Method for preparing lipoteichoic acid by using high-adhesion lactobacillus plantarum

Abstract

The invention belongs to the technical field of probiotic metabolites, and particularly relates to a method for screening high-adhesion lactobacillus plantarum and separating lipoteichoic acid of the high-adhesion lactobacillus plantarum. The invention discloses a method for preparing lipoteichoic acid by using high-adhesion lactobacillus plantarum. The method comprises the following steps: 1) selecting lactobacillus plantarum ZJ316 as high-adhesion lactobacillus plantarum; (2) extracting the high-adhesion lactobacillus plantarum through an n-butyl alcohol method to obtain a lactobacillus plantarum lipoteichoic acid crude extract; the screened high-adhesion lactobacillus plantarum has good adhesion performance in vivo and in vitro, and the prepared lipoteichoic acid has high purity and no cytotoxicity, and can be considered as a key adhesion factor of lactobacillus plantarum ZJ316.

Description

Technical Field

[0001] The invention belongs to the technical field of probiotic metabolites, and particularly relates to a method for screening high-adhesion plant lactobacillus and separating lipoteichoic acid thereof. Background Art

[0002] Probiotics refer to a mixture of single or specific microorganisms that can exist in the gastrointestinal tract as live bacteria after the host ingests a certain amount, and can regulate the composition of intestinal flora, thereby having a beneficial effect on human or animal health. Common ones include Lactobacillus, Bifidobacterium, and some Bacillus and yeast. A large number of studies have shown that probiotics that enter and exist in the human gastrointestinal tract can effectively inhibit the invasion of exogenous pathogens and remove harmful substances in the intestine. In addition, they can also protect the gastrointestinal mucosa, prevent constipation, prevent and treat diarrhea, and prevent allergic reactions. However, the adhesion of probiotic lactic acid bacteria to the intestine is an important prerequisite for its many physiological functions. Adhesion refers to the specific adhesion between bacteria and mucus or epithelial cells. The adhesion of lactic acid bacteria to intestinal epithelial cells is a necessary condition for probiotics to exist in the digestive tract for a long time, so adhesion ability is one of the important criteria for evaluating the quality of probiotics. In addition, lactic acid bacteria adhering to the intestine can also inhibit intestinal pathogens and adjust the balance of intestinal microecology by occupying space and producing antibacterial substances.

[0003] Lactobacillus plantarum is a lactic acid bacterium widely found in fermented products. It has good prebiotic function and can pass through the stomach and colonize in the intestine to play a beneficial role. It has an important impact on intestinal microorganisms and has broad application prospects in food fermentation, industrial lactic acid fermentation and medical care. Studies have shown that the lipoteichoic acid of Lactobacillus plantarum plays a certain role directly or indirectly in the adhesion process and is one of the main sources of adhesins. The cell wall of Lactobacillus plantarum is rich in lipoteichoic acid (LTA), which is a weak anionic polymer containing GroP repeating units. It mainly exists in Gram-positive bacteria and is fixed to the bacterial membrane through lipid anchors. Amphiphilic molecules, one end of which is a non-polar glycolipid, fixed to the cell membrane by a covalent bond, and the other end is a polar chain composed of polyphosphoglycerol, which extends across the periplasmic space of the cell to the peptidoglycan layer of the cell wall. Some may even pass through the peptidoglycan layer and become part of the surface antigen. Lipoteichoic acid in probiotics has effective prebiotic functions, such as anti-inflammatory, antioxidant, and anti-tumor effects. Recently, many studies have identified lipoteichoic acid as a key antigen in probiotics. As an immunomodulator, it plays a vital role in the human immune system and is expected to become the next new type of postbiotic.

[0004] The isolation of lipoteichoic acid is to completely separate it from various cellular components, such as phospholipids, proteins, polysaccharides, nucleic acids, and peptidoglycans. As mentioned earlier, the structure and composition of Gram-positive bacterial cells are complex, and a small amount of lipoteichoic acid is present in the cells. Therefore, it is not easy to isolate a certain amount of pure lipoteichoic acid from the cells. The separation of lipoteichoic acid can use organic solvent extraction, such as hot phenol, trichloroacetic acid (TCA) and n-butanol, or other extraction methods. Once the lipoteichoic acid is isolated, a purification step is used to remove impurities and obtain high purity. Due to the physical properties of the lipoteichoic acid molecule, several purification techniques can be used, including hydrophobic interaction chromatography (HIC) and ion exchange chromatography (IEC). The purified lipoteichoic acid is then characterized to confirm its identity and structural integrity.

[0005] Extensive research conducted over the years has provided valuable insights into the multifaceted roles of lipoteichoic acid in Gram-positive bacteria. Lipoteichoic acid is not only essential for microbial growth but also exhibits multiple physiological functions in humans. However, the isolation of lipoteichoic acid from microorganisms has limited the application and development of functional lipoteichoic acid. Therefore, it is particularly valuable to develop efficient lipoteichoic acid isolation conditions and improve its purity. Combining lipoteichoic acid with food substrates may provide new opportunities for functional food development and provide enhanced health benefits to consumers. Therefore, it is of great value to screen a Lactobacillus plantarum with high adhesion to obtain high-purity and structurally intact prebiotic lipoteichoic acid.

[0006] Lactobacillus plantarum ZJ316 (CCTCC No: M208077) has a corresponding disclosure in "Lactobacillus plantarum ZJ316 and its application in inhibiting Helicobacter pylori" of CN112080445A. The patent discloses that the hydrophobicity of Lactobacillus plantarum ZJ316 (65.56±1.88%) is significantly higher than that of Lactobacillus rhamnosus LGG (40.35±1.20%). It is speculated that the adhesion of L. plantarum ZJ316 to gastric epithelial cells should also be higher than that of L. rhamnosus LGG. It is possible that some kind of protein substance exists on the surface of the strain with strong hydrophobicity, thereby achieving the protective effect on the cells themselves. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a method for preparing lipoteichoic acid by utilizing high-adhesion Lactobacillus plantarum.

[0008] In order to solve the above problems, the present invention provides a method for preparing lipoteichoic acid using high-adhesion Lactobacillus plantarum, comprising the following steps:

[0009] 1) Select Lactobacillus plantarum ZJ316 as the high-adhesion Lactobacillus plantarum;

[0010] 2) Extracting the high-adhesion Lactobacillus plantarum by the n-butanol method to obtain a crude extract of Lactobacillus plantarum lipoteichoic acid.

[0011] As an improvement of the method for preparing lipoteichoic acid by using high-adhesion Lactobacillus plantarum of the present invention, the method further comprises the following steps:

[0012] 3) The crude lipoteichoic acid extract is subjected to moderate purification (hydrophobic interaction chromatography, octyl agarose gel moderate purification) to obtain a moderately purified lipoteichoic acid product;

[0013] 4) Finely purify the lipoteichoic acid intermediate purification product (ion exchange chromatography, DEAE-FF fine purification) to obtain Lactobacillus plantarum ZJ316 lipoteichoic acid (LTA).

[0014] As a further improvement of the method for preparing lipoteichoic acid using high-adhesion Lactobacillus plantarum of the present invention, step 2) is to separate lipoteichoic acid using the n-butanol method, comprising the following steps:

[0015] 2.1) Prepare bacterial mud using Lactobacillus plantarum ZJ316;

[0016] The bacterial sludge of Lactobacillus plantarum ZJ316 was resuspended in sodium citrate buffer (0.1M, pH 4.7) at 1 g: 9-11 ml (W: V), and the sludge was broken to obtain a broken cell solution;

[0017] 2.2) Add an equal volume of water-saturated n-butanol to the disrupted cell solution, mix, stir (1200r speed, 60min) at 20-22°C, centrifuge (4°C, 13000×g, 30min), collect the aqueous phase (located at the lower layer), and then centrifuge the collected aqueous phase (4°C, 13000×g, 30min) to completely remove the precipitated impurities, and the resulting aqueous phase is named as the secondary aqueous phase;

[0018] 2.3) Remove the residual organic reagent (n-butanol) in the secondary aqueous phase by rotary evaporation (35°C), dialyze with water for 3 to 5 days, and then freeze-dry (freeze-dry at -40±5°C for 48±2h) to obtain a crude lipoteichoic acid extract.

[0019] As a further improvement of the method for preparing lipoteichoic acid using high-adhesion Lactobacillus plantarum of the present invention, the crushing treatment in step 2.1) is: ultra-high pressure cell crushing: 1200 Pa, crushing can be set three times to obtain crushed cell liquid;

[0020] The dialysis in step 2.3) is specifically as follows: the secondary aqueous phase after the organic reagent is removed is placed in a 3500Da dialysis bag, placed in ultrapure water for dialysis, and the dialysis solution is replaced every 6 hours; after dialysis for 3 to 5 days (preferably 4 days), the retentate in the dialysis bag is collected.

[0021] As a further improvement of the method for preparing lipoteichoic acid using high-adhesion Lactobacillus plantarum of the present invention, the bacterial mud of step 2.1) is obtained by expanding the culture of Lactobacillus plantarum; the expanded culture of Lactobacillus plantarum is as follows: the activated Lactobacillus plantarum ZJ316 strain is inoculated into MRS liquid culture medium at a ratio of 1% (volume %), and cultured at 37°C for 20 hours; centrifuged (at 8000×g, 10 min, 4°C) to obtain the bacterial mud.

[0022] As a further improvement of the method for preparing lipoteichoic acid using high-adhesive Lactobacillus plantarum of the present invention, the step 3) of intermediately purifying the crude lipoteichoic acid extract is specifically as follows:

[0023] The crude extract of lipoteichoic acid of Lactobacillus plantarum ZJ316 was dissolved in 0.1M sodium acetate buffer at pH 4.7 containing 15vt% n-propanol for hydrophobic interaction chromatography, and eluted with a linear 15-60vt% n-propanol solution gradient. After determination by phosphate-molybdate colorimetry, the column fractions containing Lactobacillus lipoteichoic acid were combined and dialyzed with water for 48h to obtain a moderately purified product of lipoteichoic acid (i.e., a component containing Lactobacillus lipoteichoic acid was obtained).

[0024] As a further improvement of the method for preparing lipoteichoic acid using high-adhesion Lactobacillus plantarum of the present invention, the step 4) of finely purifying the lipoteichoic acid intermediate purification product is specifically as follows:

[0025] The component containing lipoteichoic acid of Lactobacillus plantarum ZJ316 was diluted with 30% n-propanol 0.1M sodium acetate buffer solution, and then subjected to ion exchange chromatography. Elution was performed using 30% n-propanol 0.1M sodium acetate buffer solution containing 0-1M sodium chloride, and the eluate was collected. The phosphorus content was determined by phosphate-molybdate colorimetry. After the lipoteichoic acid elution peak was collected, the eluate was dialyzed with water for 48 hours, and freeze-dried for 48 hours to obtain Lactobacillus lipoteichoic acid.

[0026] The invention provides a method for determining the purity and structure of lipoteichoic acid of Lactobacillus plantarum ZJ316, which comprises ultraviolet soft wavelength scanning, SDS-PAGE, agarose gel electrophoresis and Fourier transform infrared spectroscopy.

[0027] As a further improvement of the method for preparing lipoteichoic acid using high-adhesion Lactobacillus plantarum of the present invention, step 1) includes: incubating different Lactobacillus plantarum with Caco-2 cells to observe their adhesion ability, measuring the adhesion rates and strain surface properties of different Lactobacillus plantarum, screening out a high-adhesion Lactobacillus plantarum ZJ316, and observing the adhesion of the high-adhesion Lactobacillus plantarum in the mouse intestine through fluorescent probe labeling technology.

[0028] Specifically, Lactobacillus plantarum was cultured in MRS medium, then washed in sterile PBS, resuspended in DMEM, and the bacterial concentration was adjusted to 10 8 CFU / mL; incubate with Caco-2 cells cultured on slides for 2 h, wash with PBS, digest the cells with trypsin, and spread and count on MRS solid medium to screen out high-adhesion Lactobacillus plantarum.

[0029] High-adhesion Lactobacillus plantarum ZJ316 was labeled with CFDA-SE fluorescent probe, and the concentration was adjusted to an appropriate level before gavage into mice. The mice were dissected 30 min, 60 min, 180 min, 6 h, 24 h, 3 d, 5 d, and 7 d after gavage, and their digestive tracts were imaged by fluorescence in IVIS Lumina Series III.

[0030] The present invention also provides application of Lactobacillus plantarum lipoteichoic acid extracted according to the method in the adhesion of high-adhesion Lactobacillus plantarum ZJ316 to Caco-2 cells.

[0031] The present invention first screens out a strain of high-adhesion Lactobacillus plantarum and provides an efficient method for extracting lipoteichoic acid of Lactobacillus plantarum. The adhesion performance of high-adhesion Lactobacillus plantarum in vivo and in vitro is explored, lipoteichoic acid with higher purity is prepared, and the adhesion correlation between lipoteichoic acid and high-adhesion Lactobacillus plantarum is explored.

[0032] The invention firstly selects a high-adhesion Lactobacillus plantarum ZJ316 by screening a Caco-2 cell model and measuring the surface properties of the strain, observes the intestinal adhesion and colonization site of the strain in vivo, uses an ultra-high pressure cell disruption technology to disrupt the strain cells, separates lipoteichoic acid by an n-butanol method, and prepares a high-purity Lactobacillus plantarum lipoteichoic acid.

[0033] In step 1) of the present invention, screening is performed on Lactobacillus plantarum ZJ316, Lactobacillus plantarum ZFM4, Lactobacillus plantarum ZFM9, Lactobacillus plantarum ZFM55, Lactobacillus plantarum LZ206 and the like.

[0034] Compared with the existing methods for screening high-adhesion strains and separating and purifying lipoteichoic acid, the present invention has the following technical advantages:

[0035] 1. The method of the present invention screened out a highly adherent Lactobacillus plantarum ZJ316, and observed its intestinal adhesion and colonization in vivo, providing a basis for the functional study of the strain.

[0036] Specifically: In the present invention, the adhesion rate of ZJ316 to intestinal epithelial cells was determined by using a Caco-2 cell model, and the adhesion and colonization of Lactobacillus plantarum ZJ316 in the intestine of mice was explored.

[0037] 2. The method of the present invention compares four methods for separating lipoteichoic acid (LTA): hot phenol method, trichloroacetic acid method, n-butanol method and TritionX-114 method, and determines that n-butanol method is the preferred separation method for LTA by detecting the total phosphorus content. This is of great significance for the separation of lipoteichoic acid.

[0038] 3. The method of the present invention compares the effects of ultrasonic cell disruption and ultra-high pressure cell disruption on the yield of crude lipoteichoic acid extract of Lactobacillus plantarum ZJ316, and determines that the best cell disruption method for lipoteichoic acid is ultra-high pressure cell disruption (1200Pa, 3-5 times).

[0039] 4. The method of the present invention uses a Caco-2 cell model to determine the cytotoxicity of lipoteichoic acid of Lactobacillus plantarum ZJ316. The results show that there is no obvious toxicity in the concentration range of 0-200 μg / mL, which provides a basis for the functional study of lipoteichoic acid of Lactobacillus plantarum ZJ316.

[0040] 5. The method of the present invention used a Caco-2 cell model to explore the effect of lipoteichoic acid of Lactobacillus plantarum ZJ316 on the adhesion of Lactobacillus plantarum ZJ316 to Caco-2 cells. The results showed that lipoteichoic acid had a certain inhibitory effect on the adhesion of Lactobacillus plantarum ZJ316 to Caco-2 cells, indicating that lipoteichoic acid of Lactobacillus plantarum ZJ316 is a key adhesion factor of Lactobacillus plantarum ZJ316.

[0041] In summary, the high-adhesion Lactobacillus plantarum screened by the present invention has good adhesion properties both in vivo and in vitro, and the prepared lipoteichoic acid has high purity and no cytotoxicity, which can be considered as the key adhesion factor of Lactobacillus plantarum ZJ316. The present invention can provide a certain basis for the study of the adhesion mechanism of probiotic lipoteichoic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The specific implementation modes of the present invention are further described in detail below with reference to the accompanying drawings.

[0043] Figure 1 Gram staining of different Lactobacillus plantarum adhesion to Caco-2 cells;

[0044] Figure 2 Adhesion rates of different Lactobacillus plantarum to Caco-2 cells;

[0045] Figure 3 Determination of surface properties of Lactobacillus plantarum; the ordinate represents the surface properties of the strain;

[0046] Figure 4 In vivo adhesion observation of highly adherent Lactobacillus plantarum ZJ316;

[0047] Figure 5Analysis of total phosphorus content of Lactobacillus plantarum ZJ316 LTA extracted by different isolation methods;

[0048] Figure 6 The effect of crushing method on the yield of LTA crude extract;

[0049] Figure 7 Lactobacillus plantarum ZJ316 LTA purification elution profile;

[0050] Figure 7 In the figure, A is the elution diagram of Octyl-Sepharose CL-4B gel chromatography, and B is the elution diagram of DEAE-FF agarose gel chromatography.

[0051] Figure 8 UV full wavelength scan of Lactobacillus plantarum ZJ316 LTA;

[0052] Fig. 9 Electrophoresis detection diagram of Lactobacillus plantarum ZJ316 LTA;

[0053] Fig.10 Fourier transform infrared spectrum of Lactobacillus plantarum ZJ316 LTA;

[0054] Fig.11 Analysis of toxicity of Lactobacillus plantarum ZJ316 LTA to Caco-2 cells;

[0055] Fig.12 Effect of Lactobacillus plantarum ZJ316 LTA on bacterial adhesion to Caco-2 cells. DETAILED DESCRIPTION

[0056] The present invention is further described below in conjunction with specific embodiments, but the research scope of the present invention is not limited thereto:

[0057] The technical solution of the present invention is described in detail with specific embodiments below:

[0058] The culture media used in the following cases are all conventional culture media: For example:

[0059] DPBS is DPBS (1×) phosphate buffered saline.

[0060] DMEM complete medium is: DMEM basal medium + 20% FBS + 1% non-essential amino acids (L-glutamine) + 1% penicillin / streptomycin);

[0061] DMEM incomplete culture medium is: DMEM basal culture medium + 20% FBS + 1% non-essential amino acids.

[0062] The lactobacillus involved in the present invention is as follows:

[0063] Lactobacillus plantarum ZJ316 (CCTCC No: M208077) has a corresponding notice in CN112080445A "Lactobacillus plantarum ZJ316 and its application in inhibiting Helicobacter pylori";

[0064] Lactobacillus plantarum ZFM4 (CCTCC NO: M2016629) is mentioned in CN118308345A "Lactobacillus plantarum microcapsules with high folic acid production and preparation method thereof";

[0065] Rhamnosus GG (ATCC#53103) is described in the "Preparation of intermittently sterilized intact immunocompetent cells of Lactobacillus rhamnosus GG and methods for qualitative and quantitative determination thereof";

[0066] Lactobacillus plantarum ZFM9, Lactobacillus plantarum ZFM55, and Lactobacillus plantarum LZ206 were preserved in the laboratory of the inventor.

[0067] Example 1: Screening of Highly Adhesive Lactobacillus Plantarum

[0068] S1. Activation of experimental strains: First, take appropriate bacterial liquid from the glycerol tubes of Lactobacillus plantarum ZJ316, Lactobacillus plantarum ZFM4, Lactobacillus plantarum ZFM9, Lactobacillus plantarum ZFM55, Lactobacillus plantarum LZ206 and Lactobacillus rhamnosus GG, streak on MRS solid culture medium for activation, and culture at 37°C for 24 to 48 hours until single colonies with clear edges are formed. Pick single colonies and activate them twice in MRS liquid culture medium for later use.

[0069] S2. Observation of adhesion of Lactobacillus plantarum to Caco-2 cells: The activated strain was inoculated into MRS liquid medium at a ratio of 1% (volume %) and cultured at 37°C for 22 hours. Centrifuged at 8000xg for 10 minutes at 4°C and the supernatant was removed. The collected bacteria were washed twice with 1×PBS and then diluted to 1×10 8 CFU / mL, as lactic acid bacteria suspension, for later use.

[0070] The digested Caco-2 cells (human colorectal adenocarcinoma cells, No. 3115CNCB00337) were resuspended in DMEM incomplete medium at a concentration of 1 ml (10 5 / ml) cell suspension was added to a 24-well plate with a slide and incubated in a 37°C incubator for 24 hours. After the cells were completely attached to the wall, they were washed with sterile DPBS and 1 ml of 10 8CFU / mL lactic acid bacteria suspension was incubated at 37°C for 2 h, washed 5 times with DPBS to remove unadhered bacteria, fixed with pre-cooled methanol for 30 min, and then observed the adhesion of each strain under a microscope (100x) using Gram staining and photographed.

[0071] S3. Count of adhesion of Lactobacillus plantarum to Caco-2 cells: Digested Caco-2 cells were resuspended in DMEM incomplete medium at 1 mL (10 5 Cell suspension (cell / ml) was added to a 24-well plate and incubated at 37°C for 24 h. After the cells were completely attached to the wall, they were washed with sterile DPBS and 1 ml of 10 8 CFU / mL lactic acid bacteria suspension was incubated at 37°C for 2 hours. The non-adherent bacteria were removed by washing with DPBS 5 times, digested with 500 μL trypsin for 3 minutes, and terminated with 500 μL DMEM complete medium. The bacterial suspension was collected into a bacterial centrifuge tube, and after appropriate dilution, the number of attached bacteria was recorded by the plate counting method.

[0072] Adhesion rate = number of bacteria after adhesion / number of bacteria before adhesion × 100 (2)

[0073] The result obtained in step S2 is as follows Figure 1 As shown in the figure, with L. rhamnosus GG as the control, it can be seen from the figure that all six lactobacilli can adhere to Caco-2 cells, but the degree of adhesion is different. The number of adherents of ZJ316 and LGG is significantly greater than that of the other four strains. This is consistent with the results of the plate count in step S3, see Figure 2 Among them, the adhesion rate of ZJ316 was 21%, which was not significantly different from the adhesion rate of LGG strain (24%); the adhesion rates of ZFM4, LZ206, ZFM55 and ZFM9 were lower, at 1.3%, 1.2%, 0.6% and 0.75% respectively. Therefore, Lactobacillus plantarum ZJ316 was selected as a high-adhesion Lactobacillus plantarum, and the foundation was laid for the subsequent exploration of Lactobacillus plantarum adhesin.

[0074] S4. Determination of strain surface properties:

[0075] The surface properties of lactobacillus were determined according to the MATH (Microbial Adhesion To Hydrocarbons) method. The MATH method can measure both the hydrophobicity and the surface charge of the surface.

[0076] After washing by centrifugation, the cells were resuspended in 1× PBS to a concentration of about 1.0×10 8CFU / mL, and measure its absorbance at 600nm, which is recorded as A0. Take 3mL of the adjusted bacterial solution, add 1mL of xylene, chloroform or ethyl acetate respectively, and do not add to the control group (that is, 3mL of the adjusted bacterial solution is used as the control group), oscillate for 30s, pause for 10s, and then oscillate for 30s, and stand for 20min to separate the layers. Take the aqueous phase, use PBS solution as the blank control (blank solution for zeroing when measuring OD value), measure the A value at 600nm and record it, and repeat 3 tubes for each bacterial strain in parallel, and calculate the hydrophobicity of the bacterial cell surface according to formula (1).

[0077] Hydrophobicity = A0-A1 / A0×100(1)

[0078] Taking xylene as an example, A0 is the A value of the water phase obtained from the control group measured at 600nm, and A1 is the A value of the water phase obtained after adding xylene measured at 600nm;

[0079] Depend on Figure 3 It can be seen that LGG (Lactobacillus rhamnosus GG) has the strongest hydrophobicity, followed by ZJ316, and the hydrophobicity of the other four strains is relatively low; among them, LGG and ZJ316 have strong adsorption capacity (>50%) to chloroform, indicating that they have strong electron-donating ability; the adsorption capacity to ethyl acetate is relatively low, indicating that their electron-accepting ability is relatively weak; while the adsorption capacity of the other four strains to chloroform is relatively weak (<50%), indicating that their electron-donating ability is relatively weak, among which ZFM4, ZFM55 and LZ206 have strong adsorption capacity to ethyl acetate (>20%), indicating that their electron-accepting ability is relatively strong. It can also be found that the hydrophobicity and adhesion of the six strains are positively correlated, so ZJ316 is selected as the high-adhesion plant lactobacillus, and the foundation is laid for the subsequent research on adhesion properties.

[0080] S5. Observation of adhesion of Lactobacillus plantarum ZJ316 in mice: Highly adherent Lactobacillus plantarum ZJ316 was labeled with CFDA-SE fluorescent probe and the expression of 1×10 8 CFU / mice were gavaged, and the mice were dissected 30min, 60min, 180min, 6h, 24h, 3d, 5d, and 7d after gavage, and their digestive tracts were imaged with fluorescence in IVIS to observe the adhesion of Lactobacillus plantarum ZJ316 in mice.

[0081] Results from Figure 4 It can be seen that the highly adherent Lactobacillus plantarum ZJ316 can colonize in the cecum 3 hours after gavage. As time goes by, the fluorescence signal weakens and continues to adhere until the 7th day, with a weak fluorescence signal still present, indicating that Lactobacillus plantarum ZJ316 also has strong adhesion in the body.

[0082] Example 2: Determination of lipoteichoic acid separation method:

[0083] S1. Preparation of bacterial sludge:

[0084] The highly adherent Lactobacillus plantarum ZJ316 screened out in Example 1 was expanded and cultured: the activated Lactobacillus plantarum ZJ316 strain was inoculated into MRS liquid culture medium at a ratio of 1% (volume %), cultured at 37° C. for 20 h, and centrifuged at 8000×g, 10 min, 4° C. to collect the bacterial sludge for later use.

[0085] S2. Separation of lipoteichoic acid: There are 4 methods:

[0086] Method 1: Separation of lipoteichoic acid by n-butanol method:

[0087] The bacterial sludge (3 g) prepared in S1 was resuspended in sodium citrate buffer (0.1 M, pH 4.7) at a ratio of 1:10 (W:V, i.e., 1 g:10 ml), and ultrasonically disrupted at 500 W for 30 min to obtain disrupted cell fluid;

[0088] Then add water-saturated n-butanol of an equal volume to the broken cell fluid and mix, stir (1200r speed) for 60min at 20-22°C, carefully dispense into centrifuge tubes, centrifuge (4°C, 13000×g, 30min) and collect the aqueous phase (located at the lower layer), centrifuge the collected aqueous phase (4°C, 13000×g, 30min) to remove the precipitated impurities, and the resulting aqueous phase is named secondary aqueous phase and collected.

[0089] Method 2: Separation of lipoteichoic acid by hot phenol method:

[0090] The bacterial sludge prepared in S1 was resuspended in sodium citrate buffer at a ratio of 1:10 (W:V), and ultrasonically disrupted at 500W for 30min to obtain disrupted cell fluid;

[0091] Then, the broken cell solution was placed in a 65°C water bath for 40 min, and then an equal volume of 90% phenol aqueous solution preheated to 65°C was added and rapidly stirred (at a speed of 1200r) for 30 min. Then, it was placed in a 4°C refrigerator and cooled to 10°C for centrifugation (3000×g, 15 min), and the upper aqueous phase was taken.

[0092] Method 3, trichloroacetic acid method:

[0093] The bacterial sludge prepared in S1 was added to a 10% trichloroacetic acid (TCA) aqueous solution at a ratio of 1:5 (W / V), suspended evenly, and stirred overnight at 4°C with a magnetic stirrer at low speed (500r for 12 hours); centrifuged at 8000rpm at 4°C for 30min, discarded the precipitate, and collected the upper liquid, which is the extract containing teichoic acid.

[0094] Method 4: Trition-114 method:

[0095] The bacterial sludge prepared in S1 was suspended in 10 mL of pre-cooled 2% Triton TX-114 solution at a ratio of 1:10 (W:V) and stirred at low speed overnight at 4°C. The mixture was centrifuged at 5000 r / min for 30 min to remove the bacterial fragments in the lower layer and collect the upper extract. The extract was water-bathed at 50°C for 30 min. The extract was separated into two layers and the upper aqueous phase was gently collected.

[0096] S3. Determination of phosphorus content:

[0097] The secondary aqueous phase obtained by method 1, the upper aqueous phase obtained by method 2, the upper liquid obtained by method 3, and the upper aqueous phase obtained by method 4 were used as samples respectively;

[0098] Take 1 mL of the sample into a 5 mL stoppered glass tube, add 0.2 mL of potassium persulfate solution (50 g / L), and 2 , digested in a high pressure sterilizer at 120℃ for 30min. Add 0.24mL of ascorbic acid solution (100g / L) to each digestion solution and mix well. After 30s, add 0.48mL of molybdate solution and mix well. Place at room temperature for 15min and measure the absorbance at 700nm.

[0099] Preparation of phosphorus standard stock solution: Weigh 0.02195 g of potassium dihydrogen phosphate that has been dried to constant weight at 110°C and dissolve it in distilled water. Transfer it into a 50 mL volumetric flask, add 0.15 mL of nitric acid, and make up to 50 mL with distilled water to obtain a standard solution with a phosphorus concentration of 100 mg / L.

[0100] Potassium persulfate solution: Weigh 2.5 g of potassium persulfate, put it into 45 mL of distilled water, stir at 40°C until completely dissolved, transfer to a 50 mL volumetric flask, add distilled water to make up to 50 g / L potassium persulfate solution, and place it at 25°C for later use.

[0101] Molybdate solution: Dissolve 13g of ammonium molybdate in 100mL of water, and dissolve 0.35g of potassium antimony tartrate in 100mL of water. Add the ammonium molybdate solution to 300mL of sulfuric acid solution (concentrated sulfuric acid and water are mixed in a volume ratio of 1:1) under constant stirring, then add the potassium antimony tartrate solution and mix well. Store at low temperature away from light.

[0102] 100g / L ascorbic acid solution: 10g ascorbic acid, dilute to 100mL, and store in dark place.

[0103] The measured absorbance value is converted according to the conversion formula of Y=0.0566X+0.1267 to obtain the phosphorus content of the sample.

[0104] Where X is the phosphorus content (μg / mL) and Y is OD 700 The absorbance value.

[0105] The results are as follows Figure 5 As shown in the figure, the crude extract of lipoteichoic acid separated by n-butanol method has the highest phosphorus content, which can reach 20.89μg / mg, followed by the hot phenol method, in which the phosphorus content of crude extract of lipoteichoic acid separated can reach 17.96μg / mg. The crude extracts separated by TCA method and Trition-114 have lower phosphorus contents, which are 13.9μg / mg and 12.6μg / mg respectively. Therefore, the n-butanol method was selected as the method for separating lipoteichoic acid from Lactobacillus plantarum ZJ316 in the subsequent study.

[0106] S4. Optimization of cell disruption method:

[0107] 10 g of wet bacterial sludge prepared in S1 was resuspended in sodium citrate buffer at a ratio of 1:10 (W:V), and the cells were disrupted by the following three methods to obtain disrupted cell fluid;

[0108] (1) Ultrasonic cell disruption: 500w, 30min disruption once;

[0109] (2) Ultrasonic cell disruption: 500w, disrupt for 30min, collect the supernatant by centrifugation, resuspend the precipitate with sodium citrate buffer, repeat the above disruption operation twice; combine the supernatants obtained three times;

[0110] (3) Ultra-high pressure cell disruption: 1200 Pa, three times.

[0111] The broken cell solution was mixed with water-saturated n-butanol in a volume ratio of 1:1, stirred at 20-22°C for 60 min, carefully dispensed into centrifuge tubes, and centrifuged (13000 g, 30 min) to collect the aqueous phase (located at the bottom layer);

[0112] The collected aqueous phase was then centrifuged (13000×g, 30 min) to remove precipitated impurities, and the resulting phase was named the secondary aqueous phase and collected.

[0113] The residual organic reagent (n-butanol) in the secondary aqueous phase was removed by rotary evaporation at 35°C, and the extracts were dialyzed for 4 days and then freeze-dried (-40°C, 0.1 MPa) for 48 hours. The crude extracts obtained by different crushing methods were weighed and recorded.

[0114] The dialysis is specifically as follows: the secondary aqueous phase after the organic reagent is removed is placed in a 3500Da dialysis bag, placed in ultrapure water for dialysis, and the dialysis solution is replaced every 6 hours; after 4 days of dialysis, the retentate in the dialysis bag is collected;

[0115] Crude extract yield = crude extract product mass / wet weight of bacterial sludge × 100.

[0116] The results are as follows Figure 6 As shown in the figure, the yield of crude lipoteichoic acid obtained by ultrasonic disruption once was 0.49%, and the yield of crude extract increased to 1.35% after ultrasonic disruption three times. However, the yield of crude extract after ultra-high pressure cell disruption three times can reach 2.45%, which is 1.96% higher than the 2.27% in the literature "Study on Extraction and Purification Conditions of Cell Wall Lipoteichoic Acid of Probiotic Enterococcus Faecium". Therefore, ultra-high pressure cell disruption method was selected as the cell disruption method in the subsequent extraction process of lipoteichoic acid from Lactobacillus plantarum ZJ316.

[0117] Embodiment 3:

[0118] S1. Isolation of lipoteichoic acid from Lactobacillus plantarum ZJ316:

[0119] The highly adherent Lactobacillus plantarum ZJ316 screened out in Example 1 was expanded and cultured: the activated Lactobacillus plantarum ZJ316 strain was inoculated into MRS liquid culture medium at a ratio of 1% (volume %), cultured at 37° C. for 20 h, centrifuged at 8000×g, 10 min, 4° C., and the bacterial sludge was collected for later use.

[0120] S2. Separation of lipoteichoic acid by n-butanol method:

[0121] 87 g of wet bacterial sludge prepared by S1 was resuspended in sodium citrate buffer (0.1 M, pH 4.7) at a ratio of 1:10 (W:V), and ultra-high pressure cell disruption was performed at 1200 pa for three times. The resulting disrupted cell solution was mixed with an equal volume of water-saturated n-butanol, stirred at 20-22°C for 60 min, carefully dispensed into centrifuge tubes, and centrifuged at 4°C (13000×g, 30 min) to collect the aqueous phase, which was then centrifuged (4°C, 13000×g, 30 min) to remove precipitated impurities (i.e., collect the lower aqueous phase). The resulting phase was named secondary aqueous phase and collected.

[0122] The secondary aqueous phase was evaporated at 35°C to remove the organic reagent (n-butanol), dialyzed with ultrapure water for 4 days, and then freeze-dried for 48 hours for use to obtain a crude lipoteichoic acid extract. The phosphorus content of the crude lipoteichoic acid extract was 2.1335 g.

[0123] The dialysis method is the same as in Example 2.

[0124] S3. Moderate purification of lipoteichoic acid from Lactobacillus plantarum ZJ316:

[0125] According to the solid-liquid ratio of 1 g / 100 mL, 0.3 g of the crude lipid wall extract prepared by S2 was dissolved in 0.1 M sodium acetate buffer (pH 4.7) with 15% n-propanol, and then centrifuged at 13000 g for 30 min at 4°C to remove the precipitate, and then filtered with a 0.22 μm filter membrane. The entire filtrate obtained was slowly drained into an octyl agarose CL-4B chromatography column (column volume of 200 mL) as a sample, and unbound impurities were first washed away with 0.1 M sodium acetate buffer (300 mL) with 15% n-propanol, and then eluted with linear gradient eluent I;

[0126] The linear gradient eluent I is a 0.1M sodium acetate buffer solution containing 25%-65% n-propanol (n-propanol and 0.1M sodium acetate buffer solution are linearly gradient mixed by a gradient mixer, and the volume concentration of n-propanol increases from 25% to 65%), the amount of the linear gradient eluent I is 672mL in total, the flow rate is 0.5mL / min, and fractions are collected in equal parts of 8mL and the phosphorus content of each eluted fraction is detected, and a total of 84 tubes are collected. The fractions containing lipoteichoic acid (i.e., tubes 50, 51, and 80) were combined, and the organic reagent (i.e., n-propanol) was removed by rotary evaporation, and then dialyzed at 4°C for 48 hours to remove small molecular impurities (placed in a dialysis bag with a molecular weight cutoff of 3500Da, placed in ultrapure water for dialysis, and the dialysate was replaced every 6 hours; after dialysis for 48 hours, the retentate in the dialysis bag was collected; it was concentrated by rotary evaporation at 35°C to about 30% of the original volume, and then freeze-dried at -80°C for 48 hours for use, to obtain a moderately purified product of lipoteichoic acid.

[0127] The 15% n-propanol in 0.1M sodium acetate buffer (pH 4.7) refers to a mixture of n-propanol: 0.1M sodium acetate buffer (pH 4.7) in a volume ratio of 15:85. The rest can be deduced by analogy.

[0128] The results of the intermediate purification of lipoteichoic acid obtained in step S3 are as follows: Figure 7 As shown in (A), the lipoteichoic acid elution fractions of Lactobacillus plantarum ZJ316 are mainly concentrated in the 50th and 51st tubes eluted by 42% n-propanol 0.1M sodium acetate buffer solution and the 80th tube fraction eluted by 57% n-propanol 0.1M sodium acetate buffer solution, among which the phosphorus content of the 51st tube fraction can reach 30μg / mL, and the elution components are relatively concentrated, and the elution effect is good.

[0129] S4. Fine purification of lipoteichoic acid from Lactobacillus plantarum ZJ316:

[0130] According to the solid-liquid ratio of 1 g / 100 mL, the lipoteichoic acid intermediately purified product prepared in S3 was dissolved in 30% n-propanol in 0.1 M sodium acetate buffer solution (pH 4.7), and after being fully dissolved, it was filtered with a 0.22 μm filter membrane, and 60 mL of the sample was slowly drained into a DEAE-FF chromatography column (column volume of 70 mL), and then eluted with a linear gradient eluent II;

[0131] The linear gradient eluent II is a 0-1M NaCl eluent containing 0.1M sodium acetate in 30% n-propanol (NaCl is gradually increased in 0.1M sodium acetate buffer in 30% n-propanol through a gradient mixer, and the molar concentration of NaCl is from 0 to 1M); the amount of linear gradient eluent II is 456mL in total, the flow rate is 1mL / min, and the fractions are collected in equal parts of 8mL and the phosphorus content of each eluted fraction is detected, and a total of 57 tubes are collected. The fractions containing lipoteichoic acid (i.e., tubes 39-50) are combined, and the organic reagent (i.e., n-propanol) is removed by rotary evaporation, and then dialyzed at 4°C for 48h to remove small molecular impurities (the dialysis method is the same as step S3, and the cut-off molecule of the dialysis membrane is still 3500Da), and then concentrated by rotary evaporation at 35°C to about 30% of the original volume, and then freeze-dried at -80°C for 48h to obtain Lactobacillus plantarum ZJ316 lipoteichoic acid (LTA).

[0132] The lipoteichoic acid of Lactobacillus plantarum ZJ316 obtained in step S4 is as follows: Figure 7 As shown in (B), the lipoteichoic acid elution fractions of Lactobacillus plantarum ZJ316 are mainly concentrated in tubes 39-50, and the corresponding sodium chloride elution solution concentration is 0.6M-0.8M; the phosphorus content of the 41st tube fraction can reach 11μg / mL. The elution components are relatively concentrated, and the elution effect is better.

[0133] S5. UV full wavelength scanning of the purified product of lipoteichoic acid from Lactobacillus plantarum ZJ316:

[0134] The lipoteichoic acid product (LTA) of Lactobacillus plantarum ZJ316 obtained in step S4 was prepared into a 1 mg / mL aqueous solution, and a full wavelength scan was performed at a wavelength of 200-800 nm using an ultraviolet spectrophotometer.

[0135] The results are as follows Figure 8 As shown, the purified product of lipoteichoic acid of Lactobacillus plantarum ZJ316 has no obvious absorption peak at a wavelength of 200-800 nm, indicating that the product has a good purity and is free of impurities such as nucleic acid and protein.

[0136] S6. Gel electrophoresis detection of purified lipoteichoic acid product of Lactobacillus plantarum ZJ316:

[0137] The purified lipoteichoic acid product obtained in S4 and the crude lipoteichoic acid extract obtained in S2 were prepared into 1 mg / mL solutions, respectively, and detected by agarose gel electrophoresis and SDS-PAGE, respectively.

[0138] The results are as follows Fig. 9 As shown in the figure, the three groups of crude lipoteichoic acid extracts (three parallel groups were set) all contained obvious bands in the agarose gel electrophoresis and SDS-PAGE images, indicating that the crude lipoteichoic acid extracts contained a large amount of nucleic acid and protein impurities, while the three groups of lipoteichoic acid purification products did not show any bands in the agarose gel electrophoresis and SDS-PAGE images, further indicating that the purity of the purified lipoteichoic acid product of Lactobacillus plantarum ZJ316 was high and free of impurities such as nucleic acid and protein.

[0139] S7. Fourier transform infrared spectroscopy detection of purified product of lipoteichoic acid of Lactobacillus plantarum ZJ316:

[0140] Weigh 2 mg of the lipoteichoic acid product (LTA) of Lactobacillus plantarum ZJ316 obtained in step S4 and 200 mg of dry potassium bromide in an agate mortar and grind them thoroughly to make uniform granular powder, press them into a powder using a tablet press, and use a Fourier transform infrared spectrometer at 400-4000 cm -1 Infrared light scanning within the range.

[0141] The results are as follows Fig.10 As shown, 1300-650cm -1 It is the fingerprint region, and its main value lies in the characteristics of the entire molecule, which facilitates the subsequent determination of the compound. -1 Characterize the stretching vibration of the NH group of the amide, 2952.20 cm -1 Characterizes the stretching vibration of saturated CH3 and CH2 groups, 1653.46 cm -1 , 1750.41cm -1 Characterizes the stretching vibration of C=O, 1547.31 cm -1 , 1459.17cm -1 Characterizes the bending vibration of the CH group, 1399.73 cm -1 Characterizes the stretching vibration of P=O, 1223.91cm -1 , 1053.84cm -1 Characterizes the stretching vibration of CO, 836.49 cm -1 Characterize the stretching vibration of OH and P=O.

[0142] Comparative Examples: During the invention process, the present invention also conducted the following comparative tests:

[0143] The linear gradient eluent I in step S3 of the above embodiment 3 was changed from "25%-65% n-propanol in 0.1M sodium acetate buffer" to "15%-60% n-propanol in 0.1M sodium acetate buffer", and the fractions containing lipoteichoic acid were combined, and the rest was the same as the above steps 3) and 4);

[0144] The obtained purified product of lipoteichoic acid of Lactobacillus plantarum ZJ316 showed obvious absorption peaks at 260nm and 280nm at a wavelength of 200-800nm, indicating that the purity of the product was not as good as that in Example 3.

[0145] Embodiment 4:

[0146] S1. Study on the cytotoxicity of lipoteichoic acid of Lactobacillus plantarum ZJ316:

[0147] The digested Caco-2 cells were resuspended in DMEM complete medium and 100 μL (10 4 Cell suspension was added to a 96-well plate and incubated in a 37°C incubator for 24 hours. After the cells were completely attached to the wall, they were washed with sterile DPBS, and 100 μL of lipoteichoic acid product (LTA) of Lactobacillus plantarum ZJ316 obtained in step S4 of Example 3 was added to each well at concentrations of 1, 3, 10, 30, 60, 100, 150, and 200 μg / mL, respectively. After incubation at 37°C in an incubator for 24 hours, 10 μL of CCK-8 reagent was added to each well. After incubation for 30 minutes, the absorbance of each well was measured at 450 nm, and the cell survival rate was calculated according to formula (3).

[0148] Cell survival rate = Ab-Ao / Ac-Ao x100 (3)

[0149] Wherein Ab is the absorbance of the experimental well (containing Caco-2 cells, DMEM complete medium, CCK-8 solution and LTA), Ao is the absorbance of the blank well (containing DMEM complete medium and CCK-8 solution, without Caco-2 cells and LTA), and Ac is the absorbance of the control well (containing Caco-2 cells, DMEM complete medium and CCK-8 solution, without LTA).

[0150] The results are as follows Fig.11 As shown, there was no obvious cytotoxicity when the concentration of lipoteichoic acid was 0-200 μg / mL, indicating that the lipoteichoic acid of Lactobacillus plantarum ZJ316 separated and purified by the n-butanol method can be used for subsequent cell mechanism research.

[0151] S2. Effect of lipoteichoic acid of Lactobacillus plantarum ZJ316 on the adhesion of ZJ316 to Caco-2 cells:

[0152] The digested Caco-2 cells were resuspended in DMEM complete medium at a concentration of 1 mL (10 5 The cell suspension was placed in a 24-well plate with a slide and incubated in a 37°C incubator for 24 hours. After the cells were completely attached to the wall, they were washed with sterile DPBS, and 1 ml of lipoteichoic acid product (LTA) of Lactobacillus plantarum ZJ316 obtained in step S4 of Example 3 was added to each well at concentrations of 0, 10, 100, and 150 μg / mL, respectively. After incubation in an incubator at 37°C for 2 hours, the cells were washed once with sterile DPBS, and 1x10 8 cfu / ml of Lactobacillus plantarum ZJ316 for 2 h (1 ml per well, 10 8 cfu), washed 5 times with sterile DPBS to remove non-adherent bacteria, fixed the cells with pre-cooled methanol for 30 min, then stained the cell nucleus with DAPI dye, and observed the adhesion of the strains with an inverted fluorescence microscope and photographed.

[0153] The results are as follows Fig.12 As shown, with the increase of lipoteichoic acid concentration, the adhesion of Lactobacillus plantarum ZJ316 to Caco-2 cells decreased, indicating that lipoteichoic acid is one of the potential adhesion factors of Lactobacillus plantarum ZJ316.

[0154] In summary, lipoteichoic acid has no obvious toxicity to Caco-2 cells and is a key adhesion factor of Lactobacillus plantarum ZJ316, indicating that it can be used as a microecological preparation to regulate the host intestinal microecological imbalance, maintain microecological balance, prevent the invasion of exogenous pathogens, and improve the host's health level.

[0155] It is speculated that lipoteichoic acid may be the key substance for Lactobacillus plantarum ZJ316 to regulate the balance of intestinal microecology, and has an inhibitory effect on the adhesion of certain pathogenic bacteria. It is expected to become a microecological preparation in future food.

[0156] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. A method for preparing lipoteichoic acid using high-adhesion Lactobacillus plantarum, characterized in that The following steps are involved: 1) Select Lactobacillus plantarum ZJ316 as the high-adhesion Lactobacillus plantarum; 2) Extracting the high-adhesion Lactobacillus plantarum by the n-butanol method to obtain a crude extract of Lactobacillus plantarum lipoteichoic acid.

2. The method for preparing lipoteichoic acid by using high-adhesion Lactobacillus plantarum according to claim 1, characterized in that The following steps are also included: 3) performing moderate purification on the crude lipoteichoic acid extract to obtain a moderately purified lipoteichoic acid product; 4) Finely purify the lipoteichoic acid intermediate purification product to obtain Lactobacillus plantarum ZJ316 lipoteichoic acid.

3. The method for preparing lipoteichoic acid by using high-adhesion Lactobacillus plantarum according to claim 1 or 2, characterized in that The step 2) is to separate lipoteichoic acid by n-butanol method, comprising the following steps: 2.1) Prepare bacterial mud using Lactobacillus plantarum ZJ316; The bacterial sludge of Lactobacillus plantarum ZJ316 was resuspended in sodium citrate buffer at a ratio of 1 g: 9-11 ml, and the mixture was broken to obtain a broken cell solution; 2.2) Add an equal volume of water-saturated n-butanol to the disrupted cell solution, mix them, stir at 20-22°C, collect the aqueous phase after centrifugation, and then centrifuge the collected aqueous phase to completely remove the precipitated impurities. The resulting aqueous phase is named the secondary aqueous phase; 2.3) Remove the residual organic reagent in the secondary aqueous phase by rotary evaporation, dialyze with water for 3-5 days and then freeze-dry to obtain the crude extract of lipoteichoic acid.

4. The method for preparing lipoteichoic acid by using high-adhesion Lactobacillus plantarum according to claim 3, characterized in that: The crushing treatment in step 2.1) is: crushing cells under ultra-high pressure of 1200 Pa to obtain crushed cell fluid; The dialysis in step 2.3) is specifically as follows: the secondary aqueous phase after the organic reagent is removed is placed in a 3500Da dialysis bag, placed in ultrapure water for dialysis, and the dialysis solution is replaced every 6 hours; after dialysis for 3 to 5 days, the retentate in the dialysis bag is collected.

5. The method for preparing lipoteichoic acid using high-adhesion Lactobacillus plantarum according to claim 4, characterized in that: The bacterial mud in step 2.1) is obtained by expanding the culture of Lactobacillus plantarum; The Lactobacillus plantarum expansion culture comprises: inoculating the activated Lactobacillus plantarum ZJ316 strain into an MRS liquid culture medium at a volume ratio of 1%, and culturing at 37° C. for 20 hours; Centrifuge to obtain bacterial sludge.

6. The method for preparing lipoteichoic acid using high-adhesion Lactobacillus plantarum according to any one of claims 2 to 5, characterized in that The step 3) of performing moderate purification of the crude lipid wall extract is specifically as follows: The crude extract of lipoteichoic acid of Lactobacillus plantarum ZJ316 was dissolved in 0.1M sodium acetate buffer at pH 4.7 containing 15vt% n-propanol for hydrophobic interaction chromatography, and eluted with a linear 15-60vt% n-propanol solution gradient. After determination by phosphate-molybdate colorimetry, the column fractions containing Lactobacillus lipoteichoic acid were combined and dialyzed with water for 48 hours to obtain a moderately purified product of lipoteichoic acid.

7. The method for preparing lipoteichoic acid using high-adhesion Lactobacillus plantarum according to claim 6, characterized in that The step 4) of finely purifying the lipoteichoic acid intermediate purification product is specifically as follows: The component containing lipoteichoic acid of Lactobacillus plantarum ZJ316 was diluted with 30% n-propanol 0.1M sodium acetate buffer solution, and then subjected to ion exchange chromatography. Elution was performed using 30% n-propanol 0.1M sodium acetate buffer solution containing 0-1M sodium chloride, and the eluate was collected. The phosphorus content was determined by phosphate-molybdate colorimetry. After the lipoteichoic acid elution peak was collected, the eluate was dialyzed with water for 48 hours, and freeze-dried for 48 hours to obtain Lactobacillus lipoteichoic acid.

8. The method for preparing lipoteichoic acid using high-adhesive Lactobacillus plantarum according to any one of claims 1 to 7, characterized in that The step 1) comprises: incubating different plant lactobacilli with Caco-2 cells to observe their adhesion ability, determining the adhesion rates and surface properties of different plant lactobacilli, screening out a high-adhesion plant lactobacillus ZJ316, and observing the adhesion of the high-adhesion plant lactobacillus in the mouse intestine by fluorescent probe labeling technology.

9. Use of Lactobacillus plantarum lipoteichoic acid extracted according to any one of claims 1 to 8 in the adhesion of high-adhesion Lactobacillus plantarum ZJ316 to Caco-2 cells.

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