CN115068510A - Extraction method of lactobacillus lipoteichoic acid and anti-inflammatory activity application thereof - Google Patents
Extraction method of lactobacillus lipoteichoic acid and anti-inflammatory activity application thereof Download PDFInfo
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- CN115068510A CN115068510A CN202210566930.8A CN202210566930A CN115068510A CN 115068510 A CN115068510 A CN 115068510A CN 202210566930 A CN202210566930 A CN 202210566930A CN 115068510 A CN115068510 A CN 115068510A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/115—Fatty acids or derivatives thereof; Fats or oils
- A23L33/12—Fatty acids or derivatives thereof
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
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- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
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Abstract
The invention discloses a method for extracting lipoteichoic acid of lactobacillus and anti-inflammatory activity application thereof, which is characterized by comprising the following steps: (1) extracting lactobacillus by trichloroacetic acid method, TritonX-114 method or n-butanol method to obtain lactobacillus lipoteichoic acid crude substance; (2) the lactobacillus lipoteichoic acid crude body is subjected to hydrophobic chromatography and anion exchange chromatography for purification to obtain a lactobacillus lipoteichoic acid product, wherein the lactobacillus is lactobacillus plantarum, lactobacillus reuteri or lactobacillus acidophilus, and has the application in inhibiting the expression of a cell factor of an RAW264.7 cell induced by LPS, preparing an TNF-alpha and/or IL-6 inhibitor and/or an anti-inflammatory factor IL-10 secretion activator and weakening the phosphorylation of p65 induced by the LPS in a concentration-dependent manner.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an extraction method of lactobacillus lipoteichoic acid and an anti-inflammatory activity application thereof.
Background
Lactic Acid Bacteria (LAB) are a group of gram-positive bacteria that can utilize carbohydrates for fermentation to produce organic acids, and are probiotics that are widely distributed, numerous, and abundant in nature. Lactobacillus in lactic acid bacteria is widely present in fermented foods and has many physiological functions as follows: regulating intestinal flora balance, maintaining gastrointestinal health, enhancing biological barrier, regulating immune system, reducing cholesterol, resisting oxidation, lowering blood pressure, etc.
The research shows that the immunoregulation function of lactobacillus on host may be related to peptidoglycan, teichoic acid, surface protein and other substances in cell wall. Teichoic acids, including Lipoteichoic acid (LTA) and teichoic acid (WTA), are important components of the cell Wall of gram-positive bacteria, with a dry weight of up to 20-50% of the dry weight of the cell Wall of Lactobacillus. The main structure of LTA is 1, 3-chain polyphosphate glycerolic skeleton composed of 16-40 monomers, and D-alanine, lipoid, salts and glycosyl are often connected on glycerol residue, wherein the phosphate glycerol residue of the main chain of the LTA molecule of lactobacillus is mainly connected with the D-alanine. The lipid to which LTA is bound to the cell membrane via a phosphodiester bond is the hydrophobic part of the whole molecule, while phosphoglycerides constitute the hydrophilic part of the molecule, and thus LTA is also known as an amphiphilic molecule. The LTA on the surface of the cell wall directly contacts with intestinal epithelial cells to mediate the intestinal adhesion and colonization process of the lactobacillus, and plays an important role in maintaining the steady state of the intestinal microenvironment.
LTA molecules are not only connected to cell membranes, but also inserted into a peptidoglycan layer with thick cell walls, and the complex structure and components of gram-positive bacteria cells cause the low yield of LTA extraction, so that certain amount of LTA is not easy to extract. According to literature research, the methods commonly used for extracting LTA mainly comprise Trichloroacetic acid (TCA) method, TritonX-114 method, n-butanol method and the like. Wherein trichloroacetic acid is a medium strong acid, has the functions of degreasing and promoting protein precipitation, and the trichloroacetic acid is mainly used for extracting LTA through hydrolyzing covalent bonds between the LTA and peptidoglycan, thereby achieving the purpose of separation. TritonX-114 is a novel nonionic detergent widely used for preparing components of amphoteric cell membranes, and thus is used as an effective method for LTA extraction, which maintains good biological activity and high purity. N-butanol is commonly used to extract proteins, enzymes and lipids that bind more strongly to lipids or contain more non-polar side chains, and thus LTA extracted by this method often retains its integrity.
To further investigate the immunomodulatory properties of lactobacillus LTA, we selected lactobacillus plantarum: (Lactobacillus plantarum) Lactobacillus reuteri (L.), (Lactobacillus reuteri) And Lactobacillus acidophilus (Lactobacillus acidophilus) To evaluate the function of LTA in Lipopolysaccharide (LPS) -induced inflammation of RAW264.7 cells. Although the functional research of LTA has been receiving wide attention, the extraction yield is low, and in order to better perform the functional research of LTA of lactobacillus, LTA with complete structure needs to be efficiently extracted and the content of LTA needs to be accurately analyzed.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a rapid and efficient extraction method of lactobacillus lipoteichoic acid and anti-inflammatory activity application thereof, and application thereof in inhibiting cell factor expression of RAW264.7 cells induced by LPS.
The technical scheme adopted by the invention for solving the technical problems is as follows: a method for extracting lipoteichoic acid from lactobacillus comprises the following steps:
(1) extracting lactobacillus by trichloroacetic acid method, TritonX-114 method or n-butanol method to obtain lactobacillus lipoteichoic acid crude substance;
(2) and (3) purifying the crude lactobacilli lipoteichoic acid by hydrophobic chromatography and anion exchange chromatography to obtain a lactobacilli lipoteichoic acid product.
Preference is given toThe lactobacillus in the step (1) is lactobacillus plantarum (F: (B))Lactobacillus plantarum) The strain is ATCC BAA 793; lactobacillus plantarum (A)Lactobacillus plantarum) The strain is A3; l. reuteri bacterium (A), (B)Lactobacillus reuteri) The strain is DMSZ 8533; or Lactobacillus acidophilus (Lactobacillus acidophilus) The strain is CICC 6074.
Preferably, the trichloroacetic acid extraction process in the step (1) specifically comprises: adding wet lactobacillus into trichloroacetic acid solution with the concentration of 10wt% according to the mass-volume ratio of 1g:10mL, heating in water bath at 85 ℃, stirring and extracting for 2h, cooling, centrifuging the mixed emulsion liquid at 4 ℃ at 5000rpm/min for 30min, and gently collecting supernatant to obtain the crude lactobacillus lipoteichoic acid extract.
Preferably, the extraction process of the TritonX-114 method in the step (1) is as follows: dissolving TritonX-114 in 50mM Tris-HCl buffer solution with pH 6.5 to prepare TritonX-114 solution with mass concentration of 2%, and placing the TritonX-114 solution in a refrigerator at 4 ℃ for later use; adding wet lactobacillus into the TritonX-114 solution according to the mass-volume ratio of 1g to 10mL, stirring on a magnetic stirrer, and standing overnight at 4 ℃; centrifuging at 4 deg.C and 5000rpm/min for 30min to remove cell debris, collecting upper layer extract, centrifuging at room temperature and 5000rpm/min for 30min in 50 deg.C water bath to separate the extract into two layers, and collecting upper layer water phase to obtain crude extract of lactobacilli lipoteichoic acid.
Preferably, the n-butanol extraction process in step (1) comprises: adding wet lactobacillus into 0.1M sodium citrate buffer solution with pH =4.7 according to the mass-volume ratio of 1g:5mL, treating for 30min by ultrasonic waves, mixing the thallus suspension with n-butanol with the same volume, magnetically stirring for 4h at room temperature, centrifuging for 30min at 5000rpm/min at 4 ℃ to remove cell fragments, and sucking the lower aqueous phase to obtain the crude lactobacillus lipoteichoic acid extract.
Preferably, the step (2) is specifically as follows:
A. dissolving the crude extract of lactobacillus lipoteichoic acid in 0.1M ammonium acetate buffer solution containing 15vt% n-propanol at pH 4.7, performing agarose gel (Sepharose CL-4B) chromatography, performing gradient elution with linear 15-60vt% n-propanol solution, measuring by phosphate-molybdic acid colorimetry, combining column fractions containing lactobacillus lipoteichoic acid, and dialyzing with water overnight to obtain a component containing lactobacillus lipoteichoic acid;
B. diluting the component containing the lactobacillus lipoteichoic acid obtained in the step A by using an ammonium acetate buffer solution with the volume 5 times that of the component, loading the diluted component on a DEAE-52 column pre-balanced by a 0.1M ammonium acetate buffer solution with the pH =4.7 for purification, eluting by using the 1.0M ammonium acetate buffer solution with the pH =4.7, collecting eluent, measuring the phosphorus content by using a phosphate-molybdic acid colorimetric method, collecting the lipoteichoic acid elution peak, dialyzing the eluent by using water for 48h, freezing and drying to obtain a lactobacillus lipoteichoic acid product, and storing the lactobacillus lipoteichoic acid product in a refrigerator at the temperature of minus 20 ℃ for later use.
The application of the Lactobacillus lipoteichoic acid extracted by the method in preparing anti-inflammatory active agent is provided.
The application of the lactobacillus lipoteichoic acid extracted by the method in inhibiting the cytokine expression of RAW264.7 cells induced by LPS.
The Lactobacillus lipoteichoic acid extracted by the method is applied to the preparation of TNF-alpha and/or IL-6 inhibitors and/or anti-inflammatory factor IL-10 secretion activators.
The lactobacillus reuteri lipoteichoic acid extracted by the method is applied to the preparation of an LPS-induced p65 phosphorylation expression inhibitor, an LPS-induced ERK phosphorylation expression inhibitor and/or an LPS-induced JNK MAPK phosphorylation expression inhibitor.
Compared with the prior art, the invention has the advantages that: the invention relates to an extraction method of functional lactobacillus LTA and anti-inflammatory activity application thereof, which are characterized in that three common LTA extraction methods, namely a trichloroacetic acid method, a TritonX-114 method and a n-butanol method, are compared, an LTA standard substance is used as a reference, the structure of the extracted LTA is represented by means of infrared spectrum and nuclear magnetic resonance hydrogen spectrum, and the LTA content is detected by total phosphorus content and enzyme-linked immunosorbent assay (ELISA) to determine an optimal extraction method, wherein the n-butanol method is adopted for extraction. And the fact that various lactobacillus LTAs can reduce inflammatory reaction caused by LPS (low cholesterol) but have different inflammation inhibition effects is proved, the immune system is stimulated to play the anti-inflammatory effect by regulating and controlling the release of related cytokines, and a theoretical basis is provided for developing novel functional foods or medicines of lactobacillus.
Drawings
FIG. 1 shows the extraction of Lactobacillus plantarum LTA by different extraction methods 1 HNMR spectrogram;
FIG. 2 is a Fourier infrared spectrum of Lactobacillus plantarum LTA extracted by different extraction methods;
FIG. 3 is a total phosphorus content analysis of Lactobacillus plantarum LTA extracted by different extraction methods;
FIG. 4 shows ELISA analysis of Lactobacillus plantarum LTA extracted by different extraction methods;
FIG. 5 is a FT-IR spectrum of Lactobacillus LTA and Staphylococcus aureus LTA standards;
FIG. 6 shows the NMR spectra of Lactobacillus LTA and Staphylococcus aureus LTA standards;
FIG. 7 shows Lactobacillus at 5% CO 2 After incubation for 2h at 37 ℃ in the environment, adhering Caco-2 cells, staining lactobacillus with FITC, and staining Caco-2 cell nuclei with Hoechst 33342;
FIG. 8 shows the effect of LTA on LPS-induced cytotoxicity of RAW264.7 and expression of inflammatory factors TNF- α, IL-6 and IL-10. (A) Effect of different concentrations of LTA on RAW264.7 macrophage activity; (B) the influence of LTA on LPS-induced RAW264.7 macrophage inflammatory factor TNF-alpha expression; (C) the effect of LTA on LPS-induced RAW264.7 macrophage inflammatory factor IL-6; (D) the effect of LTA on LPS-induced RAW264.7 macrophage anti-inflammatory factor IL-10.
FIG. 9 (A) shows the effect of LTA on the expression of MAPK signaling pathway protein regulated by LPS-induced RAW264.7 macrophage inflammation, and immunoblotting (Western blotting) tests show that the phosphorylation levels of ERK and JNK MAPK; (B) in order to influence the LTA on the regulation of NF-kB signal channel protein expression by LPS-induced RAW264.7 macrophage inflammation, an immunoblotting experiment (Western blotting) detects the phosphorylation condition of p 65.
Detailed Description
The invention is described in further detail below with reference to the accompanying examples.
Detailed description of the preferred embodiment
Plant breast stems used in the inventionBacteria (A), (B)Lactobacillus plantarum) The strain is ATCC BAA 793; lactobacillus plantarum (A)Lactobacillus plantarum) The strain is A3; lactobacillus reuteri: (Lactobacillus reuteri) The strain is DMSZ 8533; lactobacillus acidophilus (C.acidophilus: (C.acidophilus)Lactobacillus acidophilus) The strain is CICC 6074, which is purchased from the market.
1. Different methods for extracting LTA
A. Trichloroacetic acid process
Adding a certain weight of wet bacteria into 10% trichloroacetic acid solution according to a mass concentration of 1g:10mL (w/v), placing in a water bath at 85 ℃, heating, stirring, extracting for 2h, cooling, centrifuging the mixed emulsion liquid at 4 ℃ at 5000rpm/min for 30min, and gently collecting supernatant, namely the crude extract of LTA.
B. TritonX-114 method
Preparing 2% TritonX-114 solution with 50mM Tris-HCl buffer solution with pH 6.5, placing in a refrigerator at 4 ℃ for later use, adding 10mL 2% TritonX-114 solution into 1g of wet bacteria, suspending the bacteria, stirring on a magnetic stirrer, and standing overnight at 4 ℃. Then, the cell debris was removed by centrifugation at 5000rpm/min at 4 ℃ for 30min, and the upper extract was collected. The extract is subjected to water bath at 50 ℃ for 30min, and centrifuged at 5000rpm/min at normal temperature for 30min to separate the extract into two layers, and the upper aqueous phase is collected gently to obtain the crude extract of LTA.
C. N-butanol process
According to the method, 1g of wet bacteria are suspended in 5mL of 0.1M sodium citrate buffer solution (pH 4.7), ultrasonic treatment is carried out for 30min, then the suspension is mixed with n-butanol with the same volume, magnetic stirring is carried out for 4h at room temperature, then centrifugation is carried out for 30min at 5000rpm/min at 4 ℃ to remove cell fragments, and a lower aqueous phase, namely a crude extract of LTA, is absorbed.
2. Purification of crude LTA extract
Subjecting the crude extract to hydrophobic chromatography and anion exchange chromatography. First, agarose gel (Sepharose CL-4B) chromatography was performed, and the LTA extract was dissolved in 0.1M ammonium acetate buffer (containing 15% by volume of n-propanol, pH 4.7). LTA was eluted with a linear 15-60vt% n-propanol (vol. concentration) gradient. After colorimetric phosphate-molybdic acid assay, the column fractions containing LTA were pooled and dialyzed against water overnight. The LTA-containing fraction was subjected to further DEAE-52 column purification. The DEAE-52 column is preferably pre-equilibrated with 0.1M ammonium acetate buffer (pH 4.7), the LTA component is diluted with 5 volumes of ammonium acetate buffer, loaded onto the DEAE-52 column, and eluted with 1.0M ammonium acetate buffer (pH 4.7). Collecting the eluent, measuring the phosphorus content by a phosphate-molybdic acid colorimetric method, and collecting an LTA elution peak. Finally, the eluent is dialyzed for 48h by water, frozen and dried, and stored in a refrigerator at the temperature of 20 ℃ below zero for standby.
3. LTA structural analysis
The structure of LTA was determined by Fourier transform infrared spectroscopy and nuclear magnetic resonance spectroscopy. Respectively mixing LTA standard substance of staphylococcus aureus and LTA sample of lactobacillus with KBr, and adding water at 3750-750 cm -1 Infrared spectroscopic analysis was performed on LTA samples over a range of wavelengths. A15 mg sample of LTA was dissolved in 0.5 mL ddH 2 In O, nuclear magnetic resonance detection 1 H。
4. Adhesion Capacity of Lactobacillus strains
Will be in the laboratoryL. plantarum A3、L. reuteriDMSZ 8533 andL. acidophilusCICC 6074 was cultured in MRS medium, then washed in sterile PBS for resuspension, OD adjusted 600 nm The value is 1. Lactobacillus was dissolved in 0.05 mg mL -1 FITC Green fluorescence-labeled strains were prepared in Fluorescein Isothiocyanate (FITC), by allowing to dark at 37 ℃ for 1 h, followed by 5 washes with PBS (pH = 7.2). Caco-2 cells in DMEM medium containing 20% fetal bovine serum and 1% double antibody at 37 deg.C with CO 2 Culturing in an incubator with the concentration of 5%. When Caco-2 cells grew to a monolayer, FITC-labeled strain was added to the cell well plate and incubated with Caco-2 cells for 2 h. Cells were then washed three times with PBS (pH =7.2) to remove excess staining. Finally, after fixation with 4% paraformaldehyde for 20 min, Hoechst 33342 (0.1 mg mL) was fixed with nuclear blue dye -1 ) Staining the nuclei for 15 min, and observing the fixed cells by an inverted fluorescence microscope.
5. Cytotoxicity assays
The CCK-8 kit is adopted to detect the influence of LTA on the activity of RAW264.7 cells. RAW264.7 cells were seeded in 96-well plates and incubated for 12 h, followed by 100μ LLTA (0, 25, 50 and 100 μ g mL) -1 ) The cells were treated for 18 h. Add 10. mu.L of CCK-8 reagent to each well and incubate for 6 h. The absorbance was measured using a Synergy plate reader at a wavelength of 450 nm. Cell viability was calculated as follows:
cell viability = (a-C)/(B-C) × 100%, where a is the absorbance of the experimental wells (cell-containing medium, LTA sample, CCK-8); b is the absorbance of the control well (cell-containing medium, LTA-free sample, CCK-8); c is the absorbance of blank wells (medium without cells, LTA-free samples, CCK-8).
6. Determination of cytokine content in RAW264.7 macrophages
When RAW264.7 cells reached 80% confluence in 96-well plates, LPS (500 ng mL) was used -1 ) Treatment for 10 h, then final concentrations of 0, 25, 50 and 100. mu.g mL were added -1 And incubating for 18 h. The supernatant was collected and centrifuged at 10000rpm at 4 ℃ for 10 min. TNF-alpha, IL-6 and IL-10 cytokines were detected in cell supernatants using ELISA kits according to the manufacturer's instructions. Absorbance at 450 nm was measured using a microplate reader (Thermo Fisher Scientific, USA).
7. Immunoblotting experiments
The effect of Lactobacillus LTA on proteins in the mitogen-activated protein kinase (MAPK) and nuclear factor-activated kappa-light chain (NF-. kappa.B) signaling pathway in B cells was investigated. Equivalent amounts of protein were separated by 12% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. Subsequently, the PVDF membrane was blocked with 5% Bovine Serum Albumin (BSA) for 2 h. Then, ERK, p-ERK, JNK, p-JNK, p65, p-p65, and β -actin were incubated with horseradish peroxidase (HRP) -conjugated secondary antibodies overnight at 4 ℃ in Tris-buffered saline/Tween 20 (TBS-T), respectively, and then incubated for 1 h. The immunoreactive bands were visualized using an enhanced chemiluminescence detection system (Bio-Rad, USA). Finally, the grayscale values of the individual bands were analyzed using Image J software (ChemiScope 6000 Exp).
Detailed description of the invention
1. Structural characterization of Lactobacillus plantarum LTA
The invention carries out the extraction of the LTA standard product of staphylococcus aureus and the Lactobacillus plantarum LTA extracted by the three methodsAnd (4) nuclear magnetic resonance hydrogen spectrometry. As a result, the LTA of the lactobacillus plantarum extracted by three different extraction methods is similar to the LTA standard of staphylococcus aureus 1 H NMR characteristic peak. As shown in FIG. 1, of LTA samples 1 H NMR showed 4 single peaks at 2.04 and 1.24 ppm, both assigned to different-CH groups of alanine, indicating that the LTA structure contains a modified structure of alanine. Compared with Staphylococcus aureus, the LTA sample has peaks similar in the region of 3.79-4.10 ppm due to their identical chemical environment or similar structural groups. The results show that the trichloroacetic acid method, the TritonX-114 method and the n-butanol method can effectively extract the LTA of the lactobacillus plantarum.
Fourier transform infrared spectroscopy is a common method of studying the type of molecular functional groups and their chemical environment. FT-IR at 3750 and 750 cm -1 LTA samples were analyzed in range. As shown in FIG. 2, there is no significant difference in the lengths and saturations of the alpha-D-N-acetylglucosamine, alanine, acyl fatty acid chains of LTA extracted by three different extraction methods. In its infrared spectrum at 2939 cm -1 The peak at (a) is due to the vibration of the-CH bond within the fatty acid chain. At 1655 cm −1 And 1545 cm -1 The weak peaks appearing nearby are due to the C = O and N-H bonds of α -D-N-acetylglucosamine, respectively. At 1390 cm -1 Here, the vs (COO-) adsorption peak of alanine is clearly visible. 1047 cm −1 The nearby peaks are the result of the P-O stretching vibrations. Therefore, the characteristic peaks of the LTA extracted by the Lactobacillus plantarum and the LTA extracted by the trichloroacetic acid method, the TritonX-114 method and the n-butanol method have many similar parts with the characteristic peaks of the LTA standard of the staphylococcus aureus, which indicates that the LTA extracted by the three methods is successful.
2. Analysis of Total phosphorus content in Lactobacillus plantarum LTA crude extract
FIG. 3 is a graph showing the results of total phosphorus content in LTA crude extracts extracted from Lactobacillus plantarum strains of the same weight by three methods. The characteristic element in LTA is phosphorus, so the content of LTA can be reflected to a certain extent by the content of total phosphorus. As can be seen from FIG. 3, the phosphorus content of the crude LTA extract obtained by the trichloroacetic acid method is up to 35.46mmol/L, which is higher than that of the other two methods, and the phosphorus content of the crude LTA extract obtained by the TritonX-114 method is up to 16.12 mmol/L.
3. ELISA detection analysis of LTA in crude extract of Lactobacillus plantarum LTA
FIG. 4 is a graph showing the results of ELISA detection of LTA content in crude LTA extracts from wet Lactobacillus plantarum having the same weight by three methods. As can be seen from FIG. 4, the LTA content obtained by the n-butanol method was the highest, and the average content was 72.1pg mL -1 (ii) a Secondly, TritonX-114 method; LTA obtained by trichloroacetic acid method is lowest, and average content is 15.8pg mL -1 . The result shows that the yield of LTA extracted by the n-butanol method is highest, and the method is the optimal extraction method of the LTA of the lactobacillus.
Comparing the structural characteristics of the LTA standard products, the results show that the LTA of the lactobacillus plantarum obtained by the three extraction methods has structural similarity, and the LTA extracted by the three methods can be determined to have consistent structural characteristics. By analyzing the total phosphorus content and LTA content of lactobacillus plantarum LTA, although the phosphorus content of LTA extracted by a trichloroacetic acid method is the most abundant, the LTA content detected by ELISA is not the highest, which indicates that the total phosphorus content is in certain correlation with LTA, but the total phosphorus content cannot accurately reflect the LTA level. Researches show that due to the strong acidity of trichloroacetic acid, LTA molecules can cause hydrolysis of phosphodiester bonds after contacting with trichloroacetic acid for a long time, the molecular structure of an extract is incomplete, the phosphorus content is increased, and the LTA level is reduced. The lactobacillus plantarum LTA extracted by the n-butanol method is detected by ELISA, and the content is the highest, so the n-butanol extraction method can be used as the preferred extraction method of the lactobacillus LTA.
4. Structural characterization of Lactobacillus LTA
For researching the LTA structural characteristics of lactobacillus, FT-IR method is adopted at 3750-750 cm -1 In the scanning wavelength range ofL. reuteri DMSZ 8533、L. plantarumA3 andL. acidophilusCICC 6074 carried out LTA structural analysis. As shown in FIG. 5, at 3400 cm -1 Tensile oscillations of the OH groups are visible. 2939 cm -1 The peak at (A) is the vibration of the internal-CH bond in the fatty acid chain. At 1655 cm -1 And 1545 cm -1 The weak characteristic peaks appearing nearby belong to the C = O and N-H bonds of α -D-N-acetylglucosamine, respectively. V s (COO)-) the absorption peak of the vibration contraction was located at 1390 cm -1 To (3). 1047 cm -1 The nearby peaks indicate the P-O stretching vibration. Notably, LTA from both Lactobacillus plantarum and Lactobacillus reuteri was 1545 cm -1 A clear N-H oscillation peak appears, but the Lactobacillus acidophilus LTA does not exist, which indicates that the strain is derived fromL. reuteriDMSZ 8533 andL. plantarumthe LTA of a3 has a longer saturated acyl fatty acid chain modification.
LTA was analyzed by 500 MHz high field NMR spectroscopy. As shown in fig. 6, the staphylococcus aureus LTA standard has distinct absorption peaks at ppm =0.81, 1.11, 1.24, 2.04;L. plantarumLTA of a3 has distinct absorption peaks at ppm =1.3, 1.98, 3.50, 3.79;L. reuterithe LTA of DMSZ 8533 has distinct absorption peaks at ppm =1.19, 1.98, 3.517, 3.721, 3.902;L. acidophilusLTA of cic 6074 has distinct absorption peaks at ppm =1.204, 1.863, 3.561, 3.812. The three strains of lactobacillus LTA contain glycerol monomer, N-acetylglucosamine and alanine modification group, whereinL. reuteriThe LTA of DMSZ 8533 contains more glycerol monomers, N-acetylglucosamine and alanine modifications. Therefore, there are significant differences in the length and saturation of the α -D-N-acetylglucosamine, alanine and acyl fatty acid chains of three strains of lactobacillus.
5. Adhesion properties of Lactobacillus strains to Caco-2 cells
Are respectively pairedL. reuteri DMSZ 8533、L. plantarumA3 andL. acidophilusthe CICC 6074 strain is labeled with FITC, successfully labeled thalli is observed to be green in a microscopic mode, and is incubated with Caco-2 cells for 2 hours at 37 ℃, and the cells are washed three times by PBS to remove redundant staining. Finally, after being fixed for 20 min by 4% paraformaldehyde, the cell nucleus is stained by Hoechst 33342 for 15 min, and the fixed cell nucleus morphology is observed by an inverted fluorescence microscope (the second bright spot in the black and white image is Caco-2 cell nucleus). As shown in FIG. 7, FITC-labeledL. reuteri DMSZ 8533 andL. plantarum a3 strain andL. acidophilusCICC 6074 shows a significantly stronger green fluorescence signal intensity (corresponding to the third column highlight in the black and white image, FITC labeled strain) and a stronger adhesion property.
6. Regulation effect of lactobacillus LTA on secretion of RAW264.7 cell inflammatory factor
First, the toxicity of LTA addition to RAW264.7 cells was tested using the CCK-8 kit. As can be seen in FIG. 8A, at 25, 50 and 100. mu.g mL -1 At a concentration of (a) of (b),L. reuteri DMSZ 8533、L. plantaruma3 andL. acidophilusthe LTA of CICC 6074 has no obvious influence on the cell viability, and shows that the LTA of the three lactobacillus is 0-100 mu g mL -1 In the above range, the cells were not affected by toxicity. The effect of lactobacillus LTA on LPS-induced RAW264.7 cell inflammation was examined using an ELISA kit. As shown in FIGS. 8B and C, LPS significantly promoted the production of the cytokines TNF-. alpha.and IL-6. LTA separated from the three strains remarkably inhibits TNF-alpha production induced by LPS, and especially LTA of lactobacillus plantarum and lactobacillus acidophilus can inhibit TNF-alpha production in a concentration-dependent manner. Similarly, LTA of all three lactobacilli significantly inhibited IL-6 levels. As shown in FIG. 8D, LTA of three kinds of Lactobacillus was able to activate the secretion of anti-inflammatory factor IL-10, especially LTA of Lactobacillus reuteri at concentrations of 25 and 50. mu.g mL -1 The expression of IL-10 is obviously improved. Thus, lactobacillus LTA is able to inhibit LPS-induced cytokine expression in RAW264.7 cells.
7. Lactobacillus LTA plays a role in anti-inflammatory regulation through MAPK/NF-kB signal pathway
Combining the adhesion and cytokine results, the lactobacillus reuteri LTA has better anti-inflammatory and probiotic properties, and the potential mechanism of the immunoregulatory activity mediated by the lactobacillus reuteri LTA is further explored. In the LPS-stimulated RAW264.7 cell inflammatory response studies, MAPK (ERK and JNK) and NF- κ B (p 65 and p50 subunits) were shown to be involved in inflammatory regulation. As shown in fig. 9A, LPS caused a significant increase in phosphorylation of ERK and JNK MAPK compared to the control without any treatment, while LTA significantly inhibited both expression in a dose-dependent manner. LTA had no significant effect on the non-phosphorylation levels of ERK and JNK MAPK.
NF- κ B (p 65 and p 50) is involved in LPS-induced inflammatory responses, a key downstream signaling molecule of the MyD88 signaling pathway, ultimately driving cytokine gene expression. As shown in fig. 9B, LPS significantly upregulated phosphorylation of p65, while LTA attenuated LPS-induced p65 phosphorylation in a concentration-dependent manner. This finding suggests that the MAPK and NF- κ B signaling pathways, which MyD88 is dependent on, play an important role in LTA-mediated anti-inflammatory responses.
The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Those skilled in the art should also realize that changes, modifications, additions and substitutions can be made without departing from the true spirit and scope of the invention.
Claims (10)
1. A method for extracting lipoteichoic acid of lactobacillus is characterized by comprising the following steps:
(1) extracting lactobacillus by trichloroacetic acid method, TritonX-114 method or n-butanol method to obtain lactobacillus lipoteichoic acid crude substance;
(2) and (3) purifying the lactobacillus lipoteichoic acid crude body by hydrophobic chromatography and anion exchange chromatography to obtain a lactobacillus lipoteichoic acid product.
2. The method for extracting lipoteichoic acid from lactobacillus according to claim 1, wherein the method comprises the following steps: the lactobacillus in the step (1) is lactobacillus plantarum (A)Lactobacillus plantarum) The strain is ATCC BAA 793; lactobacillus plantarum (A)Lactobacillus plantarum) The strain is A3; lactobacillus reuteri: (Lactobacillus reuteri) The strain is DMSZ 8533; or Lactobacillus acidophilus (Lactobacillus acidophilus) The strain is CICC 6074.
3. The method for extracting lipoteichoic acid from lactobacillus according to claim 2, wherein the trichloroacetic acid extraction process in step (1) comprises: adding wet lactobacillus into trichloroacetic acid solution with the concentration of 10wt% according to the mass-volume ratio of 1g:10mL, heating in water bath at 85 ℃, stirring and extracting for 2h, cooling, centrifuging the mixed emulsion liquid at 4 ℃ at 5000rpm/min for 30min, and gently collecting supernatant to obtain the crude lactobacillus lipoteichoic acid extract.
4. The method for extracting lipoteichoic acid from lactobacillus according to claim 2, wherein the BTritonX-114 extraction process in step (1) comprises: dissolving TritonX-114 in 50mM Tris-HCl buffer solution with pH 6.5 to prepare TritonX-114 solution with mass concentration of 2%, and placing the TritonX-114 solution in a refrigerator at 4 ℃ for later use; adding wet lactobacillus into the TritonX-114 solution according to the mass-volume ratio of 1g to 10mL, stirring on a magnetic stirrer, and standing overnight at 4 ℃; then centrifuging at 5000rpm/min at 4 deg.C for 30min to remove cell debris, and collecting the upper layer extract; and (3) centrifuging the upper layer extract in water bath at 50 ℃ for 30min and at the normal temperature of 5000rpm/min for 30min to separate the extract into two layers, and gently collecting the upper layer water phase to obtain the crude extract of the lipoteichoic acid of the lactobacillus.
5. The method for extracting lipoteichoic acid from lactobacillus according to claim 2, wherein the n-butanol extraction process of step (1) comprises: adding wet lactobacillus into 0.1M sodium citrate buffer solution with pH =4.7 according to the mass-volume ratio of 1g:5mL, treating for 30min by ultrasonic waves, mixing the thallus suspension with n-butanol with the same volume, magnetically stirring for 4h at room temperature, centrifuging for 30min at 5000rpm/min at 4 ℃ to remove cell fragments, and sucking the lower aqueous phase to obtain the crude lactobacillus lipoteichoic acid extract.
6. The method for extracting lipoteichoic acid from lactobacillus according to claim 2, wherein the step (2) is as follows:
A. dissolving the crude extract of lactobacilli lipoteichoic acid in 0.1M ammonium acetate buffer solution with pH of 4.7 and containing 15vt% of n-propanol, carrying out agarose gel chromatography, carrying out gradient elution by using linear 15-60vt% of n-propanol solution, combining column fractions containing lactobacilli lipoteichoic acid after the determination by a phosphate-molybdic acid colorimetric method, and dialyzing with water overnight to obtain a component containing lactobacilli lipoteichoic acid;
B. diluting the component containing the lactobacillus lipoteichoic acid obtained in the step A by using an ammonium acetate buffer solution with the volume 5 times that of the component, loading the diluted component on a DEAE-52 column pre-balanced by a 0.1M ammonium acetate buffer solution with the pH =4.7 for purification, eluting by using the 1.0M ammonium acetate buffer solution with the pH =4.7, collecting eluent, measuring the phosphorus content by using a phosphate-molybdic acid colorimetric method, collecting the lipoteichoic acid elution peak, dialyzing the eluent by using water for 48h, freezing and drying to obtain a lactobacillus lipoteichoic acid product, and storing the lactobacillus lipoteichoic acid product in a refrigerator at the temperature of minus 20 ℃ for later use.
7. Use of a lactobacilli lipoteichoic acid extracted by a method according to any one of claims 2-6 for the preparation of an anti-inflammatory active agent.
8. Use of a lipoteichoic acid of lactobacillus extracted according to any of the claims 2-6 for inhibiting LPS-induced cytokine expression in RAW264.7 cells.
9. Use of a lipoteichoic acid of lactobacillus extracted by the method of any one of claims 2 to 6 for the preparation of a TNF- α and/or IL-6 inhibitor and/or a secretion activator of the anti-inflammatory factor IL-10.
10. Use of a lipoteichoic acid of Lactobacillus reuteri extracted according to any of claims 2 to 6 for the preparation of an inhibitor of LPS-induced phosphorylation expression of p65, an inhibitor of LPS-induced phosphorylation expression of ERK and/or an inhibitor of LPS-induced phosphorylation expression of JNK MAPK.
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| CN114099555A (en) * | 2021-11-11 | 2022-03-01 | 天津科技大学 | Lactobacillus plantarum lipoteichoic acid and application thereof in inhibiting amyloid protein aggregation |
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