A method for extracting pn / pdrn from lactobacillus bulgaricus and its application in soothing anti-inflammation

Abstract

This invention discloses a method for extracting PN / PDRN from Lactobacillus bulgaricus and its application in soothing and anti-inflammatory effects. Belonging to the field of biochemistry, this method solves the problems of unstable PN / PDRN sources and susceptibility to contamination in existing technologies. In this invention, the preparation relies on alkaline conditions and lysozyme to initially dissolve the cell wall of Lactobacillus bulgaricus, and then sodium dodecyl sulfate is used to disrupt the bacterial cell membrane, causing it to lyse. The conditions are mild, and PVP is added to protect the PN structure. The final product prepared by this invention effectively improves the purity of the obtained PN / PDRN.

Description

Technical Field

[0001] This invention relates to the field of biochemistry, and more specifically to a method for extracting PN / PDRN from Lactobacillus bulgaricus and its application in soothing and anti-inflammatory effects. Background Technology

[0002] Polydeoxyribonucleases (PDRN) are naturally derived, low-molecular-weight DNA derivatives. PDRN is a small, scaffold-free, short-chain single helix with a molecular weight ranging from 50 to 1500 kDa. Currently, it is mainly extracted from the testes of trout and salmon, which are rich in nucleic acids. Its clinical applications are gradually expanding, showing significant effects in tissue repair, wound healing, anti-ischemia, and anti-inflammation. Different molecular weights of PDRN can be applied to different fields, exerting powerful anti-inflammatory, repair, and regenerative effects: 700 kDa and above are used for skin regeneration, articular cartilage regeneration, and promoting bone formation; 350–500 kDa are used in pharmaceuticals for tissue regeneration, corneal regeneration, and treatment of musculoskeletal diseases; 40–60 kDa are used in cosmetics and food for barrier repair, wrinkle improvement, and skin beautification. Based on the application potential of PDRN in biomedicine and controlled drug release, upgraded PDRN products have also developed rapidly.

[0003] Polynucleotides (PNs) are macromolecules further optimized from PDRNs, possessing a long-chain double helix structure. PNs are high-molecular-weight polymer chains composed of identical bases (purine / pyrimidine bases), sugars (ribose / deoxyribose), and phosphate subunits. Alternatively, they can be interpreted as compositions of nucleotide monomers linked together by covalent bonds. Generally, a polynucleotide molecule consists of 14 or more nucleotide monomers. PNs possess a more stable long-chain molecular structure and a three-dimensional support structure. The long-chain structure of PNs can repair dermal cell damage, enhance the regenerative capacity of senescent cells, and promote extracellular matrix regeneration. The porous structure of PNs provides excellent mechanical support. As a natural biomolecule, it exhibits good biocompatibility; its enzymatic hydrolysis products can be naturally absorbed and utilized by the human body. This degradability makes PNs one of the ideal scaffold materials in tissue engineering. In bone tissue engineering, the three-dimensional porous structure of PNs can serve as a scaffold material for bone defect repair. Its porous structure facilitates cell adhesion, proliferation, and differentiation, thereby promoting the repair and regeneration of bone defects. In cartilage tissue engineering, cartilage tissue has limited self-repair capabilities after injury, thus requiring the use of external scaffold materials for repair. PN's three-dimensional porous structure can provide a favorable cell growth environment, promoting chondrocyte growth and cartilage tissue repair. In skin tissue engineering, PN's three-dimensional porous structure can serve as a scaffold material for skin defect repair, promoting skin regeneration and repair.

[0004] The current method of obtaining PDRN mainly relies on extraction from salmon sperm cells. However, salmon resources are very limited and are prone to supply fluctuations and environmental or biological pollution risks. Summary of the Invention

[0005] This invention provides a method for extracting PN / PDRN from Lactobacillus bulgaricus and its application in soothing and anti-inflammatory effects, in order to solve the problems of unstable sources and easy contamination in the prior art of obtaining PN / PDRN.

[0006] In a first aspect, the present invention provides a method for preparing polynucleotides using *Lactobacillus bulgaricus*, comprising the following steps: collecting cell precipitate from *Lactobacillus bulgaricus* fermentation broth, washing the cell precipitate and adding an extract for resuspension to obtain a resuspension; adding lysozyme to the resuspension, stirring thoroughly, adding sodium dodecyl sulfate (SDS) and polyvinylpyrrolidone, stirring thoroughly again to obtain a lysis buffer; centrifuging the lysis buffer and collecting supernatant I; resuspending and centrifuging the precipitate sequentially and collecting supernatant II; combining supernatant I and supernatant II and adjusting their pH to 6.5–7.5 to obtain a neutral supernatant; adding RNase and proteinase K sequentially to the neutral supernatant, centrifuging and collecting the supernatant to obtain a pure supernatant; adding anhydrous ethanol to the pure supernatant and performing fractional precipitation to obtain the polynucleotide.

[0007] As one possible implementation, the fractional precipitation includes the steps of: removing the filamentous precipitate from the reaction system, drying it to obtain polynucleotides; centrifuging the remaining reaction system overnight at -20°C, collecting the precipitate, washing it, and drying it to obtain polydeoxyribonucleotides.

[0008] As one possible implementation, the extraction solution comprises: 10–20 mM Tris-HCl, 1–2 M NaCl, and 1 mM EDTA, with a pH of 8.5–9.0; the amount of lysozyme added is such that the final concentration of lysozyme in the reaction system after addition is 5–20 mg / mL; the amount of SDS added is such that the final volume concentration of SDS in the reaction system after addition is 0.5%–1%; the amount of polyvinylpyrrolidone added is such that the final volume concentration of polyvinylpyrrolidone in the reaction system after addition is 1%–2%; the average molecular weight of polyvinylpyrrolidone is 58,000, K29-30; the amount of RNase added is such that the final concentration of RNase in the reaction system after addition is 20 μg / mL; and the amount of proteinase K added is such that the final concentration of proteinase K in the reaction system after addition is 20 μg / mL.

[0009] As one possible implementation, the method for preparing the bacterial precipitate in the Lactobacillus bulgaricus fermentation broth includes the following steps: centrifuging the Lactobacillus bulgaricus fermentation broth at 4°C and 10,000g, collecting the precipitate, and obtaining the bacterial precipitate.

[0010] As one possible implementation, the lysozyme is added and thoroughly mixed under the following conditions: 37°C, 1000–1500 rpm, and a mixing time of 1–2 hours; the mixture is then thoroughly mixed again under the following conditions: 1000–1500 rpm, and a mixing time of 2–3 hours.

[0011] In a second aspect, the present invention provides a polynucleotide obtained by the method described in any possible implementation of the first aspect, wherein the polynucleotide is a polynucleotide and / or a polydeoxyribonucleotide.

[0012] As one possible implementation, the polynucleotide promotes the secretion of interleukin-10.

[0013] As one possible implementation, the polynucleotide promotes the expression of the arginase 1 gene.

[0014] As one possible implementation, the polynucleotide promotes the expression of the VEGF gene.

[0015] In this invention, the cell walls of *Lactobacillus bulgaricus* are initially dissolved under alkaline conditions and with lysozyme, and then SDS is used to disrupt the bacterial cell membrane, causing lysis. These conditions are mild. Simultaneously, PVP is added to protect the PN structure (PVP has good dispersibility and film-forming properties, acting as a stabilizer to reduce the damage to the PN structure from external factors and lower the probability of PN hydrolysis into PDRN, but it cannot completely prevent it; some will still be damaged and hydrolyzed). Therefore, in the final product prepared in this invention, the mass ratio of high molecular weight PN to low molecular weight PDRN is approximately 2:1. Furthermore, PVP can bind to the polysaccharides released after bacterial lysis, which can be removed by centrifugation, effectively improving the purity of the obtained PN / PDRN (increasing the A260 / A230 ratio).

[0016] In the preparation process of this invention, pH is closely monitored and utilized. After lysis, *Lactobacillus bulgaricus* releases a large amount of acidic contents, making the reaction system acidic. Centrifugation under these conditions removes insoluble proteins and polysaccharides. Then, before the second purification, the pH is adjusted to neutral, which is beneficial for the function of RNase and proteinase K. Subsequent centrifugation also removes proteins and polysaccharides insoluble under neutral conditions, further improving the purity of PN / PDRN (increasing the A260 / A230 ratio). Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a schematic diagram of the morphology of the final product provided in an embodiment of the present invention, wherein A represents precipitate A, B represents sample A, C represents precipitate B, and D represents sample B.

[0019] Figure 2 The results of agarose gel electrophoresis provided in the embodiments of the present invention.

[0020] Figure 3 The SEM results of sample A provided in this embodiment of the invention.

[0021] Figure 4 The SEM results for sample B provided in this embodiment of the invention.

[0022] Figure 5 The SEM results of sample E provided in this embodiment of the invention.

[0023] Figure 6 This is a statistical graph showing the IL-10 protein secretion levels of Raw264.7 cells after treatment with samples A and B, provided in this embodiment of the invention; # indicates the significance between the "model group" and the "control group". # This indicates that the p-value is less than 0.05. ## This indicates that the p-value is less than 0.01. ### * indicates P-value < 0.001; * indicates significance analysis between "sample group" and "model group", * indicates P-value < 0.05, ** indicates P-value < 0.01, *** indicates P-value < 0.001.

[0024] Figure 7 A statistical graph showing the relative expression levels of the Arg-1 gene after treating Raw264.7 cells with samples A and B, provided in an embodiment of the present invention. # This indicates the significance between the "model group" and the "control group". # This indicates that the p-value is less than 0.05. ## This indicates that the p-value is less than 0.01. ### * indicates P-value < 0.001; * indicates significance analysis between "sample group" and "model group", * indicates P-value < 0.05, ** indicates P-value < 0.01, *** indicates P-value < 0.001.

[0025] Figure 8 A statistical graph showing the relative expression levels of the VEGF gene after treating Raw264.7 cells with samples A and B, provided in an embodiment of the present invention. # This indicates the significance between the "model group" and the "control group". # This indicates that the p-value is less than 0.05. ## This indicates that the p-value is less than 0.01. ###* indicates P-value < 0.001; * indicates significance analysis between "sample group" and "model group", * indicates P-value < 0.05, ** indicates P-value < 0.01, *** indicates P-value < 0.001. Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] To address the issues of unstable PDRN sources and susceptibility to contamination in existing technologies, this invention provides a method for preparing polynucleotides / polydeoxyribonucleotides using Lactobacillus bulgaricus, and the performance of the obtained products was tested.

[0028] Through experimental verification, the method of this invention successfully obtained PN and PDRN. Furthermore, both PN and PDRN obtained by this invention can significantly promote the secretion of interleukin-10, significantly promote the expression of arginase-1, and significantly promote the expression of VEGF. Therefore, PN and PDRN obtained by this invention can be applied to anti-inflammatory and immunomodulatory functions, promote tissue repair, and promote wound healing.

[0029] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0030] Example 1

[0031] This embodiment provides a method for preparing polynucleotides using Lactobacillus bulgaricus.

[0032] 25 L of Lactobacillus bulgaricus fermentation broth was centrifuged at 4 °C and 10000 g for 30 min, and the cell pellet was collected. The obtained cell pellet was washed twice with 2000 mL of 0.01 M PBS (pH 7.2) by centrifugation at 4 °C and 10000 g for 20 min. Then, 2300 mL of extraction buffer (composition: 10 mM Tris-HCl, 1 M NaCl, 1 mM EDTA, pH 8.86) was added to resuspend the pellet, and a resuspension was obtained. Lysozyme was added to the resuspension to a final concentration of 10 mg / mL, and the mixture was stirred at 37 °C and 1000 rpm for 1 h. Then, 1% SDS and 1% PVP were added, and the mixture was stirred at 37 °C and 1000 rpm for 3 h to obtain the lysis buffer. The lysis buffer was centrifuged at 4 °C and 17700 g for 20 min, and the supernatant I was collected. Add 2300 mL of 1 M NaCl to the precipitate again, resuspend, and centrifuge at 4 °C and 17700 g for 20 min. Collect supernatant II again. Combine supernatant I and supernatant II, and determine the pH of the combined supernatant to be 3.69. Adjust the pH of the supernatant to 7.25 using 10 M NaOH. Add RNase to the supernatant to a final concentration of 20 μg / mL, stir at 37 °C and 1000 rpm for 30 min, then add proteinase K to a final concentration of 20 μg / mL, stir at 37 °C and 1000 rpm for 30 min, centrifuge at 17700 g for 20 min, and collect the supernatant. Add 4600 mL of anhydrous ethanol to the obtained supernatant, remove the filamentous precipitate with a glass rod, and temporarily store it in 200 mL of anhydrous ethanol. This precipitate is designated as precipitate A, and its morphology is as follows. Figure 1 As shown in A; the remaining liquid was incubated overnight at -20°C, then centrifuged at 4°C and 17700g for 10 min, the precipitate was collected, washed once with 200 mL of anhydrous ethanol by centrifugation, and recorded as precipitate B, its morphology as shown in A. Figure 1 As shown in C. Precipitates A and B were freeze-dried separately to obtain the final product, denoted as sample A (its morphology is as shown in Figure C). Figure 1 As shown in B), sample B (its morphology is as shown in B). Figure 1 (as shown in D).

[0033] Example 2

[0034] This embodiment provides a method for preparing polynucleotides using Lactobacillus bulgaricus.

[0035] 8.1 L of *Lactobacillus bulgaricus* fermentation broth was centrifuged at 4 °C and 10000 g for 10 min to collect the cell pellet. The cell pellet was washed twice with 400 mL of 0.01 M PBS (pH 7.4) by centrifugation at 4 °C and 10000 g for 10 min. Then, 400 mL of extraction buffer (composition: 10 mM Tris-HCl, 1 M NaCl, 1 mM EDTA, pH 8.5) was added to resuspend the pellet, yielding a resuspended solution. Lysozyme was added to the resuspended solution at a final concentration of 5 mg / mL, and the mixture was stirred at 37 °C and 1000 rpm for 2 h. Then, 1% SDS and 1% PVP were added, and the mixture was stirred at 37 °C and 1000 rpm for 3 h to obtain a lysate. The lysate was centrifuged at 4 °C and 17700 g for 30 min, and the supernatant I was collected. Add 400 mL of 1 M NaCl to the precipitate again, resuspend, and centrifuge the resuspended solution at 4 °C and 17700 g for 10 min. Collect the supernatant (II) again. Combine supernatant (I) and supernatant (II), and determine the pH of the combined supernatant to be 3.46. Adjust the pH of the supernatant to 6.9 using 10 M NaOH. Add RNase to the supernatant to a final concentration of 20 μg / mL, stir at 37 °C and 1000 rpm for 30 min, then add proteinase K to a final concentration of 20 μg / mL, stir at 37 °C and 1000 rpm for 30 min, centrifuge at 17700 g for 30 min, and collect the supernatant. Add 800 mL of anhydrous ethanol to the obtained supernatant, remove the filamentous precipitate with a glass rod, and temporarily store it in 50 mL of anhydrous ethanol; this is precipitate C. Incubate the remaining liquid overnight at -20°C, then centrifuge at 4°C and 17700 g for 30 min. Collect the precipitate, wash it once with 50 mL of anhydrous ethanol, and this is precipitate D. Freeze-dry precipitates C and D separately to obtain the final products, denoted as sample C and sample D, respectively.

[0036] Example 3

[0037] This embodiment provides another method for preparing polynucleotides using bacteria.

[0038] Centrifuge 300 mL of Lactobacillus bulgaricus fermentation broth at 4 °C and 9000 rpm for 5 min to collect the cell pellet. Wash the pellet once with 300 mL of TE buffer (TE composition: 10 mM Tris-HCl, 1 mM EDTA, pH = 8.0), centrifuge at 9000 rpm for 5 min, add 20 mL of TE buffer, resuspend and mix well. Then add lysozyme to a final concentration of 0.2 mg / mL, incubate at 37 °C for 2 h, and homogenize at 1000 bar for 3 cycles to obtain cell lysate. Add 2.5 M NaCl and 1% SDS to the cell lysate, incubate at 60 °C for 1.5 h, centrifuge at 9000 rpm and 4 °C for 5 min, collect the supernatant to obtain the salting-out solution. Add sodium acetate to a final concentration of 0.3M (diluted with 3M solution), and stir at 500 rpm for 10 min. Slowly add 2.5 times the volume of anhydrous ethanol to precipitate the precipitate for 30 min, centrifuge at 9000 rpm and 4℃ for 10 min, and discard the supernatant. Wash the precipitate once with 75% ethanol, centrifuge at 9000 rpm and 4℃ for 10 min, and discard the supernatant to obtain a wet precipitate. Add 20 mL of anhydrous ethanol to the obtained wet precipitate and stir thoroughly. Centrifuge at 9000 rpm for 10 min, and discard the supernatant (repeat the operation 3 times). Freeze-dry the precipitate overnight to obtain the PDRN product, denoted as sample E.

[0039] Example 4

[0040] This embodiment provides a performance testing experiment.

[0041] Samples A and B prepared in Example 1 were weighed, and the results showed that sample A weighed 3.88 g and sample B weighed 1.85 g. Calculations showed that the mass ratio of sample A to sample B was approximately 2:1.

[0042] The nucleic acid purity of the samples prepared in Examples 1, 2 and 3 was tested, and the results are shown in Table 1.

[0043] Table 1. Sample purity test results

[0044] Concentration (ng / μL) A260 A280 A230 A260 / A280 A260 / A230 Sample A 1003.72 20.074 11.123 8.788 1.80 2.28 Sample B 996.213 19.924 11.01 8.911 1.81 2.24 Sample C 596.362 11.927 6.284 5.679 1.90 2.10 Sample D 374.976 7.499 3.801 3.712 1.97 2.02 Sample E 319.252 6.385 3.42 3.873 1.87 1.65

[0045] A260 / A280 and A260 / A230 are indicators of nucleic acid purity; the normal value of A260 / A280 for pure PN / PDRN samples should be between 1.8 and 2.0, while the value of A260 / A230 should be greater than 2.0. As shown in Table 1, the purity of samples A and B obtained in Example 1, and samples C and D obtained in Example 2, all meet the requirements and are pure PN / PDRN samples. The purity of sample E obtained in Example 3 is slightly lower (A260 / A230 < 2, indicating polysaccharide or phenolic residues).

[0046] Samples A and B prepared in Example 1, samples C and D prepared in Example 2, and sample E prepared in Example 3 were subjected to agarose gel electrophoresis to obtain the following results: Figure 2 The results are shown in Table 2.

[0047] Table 2 Sample molecular weight

[0048] Base pairs (bp) Molecular weight (kDa) Sample A ＞10000 ＞6600 Sample B ＜100 ＜66 Sample C ＞5000 ＞3300 Sample D ＜250 ＜165 Sample E 100-250 66-165

[0049] Agarose gel electrophoresis is commonly used to separate and identify nucleic acids; its ability to separate nucleic acids is mainly based on their relative molecular weight and molecular configuration. The larger the molecular weight, the slower the gel electrophoresis. For example... Figure 2 As shown in Table 2, Sample A obtained in Example 1 and Sample C obtained in Example 2 are high molecular weight samples, while Sample B obtained in Example 1, Sample D obtained in Example 2 and Sample E obtained in Example 3 are low molecular weight samples.

[0050] The samples A and B prepared in Example 1 and the sample E prepared in Example 3 were observed by scanning electron microscopy (SEM), and the results were as follows: Figures 3-5 The results are shown. (By...) Figure 3 , Figure 4 Can Figure 5 It can be seen that sample A exhibits a porous network structure, while samples B and E do not show this structural characteristic. Therefore, samples A and C are macromolecular PN, while samples B, D, and E are small molecule PDRN.

[0051] Example 5

[0052] This embodiment provides a mechanism of action identification and testing experiment.

[0053] Interleukin-10 (IL-10) is a cytokine expressed by cells of the innate and adaptive immune systems. As a key anti-inflammatory mediator, it plays multiple roles in inflammation and immune regulation.

[0054] Arginase 1 (Arg-1) is a member of the arginase family and an anti-inflammatory factor expressed in various cell types, including erythrocytes, hepatocytes, neutrophils, smooth muscle cells, and macrophages. Arg-1 is a marker of M2 macrophages and can inhibit inflammatory responses and promote tissue repair by degrading arginine.

[0055] VEGF, or vascular endothelial growth factor, is a core factor regulating angiogenesis and vascular permeability, playing a crucial role in tissue repair and wound healing. This study investigates IL-10 protein secretion levels and the gene expression levels of Arg-1 and VEGF to evaluate its soothing and anti-inflammatory effects.

[0056] The efficacy of samples A and B prepared in Example 1, as well as the control standard, was tested. The control standard was PDRN derived from salmon. The specific steps are as follows:

[0057] Raw264.7 cells were retrieved from the cell bank and revived. Once the cells reached 70%–80% confluence in the culture flask, they were digested and counted. Cells were then cultured at a concentration of 1 × 10⁻⁶ cells / mL. 5 The cells were seeded into 6-well plates at a density of 2 mL of cell suspension per well. The seeded 6-well plates were then placed in a cell culture incubator and cultured at 37°C and 5% CO2 for 24 h. A control group and a sample group were set up, with 2 mL of drug per well and 3 replicates per group. The samples were incubated in an incubator (37°C and 5% CO2) for 24 h. For details, please refer to Table 3.

[0058] Table 36: Types of reagents added to 6-well plates

[0059]

[0060] Supernatant and total RNA from Raw264.7 cells after treatment were collected from each group. Changes in interleukin-10 (IL-10) secretion were detected by enzyme-linked immunosorbent assay (ELISA), and changes in the expression levels of arginase-1 (Arg-1) and vascular endothelial growth factor (VEGF) genes were detected by quantitative real-time PCR (qRT-PCR). The results were as follows: Figures 6-8 The results are shown.

[0061] Depend on Figure 6 The results showed that the IL-10 content in the model group was significantly increased compared with the control group, indicating that the LPS-induced Raw264.7 cell validation model was successfully established. Compared with the model group, the IL-10 content in samples A-PN-0.3125%, A-PN-0.15625%, B-PDRN-0.3125%, and B-PDRN-0.15625% was significantly increased, with increases of 783.18%, 499.43%, 1183.73%, and 525.18%, respectively.

[0062] Depend on Figure 7 It was found that, compared with the control group, the relative expression level of Arg-1 gene in the model group was significantly increased, indicating that the LPS-induced Raw264.7 cell validation model was successfully established. Compared with the model group, the relative expression levels of Arg-1 gene in samples A-PN-0.3125%, B-PDRN-0.3125%, and B-PDRN-0.15625% were significantly increased, with increase rates of 32.45%, 74.12%, and 77.97%, respectively.

[0063] Depend on Figure 8 The results showed that, compared with the control group, the relative gene expression level of Vegf in the model group was significantly increased, indicating that the LPS-induced Raw264.7 cell validation model was successfully established. Compared with the model group, the relative gene expression levels of Vegf in samples A-PN-0.3125%, A-PN-0.15625%, B-PDRN-0.3125%, and B-PDRN-0.15625% were significantly increased, with increase rates of 18.20%, 17.63%, 112.44%, and 44.44%, respectively.

[0064] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0065] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of a polynucleotide extracted from Lactobacillus bulgaricus in the preparation of a soothing and anti-inflammatory drug, characterized in that, The extraction method includes the following steps: Collect the bacterial cell precipitate from the fermentation broth of Lactobacillus bulgaricus, wash the bacterial cell precipitate, add the extract and resuspend it to obtain a resuspended solution; Lysozyme was added to the resuspended solution and stirred thoroughly. Sodium dodecyl sulfate and polyvinylpyrrolidone were then added and stirred thoroughly again to obtain the lysate. After centrifuging the lysis solution, collect supernatant I. After resuspending and centrifuging the precipitate, collect supernatant II. Combine supernatant I and supernatant II and adjust their pH to 6.5-7.5 to obtain neutral supernatant. RNase and proteinase K were added sequentially to the neutral supernatant, and the supernatant was collected after centrifugation to obtain a pure supernatant. Anhydrous ethanol was added to the purified supernatant to perform fractional precipitation and obtain the polynucleotide; The polynucleotide is a polynucleotide and / or a polydeoxyribonucleotide.

2. The application of the polynucleotide extracted from Lactobacillus bulgaricus according to claim 1 in the preparation of a soothing and anti-inflammatory drug, characterized in that, The graded precipitation includes the following steps: The filamentous precipitate in the reaction system was removed, dried, and polynucleotides were obtained. The remaining reaction system was incubated overnight at -20°C, then centrifuged to collect the precipitate, which was then washed, dried, and polydeoxyribonucleotides were obtained.

3. The application of the polynucleotide extracted from Lactobacillus bulgaricus according to claim 1 in the preparation of a soothing and anti-inflammatory drug, characterized in that, The extraction solution consisted of 10-20 mM Tris-HCl, 1-2 M NaCl, and 1 mM EDTA, with a pH of 8.5-9.

0. The amount of lysozyme added is such that the final concentration of lysozyme in the reaction system after addition is 5~20 mg / mL; The amount of sodium dodecyl sulfate added is such that, after addition, the final volume concentration of sodium dodecyl sulfate in the reaction system is 0.5%~1%; The amount of polyvinylpyrrolidone added is such that, after addition, the final volume concentration of polyvinylpyrrolidone in the reaction system is 1%~2%; The amount of RNase added is such that the final concentration of RNase in the reaction system after addition is 20 μg / mL; The amount of proteinase K added is such that the final concentration of proteinase K in the reaction system after addition is 20 μg / mL.

4. The application of the polynucleotide extracted from Lactobacillus bulgaricus according to claim 1 in the preparation of a soothing and anti-inflammatory drug, characterized in that, The method for preparing the bacterial precipitate in the Lactobacillus bulgaricus fermentation broth includes the following steps: The fermentation broth of Lactobacillus bulgaricus was centrifuged at 4°C and 10,000 g, and the precipitate was collected to obtain the bacterial precipitate.

5. The application of the polynucleotide extracted from Lactobacillus bulgaricus according to claim 1 in the preparation of a soothing and anti-inflammatory drug, characterized in that, Add the lysozyme and mix thoroughly. The mixing conditions are 37°C, 1000~1500 rpm, and the mixing time is 1~2 h. The mixture is stirred thoroughly again at 1000-1500 rpm for 2-3 hours.

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