environment. When there are drastic changes in the external temperature and pressure, the cell membrane is the first part to sense the external environmental pressure and is also the first target matrix to be damaged, playing a key role in protecting the cell from external stress and damage. Whether the lethality rate of strain cells after drying is closely related to the cell membrane components, and whether the differences in the cell membrane components of different strains will cause differences in their anti-drying ability, there is still a lack of 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 a scientific basis for the production and application of probiotics, but also contribute to the development of new drying treatment and storage technologies, improving the quality and market competitiveness of lactic acid bacteria products.

[0005] Currently, for the general technology development of probiotic powder, most are based on the optimization of auxiliary materials and process general parameters in the strain fermentation and drying links, which has a very limited improvement in the technology of high-vitality powder. The biggest defect is that it cannot be targeted, starting from the specificity of the strain cells themselves, and cannot fundamentally solve problems such as low strain survival rate and poor stability. Summary of the Invention

[0006] To solve the above technical problems, the present invention provides a method for improving the survival rate of probiotics based on cell membrane regulation. By adding lipid components during the probiotic culture process and / or as a protective agent after the probiotic culture, the freeze-drying survival rate is significantly improved, and at the same time, the later stability is also significantly improved.

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

[0008] Adding lipid components during the probiotic culture process and / or as a protective agent after the probiotic culture;

[0009] The lipid components added during the probiotic cultivation include cytidine diphosphate diacylglycerol (CDP-DAG) and / or stearic acid (C18:0);

[0010] The lipid components added as protectants after cultivation include cardiolipin (CL) and / or oleic acid (C18:1).

[0011] In the above method, preferably, the time for adding the lipid components during the probiotic cultivation is 0 - 4 hours and / or 6 - 14 hours. In the present invention, the start of probiotic inoculation for cultivation is set as 0 hour, and the lipid components can be added at 0 - 4 hours in the initial cultivation stage or 6 - 14 hours in the logarithmic growth stage of cultivation, or added simultaneously in both periods.

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

[0013] In the above method, preferably, based on the mixture obtained after adding the lipid components during the probiotic cultivation, the molar concentration of stearic acid is 5 - 200 nM; more preferably, the molar concentration of stearic acid is 20 - 100 nM, further preferably 60 - 100 nM, and further preferably 80 nM.

[0014] In the above method, preferably, based on the mixture obtained after adding the lipid components as protectants after cultivation, the concentration of cardiolipin is 0.1 - 5% w / w, more preferably 1 - 2% w / w.

[0015] In the above method, preferably, based on the mixture obtained after adding the lipid components as protectants after cultivation, the concentration of oleic acid is 0.2 - 10% w / w, more preferably 0.3 - 2% w / w.

[0016] In the above method, preferably, the genus of the probiotics includes one or a combination of two or more of Lactobacillus, Lactobacillus casei, Lactobacillus mucosae, Lactobacillus plantarum, Lactobacillus collinoides, Lactobacillus paraplantarum, Bifidobacterium, Streptococcus, Lactococcus.

[0017] In the above method, preferably, the probiotics include one or a combination of two or more of Lactobacillus paracasei, Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus gasseri.

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

[0019] The probiotic suspension obtained by adding lipid components during the probiotic cultivation process and / or adding them as a protective agent after the probiotic cultivation is subjected to a freezing treatment.

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

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

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

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

[0024] Adding lipid components as a protective agent after the probiotic cultivation, and the lipid components added as a protective agent after the cultivation include cardiolipin and / or oleic acid;

[0025] The addition of the lipid components as a protective agent after the probiotic cultivation includes the following steps:

[0026] Strain activation: Activating the probiotic strain with MRS liquid medium.

[0027] Bacterial sludge preparation: Culturing the activated probiotic strain to the late logarithmic growth phase, and centrifuging the cultured probiotic suspension to obtain bacterial sludge;

[0028] Emulsion preparation: After washing the bacterial sludge, adding a buffer solution to suspend it to obtain a bacterial suspension; mixing the bacterial suspension with a cardiolipin solution and / or an oleic acid solution evenly to obtain an emulsion (i.e., the mixture obtained after adding lipid components as a protective agent after the cultivation);

[0029] Emulsion pre-freezing: Pre-freezing the emulsion, the pre-freezing temperature is -80 °C to -45 °C, and the pre-freezing time is 4 to 12 hours;

[0030] Freeze-drying: Performing vacuum freeze-drying on the pre-frozen emulsion, the vacuum degree ≤ 20 Pa, and the temperature is -80 °C to -40 °C.

[0031] According to the embodiment of the present invention, the buffer solution is PBS buffer solution, and the volume of the buffer solution is 70 ml.

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

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

[0034] According to the embodiments of the present invention, 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 or more.

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

[0036] Adding lipid components during the cultivation of probiotics; the lipid components include cytidine diphosphate diacylglycerol (CDP-DAG) and / or stearic acid (C18:0);

[0037] The addition of the lipid components during the cultivation of probiotics includes the following steps:

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

[0039] Preparing bacterial sludge, inoculating the activated probiotic bacterial strain into a medium for cultivation, adding lipid components including stearic acid and / or cytidine diphosphate diacylglycerol at 0 - 4 hours or 6 - 14 hours of the cultivation cycle, or adding them simultaneously in both time periods, to obtain a mixed solution (i.e., the mixed solution obtained after adding lipid components during the cultivation of probiotics), culturing until the late logarithmic growth phase, and centrifuging the cultured probiotic suspension to obtain bacterial sludge;

[0040] Preparing the emulsion: After washing the bacterial sludge, adding a buffer solution to suspend it to obtain a bacterial suspension; adding or not adding cardiolipin, oleic acid or a complex of the two substances, and adding or not adding other protective carriers to the cell suspension to obtain an emulsion for standby;

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

[0042] Freeze-drying, subjecting the pre-frozen bacterial suspension to vacuum freeze-drying with a vacuum degree ≤ 10 Pa and a temperature of -80°C to -40°C.

[0043] According to an embodiment of the present invention, 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 invention, the buffer solution is PBS buffer solution, and the volume of the buffer solution is 70 ml.

[0045] According to an embodiment of the present invention, 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; in the mixture, the addition amount of the stearic acid solution is 1 - 100 μL.

[0046] According to an embodiment of the present invention, 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; in the mixture, the addition amount of the cytidine diphosphate diacylglycerol solution is 1 - 100 μL.

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

[0048] The present invention discloses a method for improving the survival rate of probiotics based on cell membrane regulation. This method breaks through the research scope of traditional optimization of freeze-drying processes (such as protectants and parameters). By means of lipidomics and the establishment of a screening method for stress-resistant related lipid molecules, key lipid molecules directly related to resistance are screened out; by supplementing key lipid components and their precursor substances in a dual manner, that is, improving the tolerance of the strain at the metabolic level, the freeze-drying resistance ability and survival rate of the strain are significantly improved.

[0049] Through KEGG pathway analysis, the present invention finds that glycerophospholipid metabolism is the core regulatory pathway; based on the membrane lipid synthesis metabolic pathway, selecting key lipid molecules cardiolipin and C18:1 as protectant supplements or their precursor substances C18:0 and CDP-DAG for strain metabolic intervention and protectants can significantly improve the survival rate of probiotics after freeze-drying. The method for improving the survival of probiotics based on cell membrane regulation provided by the present invention can improve the processing and storage stability performance of the strain by developing key technologies from the changes in the structural components of the strain cell membrane and the regulation of the cell membrane composition. Description of the Drawings

[0050] Figure 1 Shows the differences in the lipid composition of the cell membrane of K56 at different growth stages (initial growth stage and logarithmic phase) (a - c).

[0051] Figure 2 Shows the differences in the lipid composition of the cell membrane of K56 at different growth stages (initial growth stage and stationary phase) (d - f).

[0052] Figure 3 Lipid composition differences (g-i) of the biofilm of K56 at different growth stages (logarithmic phase and stationary phase).

[0053] Figure 4 Results of the survival rate of viable freeze-dried cells of Lactobacillus paracasei K56 after exogenous addition of C18:1 precursor C18:0.

[0054] Figure 5 Metabolic pathways of C18:1 and precursor substance C18:0.

[0055] Figure 6 Results of the survival rate of viable freeze-dried cells of Lactobacillus paracasei K56 obtained under freeze-drying conditions after exogenous addition of cardiolipin precursor CDP-DAG.

[0056] Figure 7 Metabolic pathways of cardiolipin (CL) and its precursor CDP-DAG.

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

[0058] Figure 9 Infrared absorption characteristics of K56, K56+C18:0, and K56+CDP-DAG, and infrared spectrograms showing changes in the peak intensity of the K56 biofilm after supplementation with exogenous substances.

[0059] Figure 10 Results of the effect of exogenous addition of C18:1 as a cryoprotectant on the survival rate of K56 strain after freeze-drying.

[0060] Figure 11 Area per lipid molecule in the control group of molecular dynamics simulation / nm 2 and results of the distance between the phospholipid molecular head groups.

[0061] Figure 12 Results of validating the interaction mechanism between C18:1 and the biofilm by molecular dynamics simulation.

[0062] Figure 13 Results of validating the interaction mechanism between CL and the biofilm by molecular dynamics simulation.

[0063] Figure 14 Results of detailed observation of the process of cardiolipin entering the biofilm by molecular dynamics simulation. Specific implementation manner

[0064] For a clearer understanding of the technical features, objectives, and beneficial effects of the present invention, the following detailed description of the technical solution of the present invention is provided, but it should not be construed as a limitation on the scope of implementation of the present invention.

[0065] Hereinafter, embodiments of the present invention will be described. It should be noted that sometimes "pre-freezing and freeze-drying" or "freeze-drying" is abbreviated as "lyophilization", "the total time of cultivation or fermentation" is abbreviated as "life cycle", "premixing treatment of the protective carrier and the bacterial sludge" is abbreviated as "emulsification", and "protective carrier" is abbreviated as "protective agent".

[0066] In a specific embodiment of the present invention, the method for improving the survival of probiotics based on cell membrane regulation provided by the present invention utilizes the key cell membrane lipid components C18:1 and cardiolipin and their precursor substances, and is achieved by directly adding a protective agent or a life cycle. It includes the following steps: strain activation, expansion culture, bacterial sludge preparation, emulsion preparation, pre-freezing, vacuum drying, etc.

[0067] In the step of strain activation, the probiotic strain is activated with MRS liquid medium. The step of strain activation is carried out at least three times or more.

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

[0069] In the step of bacterial sludge preparation, the activated probiotic strain is cultured for a certain period of time, and the cultured probiotic suspension is centrifuged to obtain bacterial sludge for standby.

[0070] In the step of emulsion preparation, either one of C18:1 and cardiolipin or a combination thereof is supplemented to the prepared bacterial sludge.

[0071] In the step of freeze-drying, the bacterial sludge or emulsion is pre-frozen in a vacuum freeze-dryer, the pre-freezing temperature is -40 - 80°C, and the vacuum is pumped, and the vacuum degree is less than 20 Pa.

[0072] In the preliminary research of the present invention, a comparative analysis of the phospholipid composition of the cell membranes of the K56 strain at different growth cycles (initial growth stage, logarithmic phase, stationary phase) was carried out, and the results are as Figure 1 in a-c, Figure 2 in d-f, Figure 3As shown in gi, after comparing the early growth, logarithmic and stable phases of K56, it was found that the phospholipid composition of the biofilm was significantly different in different growth periods of the strain. The composition characteristics of the biofilm in the early growth phase mainly revolve around phospholipid precursors. At this stage, there are many phospholipid precursors with high reactivity in the biofilm, which play a key role in the construction of the initial structure of the biofilm. When the strain reaches the logarithmic growth phase, the length and saturation of the fatty acid chains contained in the phospholipids show characteristics that are compatible with the vigorous metabolism of the bacteria. Long fatty acid chains with moderate saturation give the biofilm good fluidity, which is conducive to the exchange of substances inside and outside the cell, and meets the needs of the bacteria for the uptake of nutrients and the discharge of metabolites during the rapid proliferation of the bacteria.

[0073] The cell membrane composition changes greatly and is in the remodeling stage (0-4 h) which is the initial growth stage and the logarithmic stage (6-14 h). Therefore, the present invention sets the life cycle time of the lipid component during the probiotic culture process to the initial culture stage 0-4 h and / or the logarithmic culture stage 6-14 h.

[0074] Example 1

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

[0076] After activating Lactobacillus paracasei K56 with MRS liquid medium, the inoculum was added to 200 mL MRS broth at a 2% (v / v) inoculation rate and cultured at 37°C during the culture period. Four groups of experiments were set up:

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

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

[0079] The bacterial suspension was pre-frozen at -80°C for 6 hours.

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

[0081] It should be noted that C18:0 or CDP-DAG was added to the culture medium at 0 or 8 hours respectively, participating in the process of biofilm formation. The exogenous addition of C18:0 and CDP-DAG reduced the direct damage to lactic acid bacteria cells during the drying process. In the method of the above embodiments, the concentration of the C18:0 solution was 0.4 mM, and the addition amounts were 10, 20, and 40 μL respectively; the concentration of the CDP-DAG solution was 10 mM, and the addition amounts were 10, 20, and 40 μL respectively.

[0082] Example 2

[0083] This example provides a method for improving the freeze-drying survival rate of probiotics (Lactobacillus paracasei K56) based on cell membrane regulation, which specifically includes the following steps:

[0084] Prepare C18:1 as a solution for later use. The solution concentration is 1 mol / L, and the addition amounts are 1, 2, and 4 ml.

[0085] Using MRS liquid medium, inoculate the Lactobacillus paracasei strain into 200 mL of MRS broth at an inoculation amount of 2% (v / v), and culture it at 37 °C for 20 h. Then centrifuge the culture to harvest the cell precipitate, wash it twice and resuspend it with 70 mL of PBS buffer, and add different volumes of oleic acid.

[0086] Pre-freeze the bacterial suspension. The pre-freezing temperature is -45 °C, and the pre-freezing time is 10 hours.

[0087] Freeze-drying conditions: the cold trap temperature is -45 °C, the vacuum degree is 8 Pa, and the freeze-drying time is about 36 hours.

[0088] It should be noted that C18:1 was added to the bacterial sludge as a protective carrier and participated in the repair process of damaged biofilms. The exogenous addition of C18:1 reduced the direct damage to lactic acid bacteria cells during the drying process.

[0089] Test Example 1

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

[0091] (1) Determination of anti-freeze-drying ability

[0092] After freeze-drying, dissolve 1 g of freeze-dried bacterial powder K56-F in 10 mL of sterilized 0.85% (v / v) NaCl solution, and then serially dilute it. Inoculate the bacterial solution diluted to the final concentration on an MRS agar plate and culture it at 37 °C for 72 h. The number of colonies on the plate is between 30 and 300 CFU. Set 3 parallel plates for each dilution gradient, and calculate the survival rate:

[0093] Survival rate (%) = Number of viable bacteria after lyophilization (CFU / g) / Number of viable bacteria before lyophilization (CFU / g) × 100%.

[0094] Figure 4 This is the result of the survival rate of viable bacteria after freeze-drying of Lactobacillus paracasei K56 with the exogenous addition of C18:0, the precursor of C18:1. From Figure 4 it can be seen that: In the C18:0 experimental group, the overall level of the freeze-drying survival rate after supplementing C18:0 at 0 h in the initial stage of cultivation was higher than that of the control group. Among the three supplemented doses, the addition of 40 μL of K56 had the most significant effect on improving the survival rate, which was 43.99% (about 1.47 times). This may be because the K56 biofilm may cause biofilm remodeling by taking up C18:0 during the formation stage ( Figure 5 is the metabolic pathway of C18:1 and its precursor C18:0), thereby improving the membrane stress resistance.

[0095] Figure 6 This is the result of the survival rate of viable bacteria after freeze-drying of Lactobacillus paracasei K56 obtained under freeze-drying conditions with the exogenous addition of cardiolipin precursor CDP-DAG. From Figure 6 it can be seen that: In the CDP-DAG experimental group, from the dose of 10 μL added to 40 μL, the freeze-drying survival rate of the K56 strain showed a trend of first increasing and then decreasing. Adding the highest dose of 40 μL instead led to a decrease in the stress resistance of the strain. The content of cardiolipin in the biofilm itself is relatively small. Therefore, if the content of the membrane is too much, it may cause negative feedback regulation of the biofilm ( Figure 7 is the metabolic pathway of cardiolipin and its precursor CDP-DAG), resulting in poor stress resistance of the biofilm. For 20 μL of CDP-DAG added at 0 h, the increase in the survival rate of K56 after freeze-drying was the largest, from 31.58% to 46.24% (about 1.46 times), indicating that CDP-DAG phospholipid molecules may improve the freeze-drying resistance of the biofilm by synthesizing cardiolipin in the biofilm.

[0096] (2) Determination of lactate dehydrogenase (LDH) activity

[0097] Weigh 0.016 g of the lyophilized bacterial powder, dissolve it with 1 mL of PBS sterilized and filtered through a 0.22 μm filter, and collect the supernatant after centrifugation (10000 g, 4 °C, 10 min). The enzyme activity was carried out according to the steps of the LDH assay kit. One enzyme activity unit was defined as the production of 1 μmol of pyruvate in the reaction system, and the enzyme assay was performed in triplicate.

[0098]

[0099] During the freeze-drying process, lactic acid bacteria are subjected to stress, which causes the cell membrane to lyse and intracellular enzymes to leak out of the cell. However, it has no effect on the activities of lactic acid enzymes (such as lactate dehydrogenase and ATPase, etc.). Therefore, the degree of cell membrane damage can be indirectly reflected by measuring the activity of extracellular enzymes. As Figure 8 shown in a1 and a2 of

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

[0101] Weigh about 0.02 g of the freeze-dried bacterial powder, dissolve it with 1 mL of PBS sterilized and filtered through a 0.22 μm aqueous filter membrane, and collect the supernatant after centrifugation (10000 g, 4 °C, 10 min). The enzyme activity was measured by following the enzymatic reaction and phosphate determination reaction of the ultra-micro Na + -K + -ATP assay kit in sequence, and finally measure the absorbance of the sample at 636 nm. The enzyme assay was performed in triplicate.

[0102] Similarly, Na + -K + -ATPase is a protein in the cytoplasmic membrane bilayer that acts as a carrier and an enzyme. It catalyzes the hydrolysis of ATP to provide energy, maintains the membrane potential on both sides of the cell membrane, regulates cell osmotic pressure, and provides power for nutrient absorption. By measuring the activity of Na + -K + -ATPase, the degree of cell membrane damage can also be indirectly reflected. As Figure 8 shown in b1 and b2 of + -K + -ATPase activity decreased significantly after adding 20 μL and 40 μL of C18:0 (b1) and CDP-DAG (b2) to the K56 culture medium, indicating that the membrane integrity was effectively improved.

[0103] As Figure 8 shown in c of , the integrity of the K56 and the bacterial membranes added with C18:0 and CDP-DAG was measured by bacterial live-dead fluorescence staining. 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 that the number of live cells in K56 was relatively small. Under the PI staining channel, the proportion of red fluorescence intensity of K56 was the highest.

[0104] Therefore, in summary, the activity of lactate dehydrogenase (LDH), the activity of Na + -K + -ATPase, and the results of bacterial live / dead fluorescence staining showed that after supplementing C18:0 and CDP-DAG, the membrane integrity of K56 could be significantly improved, indirectly increasing the number of viable cells after freeze-drying of K56.

[0105] (4) Infrared spectroscopy

[0106] Appropriately weighed amounts of freeze-dried K56, K56+C18:0, and K56+CDP-DAG powders were placed in an agate mortar. They were thoroughly ground and mixed evenly according to the mass ratio of the sample to KBr powder of approximately 1:100 until a fine and homogeneous mixture of the sample and KBr powder was formed. An appropriate amount of the ground mixture was carefully placed into the die of a tablet press and pressed for 1 minute under a certain pressure (usually 8 kgf / cm 2 ), to obtain a transparent thin film sample. The Fourier transform infrared spectrometer was set with the corresponding scanning parameters, with the scanning wavenumber range of 4000 - 400 cm -1 , resolution 4 cm -1 , and scanning times 64 times to obtain the infrared spectrum.

[0107] As Figure 9 shown by the infrared spectrum, the infrared absorption characteristics of K56, K56+C18:0, and K56+CDP-DAG and the changes in the peak intensity of the K56 cell membrane after supplementing exogenous substances were presented. There was an absorption peak at approximately 3295 cm -1 for K56, K56+C18:0, and K56+CDP-DAG, which might be related to the stretching vibration of hydroxyl groups (―OH), indicating that K56 might contain compounds or groups containing hydroxyl groups. The absorption peaks at 1371 cm -1 ~1505 cm -1 might be related to the skeletal vibration of aromatic rings or certain specific C-H bending vibrations. In addition, compared with K56, the absorption peak at approximately 1666 cm -1 showed a weakened vibration and slightly different peak intensities after adding C18:0 and CDP-DAG, which might be attributed to the substitution reaction of the stretching vibration peak of the carbonyl group (C=O) with the functional groups in C18:0 and CDP-DAG, generating ester groups, ketone groups, etc., which had a significant impact on the composition of the cell membrane phospholipids. There were also vibration peaks in other wavenumber regions, such as at 1247 cm -1 , which might be related to the stretching vibration of carbon-oxygen (C―O) or other related vibration modes, further reflecting the changes in the cell membrane phospholipid structure after exogenous supplementation of 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] Determination of cryoprotective drying ability

[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 diluted bacterial solution was inoculated onto MRS agar plates and cultured at 37 °C for 72 h. The number of colonies on the plates was between 30 and 300 CFU. Three parallel plates were set for each dilution gradient, and the survival rate was calculated as follows:

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

[0113] As Figure 10 The results of the effect of exogenous addition of C18:1 as a protective agent on the survival rate of K56 strains after cryoprotective drying showed that different volumes of oleic acid (1 M) were added before freeze-drying, and the survival rate of Lactobacillus paracasei K56 increased from 31.82% to 43.95% (P < 0.05), 44.8% (P < 0.05), and 47.08% (P < 0.01) respectively, with the maximum increase of 15.25%. The main mechanisms of action of oleic acid as a cryoprotective drying agent include: (1) its surface-active properties can form a protective film around the molecules, stabilizing the protein and cell membrane structures; (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 biological membranes. These properties enable oleic acid to exhibit good protective effects under freezing and drying stress conditions. Therefore, based on the above results, it can be concluded that exogenous addition of oleic acid as a protective agent can also play a significant role in protecting the bacterial membrane during the freeze-drying process.

[0114] In addition to the above traditional detection of freeze-dried survival rate, the present invention also explored the interaction mechanism between the key lipid molecules C18:1, CL and the bacterial membrane by molecular dynamics simulation.

[0115] Molecular Dynamics (MD) simulation has become an important tool for studying the structure and function of biological membranes and the impact of external molecules on the membrane. In recent years, significant progress has been made in MD simulations 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, specifically designed for simulating large-scale biomolecular systems, especially biological membranes, proteins, nucleic acids, etc. Its coarse-grained nature enables simulations to be carried out on longer time scales and larger system sizes. By simplifying the atoms in complex molecular systems into fewer particles (usually 4 atoms are represented as 1 particle), this greatly reduces the number of particles and computational effort in the system, thus 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 Figure 11 , Figure 12 , Figure 13 shown are the results of the impact on the area per lipid molecule of the membrane and the distance between phospholipid molecular headgroups after intervention with C18:1 and CL through molecular dynamics simulation in nm 2 and. The total simulation duration for the molecular dynamics simulation is 300 ns, and the temperature change is set to gradually decrease from -20 °C to -60 °C. After the temperature changes to -60 °C, the system is allowed to equilibrate. The system reaches equilibrium at the 200th ns of the simulation duration. The data results output at this time are as Figures 11 - 13 shown. As can be seen from Figures 11 - 13 , when the temperature changes from -20 °C to -60 °C and C18:1 and CL are added to the membrane, the area per lipid molecule of the membrane (Area per lipid) decreases significantly compared to the blank membrane. This indicates that after the content of C18:1 and CL in the membrane increases, the arrangement of phospholipid molecules becomes looser, which helps the bacterial membrane resist low-temperature environments.

[0117] As shown in Table 1 are the results of the changes in the distances between the C18:1 and CL phospholipid molecular headgroups during the low-temperature freezing process of the bacterial membrane in the molecular dynamics simulation. As can be seen from Table 1, the maximum distance between the phospholipid headgroups does not change significantly, indicating that the membrane thickness remains basically unchanged.

[0118] Table 1

[0119]

[0120] As Figure 14The figure shows the detailed observation and analysis results of the process of cardiolipin entering the bacterial membrane through molecular dynamics simulation, aiming to deeply understand the interaction mechanism between cardiolipin and the bacterial membrane. At the initial moment (0 ns), cardiolipin molecules (represented by white balls) are mainly distributed in the aqueous environment (pink part) surrounding the bacterial membrane composed of DPPC (dark gray) and POPG (green). As time goes by, by 200 ns, some cardiolipin molecules begin to contact the surface of the bacterial membrane and gradually embed into 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 expands, showing that the process of its entering the bacterial membrane has a certain continuity and gradualness. At 600 ns, the distribution of cardiolipin molecules in the bacterial membrane further diffuses and begins to penetrate deeper into the bacterial membrane. By 800 ns, the distribution of cardiolipin molecules in the bacterial membrane becomes more uniform, and the depth further increases, indicating that the process of its entering the bacterial membrane is still ongoing. Finally, at 1000 ns, the cardiolipin molecules reach a relatively stable distribution state in the bacterial membrane and interact with the lipid molecules of the bacterial membrane. Therefore, the present invention speculates that cardiolipin may gradually insert into the bacterial membrane by interacting with specific binding sites on the surface of the bacterial membrane and diffuse within the membrane. This process may be affected by various factors, such as the concentration of cardiolipin molecules, the lipid composition of the bacterial membrane, and environmental conditions, etc.

[0121] As can be seen from the above test results, the method for improving the survival rate of probiotics based on cell membrane regulation of the present invention utilizes key lipid molecules directly related to resistance, and by supplementing key lipid components and their precursor substances in a dual manner, that is, improving the tolerance of strains at the metabolic level, significantly improves the freeze-drying resistance and survival rate of strains, and at the same time can significantly improve the later stability.

Claims

1. A method for improving the survival rate of probiotics based on cell membrane regulation, characterized in that The method includes: adding lipid components during the probiotic cultivation process and / or adding them as protective agents after the probiotic cultivation; The lipid components added during the probiotic cultivation process include cytidine diphosphate diacylglycerol and / or stearic acid; The lipid components added as protective agents after the cultivation include cardiolipin and / or oleic acid.

2. The method according to claim 1, wherein The time for adding lipid components during the probiotic cultivation process is 0 - 4 hours and / or 6 - 14 hours.

3. The method according to claim 1, characterized in that Based on the mixture obtained after adding lipid components during the probiotic cultivation process, the molar concentration of cytidine diphosphate diacylglycerol is 0.1 - 10 μM.

4. The method according to claim 1, characterized in that, Based on the mixture obtained after adding lipid components during the probiotic cultivation process, the molar concentration of stearic acid is 5 - 200 nM.

5. The method according to claim 1, wherein Based on the mixture obtained after adding lipid components as protective agents after the cultivation, the concentration of cardiolipin is 0.1 - 5% w / w.

6. The method according to claim 1, wherein Based on the mixture obtained after adding lipid components as protective agents after the cultivation, the concentration of oleic acid is 0.2 - 10% w / w.

7. The method according to claim 1, wherein The genus of the probiotics includes one or a combination of two or more of Lactobacillus, Lactobacillus casei, Lactobacillus mucosae, Lactobacillus plantarum, Lactobacillus collinoides, Lactobacillus paraplantarum, Bifidobacterium, Streptococcus, Lactococcus.

8. The method according to claim 1, characterized in that, The probiotics include one or a combination of two or more of Lactobacillus paracasei, Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus gasseri.

9. The method according to claim 1, characterized in that, The method further includes: subjecting the probiotic-containing suspension obtained after adding lipid components during the probiotic cultivation process and / or adding them as protective agents after the probiotic cultivation to a freezing treatment.

10. The method according to claim 9, characterized in that, The freezing treatment includes a pre-freezing treatment and a freeze-drying treatment; The temperature of the pre-freezing treatment is -80 to -45 °C, and the time is 4 - 12 hours; The conditions of the freeze-drying treatment are: the temperature is -80 to -40 °C, the vacuum degree ≤ 20 Pa, and the freeze-drying time is 20 - 40 h.

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