Culture method for improving spray drying resistance of lactic acid bacteria and application

Abstract

The invention belongs to the technical field of lactic acid bacteria culture, and particularly discloses a culture method for improving the spray drying resistance of lactic acid bacteria and application of the culture method.The method comprises the steps that in the strain culture process, exogenous phospholipid or analogues are added into a culture medium, and an exogenous supplementary membrane phospholipid culture medium is obtained; culturing the lactic acid bacteria by adopting an exogenous supplement membrane phospholipid culture medium; in the exogenous supplementary membrane phospholipid culture medium, the addition amount of exogenous phospholipid or analogues is 0.25-2.50 [mu] mol / L. According to the method, the phospholipid structure of a thallus membrane can be remodeled, and the stress resistance of the membrane is enhanced, so that the survival rate of lactic acid bacteria in the spray drying process is remarkably improved.

Description

Technical Field

[0001] This invention belongs to the field of lactic acid bacteria culture technology, specifically relating to a culture method and its application for improving the resistance of lactic acid bacteria to spray drying. Background Technology

[0002] Lactic acid bacteria, as an important functional ingredient, are widely used in the food and pharmaceutical industries. They are usually processed into powder form for convenient transportation and storage. In recent years, spray drying has become an important method for the industrial production of dried lactic acid bacteria due to its lower energy consumption, higher productivity, and more continuous production mode. However, the damage caused by dehydration and heat inactivation during spray drying can significantly affect the stability of cellular components bound to water molecules, especially cell membrane components.

[0003] Currently, industrial methods typically employ the addition of protective agents (such as sugars, proteins, or polyols) to enhance the survival rate of bacterial strains after drying. While this can improve strain resistance to some extent by forming a physical barrier to stabilize cell membrane structure, its effectiveness often depends on the concentration of the protective agent and environmental conditions; furthermore, the mechanisms of action of protective agents are mostly physical protection, and they cannot fundamentally improve the structural properties of the cell membrane. Summary of the Invention

[0004] The purpose of this invention is to provide a cultivation method and application for improving the resistance of lactic acid bacteria to spray drying, which can reshape the phospholipid structure of the bacterial cell membrane, enhance the membrane's stress resistance, and thus significantly improve the survival rate of lactic acid bacteria during the spray drying process.

[0005] To achieve the above objectives, the present invention employs the following technical solution: According to a first aspect of the present invention, a method for improving the resistance of lactic acid bacteria to spray drying is provided, comprising the following steps: During the bacterial culture process, exogenous phospholipids or similar substances are added to the culture medium to obtain exogenous supplemented membrane phospholipid culture medium; the lactic acid bacteria are cultured using the exogenous supplemented membrane phospholipid culture medium.

[0006] By employing the above technical solution, and through the exogenous supplementation of phospholipids or similar substances, the survival rate of lactic acid bacteria during spray drying is significantly improved based on the remodeling of bacterial cell membrane phospholipids. Compared to the addition of protectants, the above technical solution is suitable for the industrial production of highly active probiotic preparations.

[0007] Adding exogenous phospholipids or analogues to the culture medium allows for the regulation of bacterial membrane stress by the bacterial strain itself. This may be because the addition of exogenous phospholipids or analogues alters the composition and content of fatty acids during bacterial cell membrane synthesis, thereby increasing the stability of the bacterial cell membrane bilayer structure and thus enhancing the strain's resistance to stress. Alternatively, the addition of exogenous phospholipids or analogues may encourage the strain to generate various phospholipid end products through different synthetic pathways, increasing the content of key phospholipid components and contributing to the increase in the length of phosphatidyl acyl chains in the bacterial cell membrane (i.e., increasing the content of long-chain acyl chains in the bacterial cell membrane phospholipids) and the content of saturated fatty acids in the bacterial cell membrane. This increases the rigidity of the cell membrane, enhances the strain's tolerance to high-temperature drying stress, and ultimately improves the survival rate of the strain after heat drying.

[0008] According to one embodiment of the present invention, the lactic acid bacteria is Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus plantarum, or Streptococcus thermophilus.

[0009] Furthermore, the lactic acid bacteria is *Lactobacillus delbrueckii* subsp. *bulgaricus* L4-2-12. *Lactobacillus delbrueckii* L4-2-12 was deposited on August 19, 2025, at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 20251858, located at the Wuhan University Collection Center, Luojia, Wuchang District, Wuhan, 430072, China.

[0010] According to one embodiment of the present invention, in the exogenous supplemented membrane phospholipid culture medium, the amount of exogenous phospholipid or similar substances added is 0.25-2.50 μmol / L.

[0011] Preferably, in the exogenous phospholipid supplementation culture medium, the amount of exogenous phospholipid or similar substance added is 0.50-2.00 μmol / L.

[0012] According to one embodiment of the present invention, the exogenous phospholipid or analogue is one or more of phosphatidic acid (PA), cardiolipin (CL), phosphatidylcholine (PC) or cytidine diphosphate diacylglycerol (CDP-DAG).

[0013] Furthermore, the exogenous phospholipid or analogue is CDP-DAG.

[0014] According to one embodiment of the present invention, the culture medium for the strain is MRS medium; the culture temperature is 35-40 °C; the culture time is 12-24 h; and the bacterial concentration at the end of the culture is 10. 9 -10 10 CFU / mL.

[0015] According to one embodiment of the present invention, an activation culture step is further included before strain culture; In the activation culture step, the culture medium is MRS liquid medium; the inoculum size is 2-4%; the culture temperature is 35-40℃; and the culture time is 18-24 h. After activation culture and at least two consecutive passages, the culture was inoculated into MRS medium for expansion culture.

[0016] According to one embodiment of the present invention, during the expansion culture of the strain, exogenous phospholipids or similar substances are added to the culture medium from 0 to 10 hours; the exogenous phospholipids or similar substances can be added all at once or in batches.

[0017] According to a second aspect of the present invention, a method for preparing a probiotic preparation is provided, wherein lactic acid bacteria are cultured using the above-described culture method; the method includes the following steps: After centrifuging the bacterial culture obtained from the strain, the bacterial sludge was collected and washed with a washing solution. The washed bacterial sludge is spray-dried to obtain a probiotic preparation.

[0018] According to one embodiment of the present invention, during the process of culturing lactic acid bacteria, the centrifugation speed is 4000-6000 rpm, the time is 10-15 min, and the temperature is 2-8 ℃.

[0019] According to one embodiment of the present invention, the washing solution is a sterile NaCl solution; the concentration of the sterile NaCl solution is 0.5-1% (w / v).

[0020] According to one embodiment of the present invention, during the spray drying process of the washed bacterial sludge, the inlet air temperature is 100-120±1 °C; the outlet air temperature is 60-80±1 °C; and the feed rate is 1-5 mL / min.

[0021] According to a third aspect of the present invention, a probiotic preparation is provided, which is prepared by culturing lactic acid bacteria using any of the above-described culturing methods, or by preparing any of the above-described probiotic preparation methods.

[0022] Compared with the prior art, the present invention has at least the following beneficial effects: 1. This invention provides a method for intervening in the formation process of lactic acid bacteria biofilms by supplementing with exogenous phospholipids or analogues. By reshaping the phospholipid structure of the bacterial biofilm, the stress resistance of the biofilm is enhanced, thereby improving the survival rate of the strain after spray drying. Supplementation with exogenous phospholipids or analogues can regulate the phospholipid composition of the biofilm, increasing the content of key phospholipids phosphatidylcholine and cardiolipin, the proportion of long-chain acyl chains, and the content of saturated fatty acyl groups, thereby improving the rigidity of the biofilm and enhancing the strain's tolerance to high-temperature drying stress.

[0023] 2. This invention significantly reduces the leakage of lactate dehydrogenase and Na+ into the extracellular space by supplementing with exogenous phospholipids or similar substances. + -K + -ATPase effectively maintains the integrity of the cell membrane, which helps improve the survival rate of the strain after spray drying.

[0024] 3. This invention can fundamentally solve the problem of poor strain resistance by regulating the strain itself, reduce or avoid the use of protectants in the spray drying process, lower the requirements for spray drying process conditions, and is environmentally friendly, making it suitable for the industrial production of highly active probiotic preparations. Attached Figure Description

[0025] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 Here are SEM images of Lactobacillus bulgaricus L4-2-12, which are involved in this invention; Figure 2 The survival rate test results of strain L4-2-12 in the probiotic powders obtained in Examples 1-3 and the comparative examples of the present invention; Figure 3 The results of LDH activity determination of the probiotic preparations obtained in Example 2 and the comparative example of this invention; Figure 4 Na is the probiotic preparation obtained in Example 2 and the comparative example of the present invention. + -K + - Results of ATPase activity assay; Figure 5 This is a schematic diagram illustrating how CDP-DAG generates various phospholipid end products through different synthetic pathways during the biosynthesis of endogenous membrane phospholipids. Figure 6 The results of determining the relative PI content in the bacterial film of the probiotic preparations obtained in Example 2 and the comparative example of the present invention; Figure 7 The results of the determination of the relative contents of PS, PE and PC in the bacterial films of the probiotic preparations obtained in Example 2 and the comparative example of the present invention; Figure 8 The results of the determination of the relative contents of PG and CL in the bacterial films of the probiotic preparations obtained in Example 2 and the comparative example of the present invention; Figure 9 The results show the relative CL content of different acyl chains with total carbon number in the bacterial films of the probiotic preparations obtained in Example 2 and the comparative example of this invention. Figure 10 The results show the determination of PC content in the bacterial films of probiotic preparations prepared in Example 2 and the comparative example of this invention, with different total carbon numbers of acyl chains. Figure 11 The results show the relative FA content of different acyl chains in the bacterial films of the probiotic preparations obtained in Example 2 and the comparative example of this invention. Figure 12 The results show the relative content of CL with different degrees of unsaturation in the bacterial films of the probiotic preparations obtained in Example 2 and the comparative example of this invention. Figure 13 The results show the relative content of PC with different degrees of unsaturation in the bacterial films of the probiotic preparations obtained in Example 2 and the comparative example of this invention. Figure 14 The results show the relative content of FA with different degrees of unsaturation in the bacterial films of the probiotic preparations obtained in Example 2 and the comparative example of the present invention. Detailed Implementation

[0026] The present invention will now be described in detail with reference to the accompanying drawings and embodiments. It should be noted that, unless otherwise specified, the embodiments and features described herein can be combined with each other.

[0027] The following detailed description is exemplary and intended to provide further detailed explanation of the invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used in this invention is for describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention.

[0028] Example 1 This embodiment provides a method for improving the resistance of lactic acid bacteria to spray drying, and a method for preparing probiotic preparations by spray drying of lactic acid bacteria cultured using this method. The lactic acid bacteria involved is *Lactobacillus bulgaricus* L4-2-12 (hereinafter referred to as strain L4-2-12), which was deposited at the China Center for Type Culture Collection on August 19, 2025, with accession number CCTCC NO: M 20251858, located at Wuhan University Collection Center, Luojia, Wuchang District, Wuhan, 430072, China. SEM images of strain L4-2-12 can be found [link to SEM image]. Figure 1The culture medium was MRS liquid medium, purchased from Qingdao Haibo Co., Ltd.

[0029] (a) Culture of Lactobacillus bulgaricus L4-2-12 1) Activation culture: Inoculate strain L4-2-12 at a 2% inoculum into MRS liquid medium preheated to 37 ℃ and activate it at 37 ℃ for 24 h. Subculture twice to restore the viability of the strain.

[0030] 2) Scale-up culture: Fresh bacterial suspension of strain L4-2-12 obtained from activation culture was inoculated into MRS liquid medium at a 2% inoculum for scale-up culture and incubated aerobically at 37 ℃ for 20 h. At the end of the culture, the bacterial suspension concentration was 10... 9 -10 10 CFU / mL.

[0031] At the initial stage of cultivation (0 h), exogenous phospholipids or similar substances were added to the culture medium to obtain exogenous membrane-supplemented phospholipid culture medium. In this example, the exogenous phospholipid or similar substance was CDP-DAG. For distinction, Lactobacillus bulgaricus L4-2-12 cultured on exogenous membrane-supplemented phospholipid culture medium supplemented with CDP-DAG is designated as strain L4-2-12. CDP-DAG The amount of CDP-DAG added was 0.5 μmol / L, and CDP-DAG was purchased from Sigma-Aldrich Shanghai Trading Co., Ltd.

[0032] In other embodiments, the activation culture time can be set to 18-24 h, the culture temperature can be set to 35-40 ℃, and the inoculum size can be set to 2-4%.

[0033] In other embodiments, the expansion culture time can be set to 12-24h according to the actual situation; the culture temperature can be set to 35-40℃; the inoculum amount can be set to 2-4%; and the CDP-DAG addition time is a suitable time such as 2h, 4h, 6h, 8h or 10h after the start of the expansion culture of lactic acid bacteria.

[0034] In other embodiments, during the scaling-up process, the exogenous phospholipid or analogue may be one or more of PA, CL, PC, or CDP-DAG.

[0035] (II) Preparation of probiotic preparations Centrifuge the bacterial culture of strain L4-2-12 after step (1) expansion culture to collect bacterial sludge. The centrifugation speed is 6000 rpm, the time is 15 min, and the temperature is 4℃.

[0036] Then, the bacterial sludge was washed three times with a sterile NaCl solution at a concentration of 0.85% (w / v).

[0037] The washed bacterial sludge is spray-dried to obtain a probiotic preparation.

[0038] During the spray drying process, the washed bacterial sludge was first uniformly resuspended in a protectant solution using a vortex mixer. The protectant solution consisted of 20% (w / v) maltodextrin and 10% (w / v) whey protein isolate. The volume ratio of the protectant solution to the MRS liquid medium used in the expansion culture of strain L4-2-12 was 1:4.

[0039] The spray drying process conditions are as follows: inlet temperature 120±1°C, outlet temperature 60±1°C, feed rate 5 mL / min, atomizing air flow rate 600 L / h, and stirring speed 600 rpm.

[0040] In other embodiments, during the collection of bacterial sludge, the centrifugation speed can be set to 4000-6000 rpm, the centrifugation time can be set to 10-15 min, and the centrifugation temperature can be set to 2-8℃. In other embodiments, during the spray drying process, the inlet air temperature can be set to 100-120±1 °C; the outlet air temperature can be set to 60-80±1 °C; and the feed rate can be set to 1-5 mL / min.

[0041] Example 2 The difference between this embodiment and Embodiment 1 is that: In the cultivation of Lactobacillus bulgaricus L4-2-12, in step 2), the amount of CDP-DAG added was 1.00 μmol / L.

[0042] All other steps and conditions are the same.

[0043] Example 3 The difference between this embodiment and Embodiment 1 is that: During the cultivation of Lactobacillus bulgaricus L4-2-12, in step 2), the amount of CDP-DAG added was 2.00 μmol / L.

[0044] All other steps and conditions are the same.

[0045] Comparative Example The difference between this comparative example and Example 1 is that: In the culture of Lactobacillus bulgaricus L4-2-12, CDP-DAG is not added in step 2).

[0046] All other steps and conditions are the same.

[0047] Test Example 1 Survival rate determination The probiotic powder preparations obtained in Examples 1-3 and the comparative example were weighed and dissolved in 0.85% (w / v) sterile NaCl solution, and vortexed for 1 min to ensure complete dissolution; the mass-to-volume ratio of probiotic powder to sterile NaCl solution was 100 mg: 1 mL. The viable cell count was determined using the MRS agar plate counting method. The survival rate of strain L4-2-12 after drying was calculated using the following formula: Survival rate (%) = ×100%.

[0048] The survival rate increase of strain L4-2-12 in the probiotic powder preparations obtained in Examples 1-3 and the comparative examples is shown in the following figures. Figure 2 Combining Figure 1 As shown, the survival rates of strain L4-2-12 in the probiotic preparations obtained in the comparative example and Examples 1-3 were 0.52% ± 0.06%, 1.82% ± 0.24%, 1.77% ± 0.10%, and 0.45% ± 0.05%, respectively. It is evident that, compared to the comparative example, the survival rates of strain L4-2-12 in the probiotic preparations obtained in Examples 1 and 2 were increased by 3.5 times and 3.4 times, respectively; indicating that adding an appropriate amount of CDP-DAG to the culture medium during the culture stage (especially the initial culture stage) helps improve the resistance of lactic acid bacteria to spray drying and increases the survival rate of the strain after spray drying. The survival rate in Example 3 decreased compared to the comparative example, which may be due to precursor overload disrupting membrane homeostasis. Therefore, it can be inferred that the addition of an appropriate amount of CDP-DAG plays a role in membrane phospholipid synthesis and stress response.

[0049] Test Example 2 Lactate dehydrogenase (LDH) activity assay The probiotic powders prepared in Example 2 and the comparative example were reconstituted in 0.85% (w / v) sterile NaCl solution, with a mass-to-volume ratio of probiotic powder to sterile NaCl solution of 16 mg:1 mL. After centrifugation at 10,000 g for 10 min, the supernatant was collected, and LDH activity was determined by visible spectrophotometry using an LDH detection kit at 37 °C. The LDH detection kit was purchased from Nanjing Jiancheng Biotechnology Co., Ltd.

[0050] Enzyme activity units are defined as the amount of enzyme that produces 1 μmol of pyruvate per gram of tissue protein reacting with the substrate for 15 min at 37 °C. Absorbance was measured spectrophotometrically at 440 nm, with three replicates for each sample. Results are shown below. Figure 3 .

[0051] Disruption of cell membrane integrity leads to the leakage of cytoplasmic contents and membrane-associated enzymes into the extracellular space. Therefore, detecting the activity of leaking enzymes in probiotic preparations can quantitatively assess the degree of cell membrane damage, providing a reliable indirect evaluation method. Lactate dehydrogenase, a commonly used intracellular enzyme, is frequently employed to assess cell membrane integrity.

[0052] like Figure 3 As shown in Example 2, the addition of CDP-DAG to the culture medium at a concentration of 1.00 μmol / L significantly reduced the extracellular LDH activity of strain L4-2-12 (P<0.05). Compared with the control group, the extracellular LDH activity decreased from 6.71 U / L to 2.24 U / L, showing a significant difference. This indicates that the addition of exogenous phospholipids or similar substances during strain cultivation improved the integrity of the bacterial membrane and prevented more LDH from leaking into the extracellular space.

[0053] Test Example 3 Na + -K + -ATPase activity assay The probiotic powders prepared in Example 2 and the comparative example were reconstituted in 0.85% (w / v) sterile NaCl solution, with a mass-to-volume ratio of probiotic powder to sterile NaCl solution of 10 mg: 1 mL. After centrifugation at 10,000 g for 10 min, the supernatant was collected and subjected to ultra-micro NaCl treatment. + -K + -The ATP kit uses spectrophotometry to determine its activity. Trace amounts of Na... + -K + - The ATP kit was purchased from Nanjing Jiancheng Biotechnology Co., Ltd.

[0054] Enzyme activity units are defined as: the production of 1 μmol of inorganic phosphorus per milligram of tissue protein per hour from the breakdown of ATP, i.e., μmol Pi / mg protein / hour. The absorbance of the samples was measured at 636 nm using a spectrophotometer, with three replicates for each sample. See [link to results]. Figure 4 .

[0055] Na + -K + -ATPase is a membrane-integrated protein whose main function is to provide energy for ion transmembrane transport by catalyzing the hydrolysis of ATP, thereby maintaining cell membrane potential, regulating osmotic pressure, and providing the driving force for nutrient absorption. Figure 4 Compared to the comparative example, the probiotic powder obtained in Example 2 has a lower Na content. + -K +-ATPase activity decreased significantly from 0.30 U / L to 0.16 U / L, indicating that strain L4-2-12 treated with CDP-DAG maintained better membrane integrity after spray drying. This suggests that the addition of exogenous phospholipids or similar substances during strain cultivation may enhance the resistance of bacterial membranes, which is speculated to be related to the remodeling of the lipid composition of the bacterial membrane.

[0056] Test Example 4 Biofilm lipidomics analysis The probiotic preparations obtained in Example 2 and the comparative example were further analyzed using UHPLC-MS / MS to examine the effect of supplementing with exogenous phospholipids or analogues on the bacterial film composition. The specific methods are as follows: Pretreatment: The probiotic powders prepared in Example 2 and the comparative example were placed in grinding tubes, and ammonium carbonate butanol-methanol solution (1:1, v / v) and 10 μL of internal standard were added, along with 2 steel balls. After homogenization for 5 min, the mixture was vortexed for 30 s, followed by sonication at room temperature for 1 h. After centrifugation at 13,000 g for 10 min, the supernatant was collected for later use. Information regarding the composition, brand, or manufacturer of the internal standard is provided in Table 1.

[0057] Table 1 Internal Standard Related Information

[0058] The concentration of the ammonium carbonate butanol-methanol solution was 10 mM; the mass-to-volume ratio of the probiotic preparation powder to the ammonium carbonate butanol-methanol solution was 2 mg:19 μL; and the volume ratio of the ammonium carbonate butanol-methanol solution to the internal standard was 19:1.

[0059] Lipid separation: Lipids in the pretreatment solution were separated using a Nexera X2 LC-30AD ultra-high performance liquid chromatography system. The mobile phase composition was as follows: Solvent A: 50% acetonitrile (containing 10 mM ammonium acetate, pH 8.0); Solvent B: 100% acetonitrile. The gradient elution program was as follows: 0–0.1 min: 90% B; 0.1–5 min: B linearly decreased from 90% to 65%; 5–5.3 min: B linearly decreased from 65% to 0%; 5.3–7.2 min: 0% B; 7.2–7.4 min: B linearly increased from 0% to 90%; 7.4–10 min: 90% B; Chromatographic conditions: column temperature 35 ℃, flow rate 300 µL / min, injection volume 6 µL.

[0060] Mass spectrometry analysis: A Triple Quad 6500+ mass spectrometer (AB SCIEX) was used for detection in both positive and negative ion modes. Each sample was injected three times using different mass spectrometry methods. The ion source parameters were set as follows: ion spray voltage: +5500 V (positive ion mode) / -4500 V (negative ion mode); ion source temperature: 400 ℃; ion source gas 1: 55 psi; ion source gas 2: 60 psi; curtain gas: 35 psi; collision gas: 8 psi. The declustering potential and collision energy parameters were optimized for different lipid classes to ensure detection sensitivity.

[0061] The peak areas were extracted and integrated using Multiquant software. Lipid identification and quantification were performed using the internal standard method by comparing the retention times with lipid standards. The lipid concentration calculation formula is as follows: C = ; Where C represents lipid concentration in ng / mg; R represents the peak area ratio of the sample to the internal standard; MIS represents the amount of internal standard added; and V represents the sample volume. All statistical analyses and bioinformatics processing were performed using the R programming language. Lipid nomenclature followed the LIPIDMAPS rules (https: / / doi.org / 10.1194 / jlr.S120001025).

[0062] In the biosynthesis of endogenous membrane phospholipids, CDP-DAG is a key metabolic branching intermediate, generated from phosphatidic acid (PA) and cytidine triphosphate via CDP-DAG synthase. CDP-DAG can generate various phospholipid end products through different synthetic pathways. Specifically, CDP-DAG participates in three major lipid synthesis pathways, generating phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylglycerol (PG), and cardiolipin (CL), respectively. See [link to relevant documentation]. Figure 5 The synthesis of CDP-DAG is evolutionarily conserved, and the CDP-DAG-dependent phosphatidylserine (PS) biosynthesis pathway is unique to prokaryotes and absent in eukaryotes. This test example uses targeted lipidomics to comprehensively analyze the changes in the phospholipid composition of the bacterial membranes of the probiotic preparations obtained in Example 2 and the comparative examples, in order to further elucidate the regulatory mechanism of CDP-DAG on lipid synthesis metabolism after entering the bacterial membrane. The detection results are shown below. Figures 6-8 .

[0063] Figure 6 The results show the determination of PI content in the bacterial film of the probiotic preparations obtained in Example 2 and the comparative example. Figure 7The results show the determination of PS, PE, and PC content in the bacterial films of the probiotic preparations obtained in Example 2 and the comparative example. Figure 8 The results of PG and CL content determination in the bacterial films of the probiotic preparations obtained in Example 2 and the comparative example are shown below. Figure 5 The three pathways for the formation of phospholipid end products from CDP-DAG are shown.

[0064] Combination Figures 5-8 The PI content synthesized via the first pathway in the bacterial films of the probiotic preparations obtained in the comparative example and Example 2 was 0.20 nmol / mg and 0.12 nmol / mg, respectively, which was 40% lower than the PI content in the comparative example and Example 2. In the second pathway, compared with the comparative example, the PS level in the bacterial film of the probiotic preparation obtained in Example 2 decreased significantly, while PE remained stable and PC increased, indicating that PC preferential synthesis may be at the expense of PS. In the third pathway, compared with the comparative example, the PG level in the bacterial film of the probiotic preparation obtained in Example 2 decreased by 87.95 nmol / mg, while CL increased by about 0.24 nmol / mg, indicating that CDP-DAG promoted the conversion of PG to downstream CL.

[0065] PIs are minor components of the cell membrane and may participate in regulating electrostatic interactions and intercellular signaling. CLs are lipids present in small amounts but highly stable within the membranes of several Gram-positive bacteria. They are formed by the condensation of two PG molecules under the action of cardiolipin synthases ClsA / B. Together with PGs, CLs constitute one of the most abundant anionic phospholipids in bacterial membranes. CLs are typically present during the bacterial growth phase, but accumulate in large quantities at the onset of the stationary phase, supporting their role in survival under stress.

[0066] Kato et al. constructed Lactobacillus gasseri JCM1131T ΔclsAΔclsB The mutant demonstrated the relationship between CL and bile acid resistance. Another study showed that increased CL content expands the volume of the phospholipid head group, thereby offsetting the toluene-induced acyl chain volume expansion and contributing to bilayer stability. Therefore, in Example 2, the addition of exogenous phospholipids or similar substances increased the CL content in the bacterial film, which helps improve the stability of the bacterial bilayer structure and enhances the cell's resistance to stress during spray drying.

[0067] Furthermore, membrane fluidity is primarily determined by two key structural parameters of the phosphatidyl chain: carbon chain length and degree of unsaturation. Membrane fluidity is closely related to the packing density of the phosphatidyl chain. Typically, increasing the average length of the phosphatidyl chain is an important adaptive mechanism of strain phospholipids in response to high-temperature stress.

[0068] like Figures 9-11As shown, compared with strain L4-2-12 in the comparative example, strain L4-2-12 obtained in Example 2 by adding CDP-DAG to the culture medium... CDP-DAG The content of acyl chains with a total of 74 carbon atoms in the CL group of the membrane was significantly increased. A similar pattern was also observed in PC, in strain L4-2-12 of Example 2. CDP-DAG The content of acyl chains with a total of 34 carbon atoms increased. Furthermore, for FA (fatty acyl groups), strain L4-2-12... CDP-DAG The results showed a higher level of acyl chains with a total carbon number of 20 atoms, indicating that the addition of CDP-DAG significantly promoted the content of long-chain acyl chains in phospholipids.

[0069] An increase in the content of saturated fatty acids in the cell membrane may improve membrane rigidity, thereby enhancing the survival rate of strains after heat drying. Regulating the unsaturation of acyl chains is one of the most common mechanisms for regulating membrane fluidity. For example... Figures 12-14 As shown, compared with strain L4-2-12 in the comparative example, strain L4-2-12 obtained in Example 2 by adding CDP-DAG to the culture medium... CDP-DAG The content of monounsaturated acyl chains in CL and PC within the bacterial film was significantly increased, as was the content of diunsaturated acyl chains. Notably, the total content of unsaturated acyl chains increased significantly by 57.26%, from 69.09 nmol / mg to 108.65 nmol / mg, indicating that CDP-DAG helps promote the accumulation of unsaturated acyl chains in phospholipids, thereby increasing the rigidity of the bacterial film and indirectly enhancing its resistance to spray drying.

[0070] In summary, adding peripheral phospholipids or similar substances to the culture medium during the cultivation of lactic acid bacteria can reshape the phospholipid structure of the bacterial membrane, enhance the membrane's stress resistance, and thus significantly improve the survival rate of lactic acid bacteria during spray drying. It should be noted that the cultivation method for improving the spray drying resistance of lactic acid bacteria provided by this invention is also applicable to other lactic acid bacteria species, such as *Lactobacillus plantarum* and *Streptococcus thermophilus*.

[0071] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A method for cultivating lactic acid bacteria to improve their resistance to spray drying, comprising: During the bacterial culture process, exogenous phospholipids or similar substances are added to the culture medium to obtain exogenously supplemented membrane phospholipid culture medium; The lactic acid bacteria were cultured using an exogenously supplemented membrane phospholipid culture medium.

2. The cultivation method according to claim 1, characterized in that, The lactic acid bacteria are Lactobacillus bulgaricus, Lactobacillus plantarum, or Streptococcus thermophilus.

3. The cultivation method according to claim 1, characterized in that, In exogenous phospholipid supplementation culture medium, the amount of exogenous phospholipids or similar substances added is 0.25-2.50 μmol / L; The exogenous phospholipid or analogue is one or more of phosphatidic acid, cardiolipin, phosphatidylcholine, or cytidine diphosphate diacylglycerol.

4. The cultivation method according to claim 1, characterized in that, The exogenous phospholipid or analogue is cytidine diphosphate diacylglycerol.

5. The cultivation method according to claim 1, characterized in that, The strain was cultured using MRS medium at a temperature of 35-40 °C for 12-24 hours. At the end of the culture, the bacterial concentration was 10%. 9 -10 10 CFU / mL.

6. The cultivation method according to claim 1, characterized in that, The process includes an activation culture step before the strain is cultured. In the activation and culture step, the culture medium is MRS liquid medium; the inoculum size is 2-4%; the culture temperature is 35-40 ℃; and the culture time is 18-24 h. After activation culture and at least two consecutive passages, the culture was inoculated into MRS medium for expansion culture.

7. A method for preparing a probiotic preparation, comprising culturing lactic acid bacteria using the culture method described in claim 1; including the following steps: After centrifuging the bacterial culture obtained from the strain, the bacterial sludge was collected and washed with a washing solution. The washed bacterial sludge is spray-dried to obtain a probiotic preparation.

8. The method for preparing the probiotic preparation according to claim 7, characterized in that, During the process of collecting bacterial sludge after centrifuging the bacterial culture obtained from the strain, the centrifugation speed is 4000-6000 rpm, the time is 10-15 min, and the temperature is 2-8℃. During the washing of the bacterial sludge using a washing solution, the washing solution is a sterile NaCl solution.

9. The method for preparing the probiotic preparation according to claim 7, characterized in that, During the spray drying process of the washed bacterial sludge, the inlet air temperature is 100-120±1 °C; the outlet air temperature is 60-80±1 °C; and the feed rate is 1-5 mL / min.

10. A probiotic preparation, prepared by culturing lactic acid bacteria using the culture method described in any one of claims 1-6, or prepared using the probiotic preparation preparation method described in any one of claims 7-9.

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