KR20200121096A - Solvent for Extracting Lipopolysaccharide and Method for Lipopolysaccharide Extraction Using Thereof - Google Patents

Abstract

The present invention relates to a solvent for extracting lipopolysaccharides (LPS), which can be used to efficiently extract LPS from Gram-negative bacteria, and to a LPS extraction method using the same. The solvent for LPS extraction is effective in extracting a complete form of LPS from Gram-negative bacteria, and when the solvent for LPS extraction is used, LPS having high immune cell activity can be more easily extracted. Also, it is possible to obtain an LPS extract that is helpful in improving the immunity of mammals including humans.

Description

Solvent for Extracting Lipopolysaccharide and Method for Lipopolysaccharide Extraction Using Thereof}

The present invention relates to a solvent for extracting LPS that can be used to efficiently extract lipopolysaccharide (LPS) from Gram-negative bacteria and a method for extracting lipopolysaccharide using the same.

LPS is one of the major constituents in the outer membrane of Gram-negative bacteria, provides an extracellular physical barrier that responds to and interacts with nature, and is recognized by the immune system as a marker for invading bacterial pathogens (Rosenfeld and Shai, 2006) . LPS is a highly modified glycolipid consisting of lipid A, non-repeating core oligosaccharide, and variable O-antigen polysaccharide (O-PS). Lipid A, a hydrophobic anchor of LPS, has been shown to be a cause of inflammation (Poltorak et al., 1998; Hoshino et al., 1999; Aderem and Ulevitch, 2000). On the other hand, O-PS residues vary according to their constituent components and lengths, and are characterized by the serotype of the pathogen (Raetz and Whitfield, 2002) and are also important in stimulating immune cells (Plevin et al., 2016). . As information on the bacterial genome increases, the consensus gene for lipid A and core oligosaccharide biosynthesis is better understood, but the molecular mechanisms for mutation of the O-PS site are not well known.

In order to extract various types of LPS from bacteria, many methods have been developed. One of the most widely used methods is the hot phenol-water method, which can be performed with high yield and short processing time (Westphal et al., 1952). However,'rough' LPS (R-LPS) containing less lipophilic heptose is difficult to extract with phenol (Kasai and Nowtny, 1967). According to previous studies, the hydrophilic or lipophilic properties of LPS influence the extraction efficiency with organic solvents (Hickman and Ashwell, 1966; Kasai and Nowtny, 1967; Raff and Wheat, 1968). Galanos et al. (1969) developed an alternative method for extracting R-LPS using a solvent mixture of phenol, chloroform, and petroleum ether, but the wild type'smooth' LPS (S-LPS) and certain impurities were also developed. There was a problem of extraction. Until now, depending on the purpose of extraction, butanol (Morrison and Leive, 1975), 2% SDS-0.1 M EDTA-0.25 M MgCl ₂ solution (Darveau and Hancock, 1983), MgCl ₂ -Triton X-100 solution (Uchida and Mizushima, 1987) ), boiling water (Eidhin and Mouton, 1993), methanol (Nurminen and Vaara, 1996), and chloroform-methanol (Henderson et al., 2013) to extract LPS. However, there is no currently available method for a method of extracting all kinds of LPS from strains of different species, or a method of extracting LPS from the same species under different environmental conditions. In addition, questions still remain about extraction efficiency, quality and equivalence in biological testing. It is important to first understand the functions and limitations of commonly used extraction methods in order to know what kind of LPS is preferred during extraction and whether it changes with the development of new methods.

The required quality and biological activity of LPS depend on the extraction solvent used and on the progress of the purification process. The degree of hydrophilicity or lipophilicity of LPS is a major issue, and thus more or less may be extracted. Liquid-liquid phase extraction is controlled by the ionic strength of the polysaccharides constituting the LPS and the surrounding environment such as pH, salt concentration, and temperature. Moreover, contamination by macromolecules such as nucleic acids and proteins degrades quality and limits their reliable use in biological applications. Enzymatic digestion may be performed before or after LPS extraction using an extraction solvent in order to remove nucleic acids and proteins for quality improvement.

The present invention is a solvent for separating LPS from Gram-negative bacteria, a pH consisting of 30 to 50% of phenol, 0.5 to 2 M of guanidine thiocyanate, 0.1 to 0.8 M of ammonium thiocyanate, and 3 to 10% of glycerol An object of the present invention is to provide a solvent for LPS extraction comprising a sodium acetate solution of 4.5 to 5.5 or a Tris/HCl solution of pH 7.5 to 8.5.

Here, the sodium acetate solution may include 0.05 to 0.5 M sodium acetate. In addition, the Tris/HCl solution may contain 0.05 to 0.5 M of Tris/HCl.

In addition, the present invention comprises the steps of: a) preparing a first sample solution by contacting the LPS extraction solvent with a sample containing Gram-negative bacteria; And b) subjecting the first sample solution to heat treatment at 20 to 70° C. for 5 to 50 minutes.

Here, after the step b), c) preparing a second sample solution by adding chloroform to the heat-treated first sample solution; d) separating the aqueous phase by centrifuging the second sample solution; And e) adding ethanol to the aqueous phase to precipitate LPS.

Step b) may be heat-treating the first sample solution at 20 to 40°C, or 50 to 70°C.

<LPS extraction solvent>

According to one embodiment of the present invention, the present invention provides a solvent for extracting LPS comprising a sodium acetate solution of pH 4.5 to 5.5 or a Tris/HCl solution of pH 7.5 to 8.5 as a solvent for separating LPS from Gram-negative bacteria do.

More specifically, the solvent for LPS extraction is sodium having a pH of 4.5 to 5.5 consisting of 30 to 50% of phenol, 0.5 to 2 M of guanidine thiocyanate, 0.1 to 0.8 M of ammonium thiocyanate and 3 to 10% of glycerol. It may be an acetate solution. At this time, the sodium acetate solution may have a final pH of 4.5 to 5.5 by including 0.05 to 0.5 M sodium acetate.

Alternatively, the solvent for LPS extraction is Tris/HCl of pH 7.5 to 8.5 consisting of 30 to 50% of phenol, 0.5 to 2 M of guanidine thiocyanate, 0.1 to 0.8 M of ammonium thiocyanate, and 3 to 10% of glycerol. It can be a solution. At this time, the Tris/HCl solution may have a final pH of 7.5 to 8.5 by including 0.05 to 0.5 M of Tris/HCl.

The gram-negative bacteria include LPS as a major constituent of the outer membrane, and LPS is composed of lipid A having endotoxin activity, O-PS, etc. consisting of a polysaccharide acting as an antigen of the strain. When such gram-negative bacteria are introduced into mammals, including humans, or when LPS isolated from gram-negative bacteria is injected into mammals, including humans, it is known that an immune response occurs by LPS in the body of the mammal.

In the extraction solvent of the present invention, phenol is used to separate or extract LPS from the cell membrane of Gram-negative bacteria. At this time, phenol denatures proteins and dissolves lipids to destroy cell membranes. These phenols may be contained in 30 to 50% of the total volume based on sodium acetate solution or Tris/HCl solution.

The guanidine thiocyanate and ammonium thiocyanate are provided as a chaotrope to inhibit hydrogen bonding of water molecules and denature polymers such as proteins and nucleic acids. Destruction and induces the separation of LPS from the cell membrane. Such guanidine thiocyanate may be contained in a total concentration of 0.5 to 2 M based on sodium acetate solution or Tris/HCl solution. In addition, ammonium thiocyanate may be contained in a total concentration of 0.1 to 0.8 M based on sodium acetate solution or Tris/HCl solution.

The glycerol is used as a solubilizer that increases the solubility of phenol. This glycerol may be contained in an amount of 3 to 10% of the total volume based on sodium acetate solution or Tris/HCl solution.

The sodium acetate and Tris/HCl play a crucial role in extracting LPS by adjusting the acidity of the solvent for extracting LPS, and are used to increase the yield of LPS. The sodium acetate may be contained in a final concentration of 0.05 to 0.5 M based on the sodium acetate solution, and accordingly, the pH of the final sodium acetate solution (a solvent for LPS extraction) may be adjusted to 4.5 to 5.5. In addition, Tris/HCl may be contained in a final concentration of 0.05 to 0.5 M based on the Tris/HCl solution, and the pH of the final Tris/HCl solution (solvent for LPS extraction) may be adjusted to 7.5 to 8.5.

Such a solvent for LPS extraction can be conveniently used to obtain a high yield of LPS from Gram-negative bacteria compared to a solvent used for conventional LPS extraction.

<LPS extraction method>

According to another embodiment of the present invention, the present invention provides an LPS extraction method for extracting LPS from Gram-negative bacteria. More specifically, a) preparing a first sample solution by contacting a sample containing Gram-negative bacteria with the solvent for extracting LPS as described above; And b) heat-treating the sample solution at 20 to 70°C for 5 to 50 minutes. Thereafter, c) preparing a second sample solution by adding chloroform to the heat-treated first sample solution; d) separating the aqueous phase by centrifuging the second sample solution; And e) adding ethanol to the aqueous phase to precipitate LPS.

Step a) is a process of mixing Gram-negative bacteria and a solvent for extracting LPS. The Gram-negative bacteria may be cultured under appropriate culture conditions and pretreated to extract LPS. Since the solvent for extracting LPS is the same as described above, it is omitted to avoid redundant description. A sample containing such a gram-negative bacteria and a solvent for extracting LPS may be mixed (contacted) to prepare a primary sample solution.

Step b) is a process of reacting the first sample solution at 20 to 70°C for 5 to 50 minutes. At this time, the reaction temperature may be 20 to 40 ℃, or 50 to 70 ℃.

Steps c) and d) are a process of preparing a second sample solution by adding chloroform to the heated first sample solution, and then centrifuging it to separate an aqueous phase containing LPS. The chloroform is used to separate an aqueous phase containing only LPS without phenol from the primary sample solution.

In step e), ethanol is added to the aqueous phase to precipitate LPS to obtain a pellet. The ethanol serves to precipitate LPS contained in the aqueous phase. Through this process, only LPS isolated from Gram-negative bacteria can be obtained.

This LPS extraction method is simpler than the conventional LPS extraction method, and a high yield of LPS extract can be obtained from Gram-negative bacteria. The LPS extract contains fewer incomplete forms of LPS such as truncated LPS and R-LPS, but contains a lot of complete forms of S-LPS, and inflammation-causing cytokines such as TNF-α, etc. for immune cells of mammals including humans Can increase the production of.

The solvent for LPS extraction according to the present invention is effective in extracting the complete form of LPS from Gram-negative bacteria, and when such an LPS extraction solvent is used, LPS with high immune cell activity can be more easily extracted, and mammalian including humans It is possible to obtain an LPS extract that helps improve animal immunity.

1 is an experimental result according to an embodiment of the present invention; (A) This is a photograph of LPS samples according to each extraction method separated into an aqueous phase (A layer) and a buffy coat (B layer). The crude LPS extracted from Salmonella Typhimurium 14028S, Acinetobacter baumannii ATCC19606, Escherichia coli W3110 was analyzed by SDS PAGE (B) Coomassie blue, (C) Alcian blue, (D) Silver ( This is a picture of a gel dyed with silver). 10 μL of crude LPS extract was used equally, and 10-15% acrylamide gel was used.
2 is an experimental result according to an embodiment of the present invention; Gel photo of (A) S. Typhimurium 14028S, (B) A. baumannii ATCC19606, (C) E. coli W3110, and then purified LPS extract treated with nucleic acid and protease was analyzed by SDS PAGE. to be. The purified LPS extract was repeated 3 times using the same 200 μg (dry weight). For the LPS extract of S. Typhimurium 14028S, a 10% acrylamide gel was used to distinguish the S-LPS band with O-PS, and R-LPS, truncated for the LPS extract of A. baumannii ATCC19606 and E. coli W3110. A 15% acrylamide gel was used to distinguish the LPS bands. The gel was stained with Coomassie Blue, Alcian Blue and Silver.
3 is an experimental result according to an embodiment of the present invention; It is a gel picture of SDS PAGE analysis of LPS extract purified by treatment with nucleic acid and protease after extraction from S. Typhimurium 14028S, A. baumannii ATCC19606, E. coli W3110 by each T-sol method. 200 μg (dry weight) of the purified LPS extract was equally used. For the LPS extract of S. Typhimurium 14028S, a 10% acrylamide gel was used to distinguish the S-LPS band with O-PS, and R-LPS, truncated for the LPS extract of A. baumannii ATCC19606 and E. coli W3110. A 15% acrylamide gel was used to distinguish the LPS bands. The gel was stained with Coomassie Blue, Alcian Blue and Silver.
4 is an experimental result according to an embodiment of the present invention; These are the standard curves of LPS extracts extracted and purified by hot phenol-water method and T-sol (pH 8, 60°C) method. Each curve represents the absorbance value measured at 204 nm by diluting LPS twice with 1×PBS.
5 is an experimental result according to an embodiment of the present invention; It shows the activation of RAW264.7 cells by the purified LPS extract extracted by hot phenol-water method and T-sol (pH 8, 60°C) method. (A) 1 μg mL ^-1 of LPS extracted from E. coli W3110 by the T-sol (pH 8, 60°C) method was treated with RAW264.7 cells, and 4 hours later compared with the control without LPS treatment with a phase contrast microscope This is a photograph of a mononuclear cell and a macrophage type. (B) To calculate the effective dose (ED50) and maximum level (Emax) for the differentiation of monocytes after 4 hours by treating RAW264.7 cells with LPS extracted from E. coli W3110 at various concentrations by each method. This is a nonlinear regression graph. The percentage (%) of macrophages in which RAW264.7 cells were activated is expressed as the mean value obtained from 3 replicate experiments and the standard deviation thereof. (C) To calculate the effective dose (ED50) and the maximum level (Emax) for the stimulation of TNF-α expression after 4 hours by treating the LPS extracted from E. coli W3110 at various concentrations on RAW264.7 cells by each method. This is a nonlinear regression graph. The concentration of TNF-α was measured using an ELISA kit.

Hereinafter, with reference to the accompanying drawings will be described in more detail the solvent for LPS extraction of the present invention and the LPS extraction method using the same. However, these descriptions are provided by way of example only to aid understanding of the present invention, and the scope of the present invention is not limited by these exemplary descriptions.

1. Materials and methods

1-1. Strains and culture

Salmonella enterica serovar Typhimurium strain 14028S producing S-LPS (Marshall and Gunn, 2015) and R-LPS (Richie et al., 2016), Acinetobacter baumannii strain ATCC 19606 were used, Escherichia coli strain W3110 having a truncated LPS form (Wang et al., 2015) was used.

In order to extract LPS, S. Typhimurium 14028S, A. baumannii ATCC 19606 and E. coli W3110 were mixed at 37° C., aerobic conditions for 3 hours in 1 L of LB liquid medium (Luria-Bertani broth) OD _{600 nm} = ~ 0.5 It was cultured to be Each cell was recovered using a Combi-514R centrifuge (manufactured by Hanil, Korea) at 4,000 rpm (3,515 × g ) at 4° C. for 15 minutes, and this was used to extract LPS.

To measure immunostimulatory activity, rat RAW264.7 cells were treated in an incubator at 37° C., 5% CO ₂ with 5% bovine serum and endotoxin-free (with antibiotics (penicillin G 100 U and streptomycin 10 μg/mL)). endotoxin-free) RPMI1640 culture medium.

1-2. LPS extraction

LPS was extracted from cells of each strain by using conventional LPS extraction methods and T-sol, which is a solvent for LPS extraction according to the present invention, and repeated experiments were performed three times for each of the three bacterial strains.

① Hot phenol-chloroform method

Glass beads (ø 3 mm) were put and vortexed, and the cells were suspended in 15 mL of distilled water. 10 mL of 90% phenol was added thereto, vortexed, and incubated at 60° C. for 30 minutes in a dry heat chamber. The aqueous phase was transferred to a new container by centrifuging at 4,000 rpm for 20 minutes to separate the phases. The aqueous solution was adjusted to pH 5 using 3 M sodium acetate so that the final concentration was 0.15 M. Two times the ethanol was added and incubated overnight at -20°C to precipitate LPS. The supernatant was removed by centrifugation at 4,000 rpm for 20 minutes, and the recovered pellet was dried in air.

② Water-soluble phenol-chloroform method

The cells were suspended in TEA buffer, glass beads were added, vortexed, and mixed with 9 mL of alkaline buffer. Incubated at 60° C. for 70 minutes in a dry heater. To this, 10 mL of phenol-chloroform (1:1) was added and vortexed for at least 30 minutes. It was incubated at 60° C. for 30 minutes in a dry heater and then cooled at room temperature. The aqueous phase was transferred to a new container by centrifuging at 4,000 rpm for 20 minutes to separate the phases. The aqueous solution was adjusted to pH 5 by adding 3 M sodium acetate so that the final concentration was 0.3 M. Two times the ethanol was added and incubated overnight at -20°C to precipitate LPS. The supernatant was removed by centrifugation at 4,000 rpm for 20 minutes. The pellet was dissolved in 50 mM Tris/HCl (pH 8) and 100 mM sodium acetate and incubated overnight at -20° C. to reprecipitate LPS by adding twice as much ethanol. The supernatant was removed by centrifugation at 4,000 rpm for 20 minutes, and the recovered pellet was dried in air.

③ Water-soluble phenol-diethyl ether method

Glass beads were added and vortexed to suspend the cells in 15 mL of SDS buffer. It was boiled for 15 minutes. 2 mL of a cold phenol solution (ice-cold Tris-saturated phenol) was added, and incubated at 60° C. for 30 minutes in a dry heater, and cooled at room temperature. 10 mL of diethyl ether was added and vortexed for a minimum of 30 minutes. After centrifugation at 4,000 rpm for 20 minutes, the aqueous phase was transferred to a new container. The aqueous solution was adjusted to pH 5 by adding 3 M sodium acetate so that the final concentration was 0.15 M. Three times the ethanol was added and incubated overnight at -20°C to precipitate LPS. The supernatant was removed by centrifugation at 4,000 rpm for 20 minutes, and the recovered pellet was dried in air.

④ ~ ⑦ T-sol method

Glass beads were added and vortexed to suspend the cells in 20 mL of T-sol (pH 5 or pH 8). This was incubated at room temperature (25°C) or dry heat (60°C) for 30 minutes. To this, 6 mL of chloroform was added and vortexed for at least 30 minutes. The aqueous phase was transferred to a new container by centrifuging at 4,000 rpm for 20 minutes to separate the phases. Three times the ethanol was added and incubated overnight at -20°C to precipitate LPS. The supernatant was removed by centrifugation at 4,000 rpm for 20 minutes, and the recovered pellet was dried in air.

Then, the dried pellet was dissolved in 200 uL of a digestion buffer (10 mM MgSO ₄ and 1 mM CaCl ₂ in 40 mM Tris/HCl, pH 8). To remove nucleic acids and proteins, RNase A 20 μg mL ^-1 and DNase I 5 U mL ^-1 were added thereto, followed by incubation at 37° C. for 1 hour. 1% SDS and protease K (proteinase K) 100 μg mL ^-1 were further added, and incubated at 60° C. for 1 hour. Three times the ethanol was added and incubated overnight at -20°C to precipitate LPS. The supernatant was removed by centrifugation at 4,000 rpm for 20 minutes, and the recovered pellet was dried in air.

1-3. LPS spectral quality evaluation

For the LPS extract, the UV spectrum was analyzed using DS-11FX Spectrophotometer/Fluorometer (manufactured by DeNovix, USA). The main macromolecules in the aqueous phase separated from the phenol phase are polysaccharides, nucleic acids and proteins. These polymers show distinct UV spectra with different absorption peaks at 204 nm (carbonyl and carboxyl bonds), 260 nm (nucleotide base), and 280 nm (aromatic amino acids). According to previous studies, both LPS and polysaccharides are detected at ~204 nm, and their peaks vary widely depending on chemical structure, pH, dissolved CO ₂ and the presence of salts (Kobayashi et al., 1986; Perdomo and Montero, 2006; Kaijanen et al., 2015). In addition, nucleic acids and proteins also show strong absorbance at 204 nm, and generally, absorbance at 260 nm and 280 nm. The three wavelengths of the major polymers in the aqueous phase are useful for preliminary measurement and detection of impurities as well as purified substances. The absorbance ratio of the complementary wavelength for nucleic acids ( b = A 204 / A 260) and protein ( c = A 204 / A 280) is the spectral quality index of LPS between [0, 1] as follows ( quality index) was used to derive:

Quality index of LPS = ( a × A 204)/{( a × A 204) + ( b × A 260) + ( c × A 280)}

The parameter a for the absorbance of LPS at 204 nm is determined by estimating the predicted quality index as one eigenvalue as the pure LPS approaches zero at 260 nm and 280 nm (Perdomo and Montero, 2006). It was calculated as 7.62 from the UV spectrum of purified LPS obtained from the strain. The absorbance ratios b and c of nucleic acid and protein in the denominator were measured based on the results of herring sperm DNA ( A 204/ A 260 = 4.76) and bovine serum albumin ( A 204/ A 280 = 102.05).

1-4. SDS PAGE analysis

In order to confirm the presence of the protein contained in LPS, it was analyzed by SDS PAGE (10-15% polyacrylamide gel). After photographing the gel stained with Coomassie brilliant blue R-250, it was washed with 50% acetonitrile and 100 mM ammonium hydrocarbon (pH 8) to remove Coomassie blue bound to the protein. The bleached gel was rinsed with distilled water and stained with Alcian blue and silver. The amount of LPS is measured by the dried weight of each sample, or the concentration is continuously adjusted with 1×PBS (NaCl 8 g, KCl 0.2 g, Na ₂ HPO ₄ 1.44 g, KH ₂ PO ₄ 0.24 g in DW 1 L). It was diluted and measured using a standard absorbance curve measured at 204 nm.

1-5. Endotoxin test and bioassay test

The endotoxin unit (EU) of LPS was measured using Limulus amebocyte lysate (LAL) Kinetic-QCLTM reagent and E. coli O55:B5 endotoxin (manufactured by Lonza, USA) according to the manual.

RAW264.7 cells were treated with 0.2 μm membrane-filtered LPS (in 1×PBS) at various concentrations and incubated for 4 hours, and then the morphology of monocytes and macrophages was examined under a phase contrast microscope (Olympus CKX41). The cells were photographed with an IMTcam3 camera and IMT iSolution Lite version 26.1 (IMT i-Solution, Canada). The concentration of TNF-α (tumor necrosis factor-alpha) in the cell culture was measured with a TNF-α ELISA kit (manufactured by Abcam, UK).

1-6. statistics

All experiments were repeated three times independently for each strain, and the result values were described as mean and standard deviation (SD). The statistical difference between the experiments was measured with a two-tailed t-test, and a P value <0.05 was considered significant.

2. Results

2-1. Quality evaluation of crude LPS extract

In order to evaluate the quality of LPS extracted by 7 extraction methods with different organic solvent, pH, temperature conditions, etc., different types of LPS were extracted from three bacterial strains and compared. The same amount of crude LPS extract was analyzed by SDS PAGE and stained with Coomassie Blue, Alcian Blue and Silver. The results are shown in FIG. 1.

Referring to FIG. 1, in Coomassie blue staining, it was confirmed that the phenol-chloroform and phenol-diethyl ether methods extracted low-quality LPS due to protein contamination regardless of the strain type. In Alcian Blue staining, it was confirmed that the bands of polysaccharides contained in the crude LPS extract appeared very diversely depending on the strain, pH, and heating temperature. In silver staining, the hot phenol-water and T-sol methods were found to be effective in reducing sample contamination by proteins.

In addition, UV spectrum analysis was performed on the crude LPS extract, and the results are shown in Table 1 below.

E. coli
W3110 S. Typhimurium 14028S A. baumannii
ATCC 19606 ① Hot phenol-water 0.413±0.016 0.400±0.011 0.365±0.065 ② Water-soluble phenol-chloroform 0.279±0.006 ^a 0.305±0.060 0.275±0.058 ③ Water-soluble phenol-diethyl ether 0.325±0.084 0.372±0.041 0.375±0.056 ④ T-sol (pH 5, 25℃) 0.523±0.017 ^abc 0.653±0.063 ^abc 0.583±0.156 ⑤ T-sol (pH 5, 60℃) 0.559±0.065 ^c 0.646±0.115 ^bc 0.592±0.046 ^abc ⑥ T-sol (pH 8, 25℃) 0.470±0.086 0.565±0.089 ^bc 0.571±0.054 ^abc ⑦ T-sol (pH 8, 60℃) 0.527±0.067 ^c 0.535±0.034 ^abc 0.633±0.142 ^abc *a, b, and c mean that there is a significant difference compared with the hot phenol-water, water-soluble phenol-chloroform, and water-soluble phenol-diethyl ether methods, respectively (P <0.05)

As shown in Table 1, it was confirmed that the T-sol method of ④ to ⑦ produced an overall high quality LPS extract compared to the hot phenol-water method. And it was confirmed that the crude LPS extract extracted by the T-sol method had similar UV spectrum results regardless of the pH and heating temperature of the extraction solvent.

2-2. Quality evaluation of purified LPS extract after enzymatic digestion

To evaluate the quality of purified LPS, LPS was lyophilized after digestive enzyme treatment, and the yield of dried LPS was measured. The results are shown in Table 2 below.

E. coli
W3110 S. Typhimurium 14028S ^{A. baumannii}
ATCC 19606 ① Hot phenol-water 8.253±4.368 9.427±5.396 4.564±2.073 ② Water-soluble phenol-chloroform 6.574±3.677 6.165±4.414 2.592±0.934 ③ Water-soluble phenol-diethyl ether 17.92±9.292 20.84±0.940 ^b 5.682±0.601 ^b ④ T-sol (pH 5, 25℃) 3.572±3.510 6.662±3.723 ^c 1.467±0.600 ^c ⑤ T-sol (pH 5, 60℃) 8.403±1.332 11.11±4.431 ^c 2.863±1.468 ⑥ T-sol (pH 8, 25℃) 1.999±0.878 ^e 9.706±3.381 ^c 2.174±1.568 ^c ⑦ T-sol (pH 8, 60℃) 9.757±3.009 ^f 12.00±0.818 ^c 6.428±3.811 *The value obtained from 3 repeated experiments is expressed as mean ± standard deviation.
* b, c, e, and f are significant differences compared to the water-soluble phenol-chloroform, water-soluble phenol-diethyl ether, T-sol (pH 5, 60℃), and T-sol (pH 8, 25℃) methods, respectively. Means there is (P <0.05)

As shown in Table 2, it was confirmed that the water-soluble phenol-diethyl ether method can obtain the LPS extract in high yield compared to the methods of ①, ②, ④ to ⑦.

However, as a result of SDS PAGE analysis of the purified LPS extract (see FIG. 2), it was confirmed that even though the water-soluble phenol-diethyl ether method was treated with a protease, a large amount of protein remained in the LPS extract. On the other hand, it was confirmed that the T-sol method of ④ to ⑦ contains less protein in the LPS extract than the conventional method of ① to ③.

In addition, UV spectrum analysis was performed on the purified LPS extract, and the results are shown in Table 3 below.

E. coli
W3110 S. Typhimurium 14028S A. baumannii
ATCC 19606 ① Hot phenol-water 0.831±0.009 0.836±0.009 0.811±0.017 ② Water-soluble phenol-chloroform 0.300±0.021 ^a 0.795±0.031 0.829±0.011 ③ Water-soluble phenol-diethyl ether 0.359±0.007 ^ab 0.356±0.039 ^ab 0.407±0.045 ^ab ④ T-sol (pH 5, 25℃) 0.832±0.022 ^bc 0.846±0.016 ^c 0.794±0.040 ^c ⑤ T-sol (pH 5, 60℃) 0.837±0.010 ^bc 0.849±0.013 ^c 0.814±0.016 ^c ⑥ T-sol (pH 8, 25℃) 0.793±0.019 ^bce 0.844±0.009 ^c 0.858±0.025 ^c ⑦ T-sol (pH 8, 60℃) 0.845±0.005 ^bcf 0.855±0.006 ^ac 0.830±0.021 ^c *a, b, c, e, and f are hot phenol-water, water-soluble phenol-chloroform, water-soluble phenol-diethyl ether, T-sol (pH 5, 60℃), T-sol (pH 8, 25℃) Means a significant difference compared to the method (P <0.05)

As shown in Table 3, in the water-soluble phenol-chloroform and water-soluble phenol-diethyl ether methods, the absorbance ratio of A204/A280 was measured high due to contamination of the protein, and it was confirmed that low-quality LPS extract was produced. On the other hand, it was confirmed that the hot phenol-water and T-sol methods produce high quality LPS extracts. In particular, it can be seen that it is efficient to extract S-LPS from S. Typhimurium 14028S using the T-sol method of ④ to ⑦.

In order to compare the quality of the LPS extract extracted by the T-sol method, the LPS extract extracted and purified by the T-sol method was analyzed by SDS PAGE and stained with Coomassie Blue, Alcian Blue, and Silver. The results are shown in FIG. 3.

Referring to Figure 3, it was confirmed that the LPS extract obtained from A. baumannii ATCC 19606 and E. coli W3110 contains some incomplete LPS such as truncated LPS and R-LPS.

When the results of Table 3 and FIG. 3 are summarized, it can be seen that some incomplete LPS may be included in the LPS extract extracted by the T-sol method, but this does not significantly affect the quality of the LPS extract. In addition, it can be seen that the T-sol method is more efficient in extracting S-LPS having an O-PS side chain.

On the other hand, in order to measure the endotoxin unit (EU) of the purified LPS extract, an LAL test was performed. The results are shown in Table 4 below.

E. coli
W3110 S. Typhimurium 14028S A. baumannii
ATCC 19606 ① Hot phenol-water 212±221 490±247 43±49 ② Water-soluble phenol-chloroform 1,847±1,274 710±32 976±324 ③ Water-soluble phenol-diethyl ether 173±20 ^b 627±123 32±15 ^b ④ T-sol (pH 5, 25℃) 7±5 ^b 1,341±720 12±16 ^b ⑤ T-sol (pH 5, 60℃) 2±0.6 ^b 844±242 28±44 ^b ⑥ T-sol (pH 8, 25℃) 51±58 ^b 1,003±165 ^abc 19±30 ^b ⑦ T-sol (pH 8, 60℃) 7±6 ^b 825±110 19±21 ^b *Indicates the endotoxin unit (EU/g) of LPS obtained from 4 repeated experiments for each strain as mean ± standard deviation
*a, b, and c mean that there is a significant difference compared with the hot phenol-water, water-soluble phenol-chloroform, and water-soluble phenol-diethyl ether methods, respectively (P <0.05)

As shown in Table 4, the LPS extract extracted by the T-sol method of ④ to ⑦ from S. Typhimurium 14028S was found to have superior values compared to the LPS extract extracted by the conventional method of ① to ③. On the other hand, endotoxin units (EU) extracted from E. coli W3110 and A. baumannii ATCC 19606 and contained in the LPS extract containing incomplete LPS such as R-LPS and truncated LPS were significantly less than the detection limit of the LAL test method used. It was found to decrease.

In order to confirm the incomplete LPS contained in the LPS extract, the absorbance values measured at 204 nm of the LPS extract extracted by hot phenol-water and T-sol (pH 8, 60° C.) were compared. The results are shown in FIG. 4.

Referring to FIG. 4, the slope of the standard curve of each LPS extract did not differ between extraction methods, but it was confirmed that there was a difference between strains. From this result, it can be seen that even though the extraction methods are different, the LPS extracts extracted by the two methods from one strain have similar characteristics of polysaccharides.

2-3. RAW264.7 cell activity

To measure the immunostimulatory effect of the LPS extract extracted by hot phenol-water and T-sol (pH 8, 60°C) method, various concentrations of LPS (0 ~ 10 ug/mL) were added to rat RAW264.7 cells. Exposed for hours. The results are shown in Figure 5 and Table 5 below.

E. coli
W3110 S. Typhimurium
14028S A. baumannii
ATCC 19606 ED50 Emax ED50 Emax ED50 Emax ① Hot phenol-water 247±92 64±4.1 950±657 49±5.3 7241±3396 62±5.5 ⑦ T-sol (pH 8, 60℃) 138±85 70±5.6 105±34 52±2.9 180±50 59±3.4 *The effective dose of LPS (ED50, ng/mL) and the maximum level of RAW264.7 macrophages (Emax, %) were calculated by logistic regression analysis of the minimum threshold, and the values obtained by repeating three experiments for each strain Is expressed as the mean ± standard deviation

Referring to FIG. 5A as an example, it was confirmed that when LPS extracted from S. Typhimurium 14028S was treated, RAW264.7 cells were rapidly differentiated into macrophages compared to the case where LPS was not treated.

Referring to FIG. 5B as an example, the LPS extracts extracted from S. Typhimurium 14028S by each method differentiate RAW264.7 cells, which are monocytes, into macrophages, and the effect of stimulating RAW264.7 cell activity according to the extraction method Confirmed to be different. In addition, as shown in Table 5, it was confirmed that the ED50 value of LPS was significantly different depending on the extraction method, while the macrophage activity effect at the maximum LPS concentration (> 1 μg mL ^-1 ) was similar.

As a result, it was found that the T-sol method extracts S-LPS specific for RAW264.7 cell activity at a higher level than the hot phenol-water method, which extracts more incomplete LPS such as R-LPS and truncated LPS. I can. These results indicate that LPS with O-PS side chain plays an important role in the activation of monocytes.

Referring to the example of C of FIG. 5 and Table 6 below, it was confirmed that the LPS extract extracted from S. Typhimurium 14028S by each method had a similar tendency to express TNF-α. However, it was confirmed that the LPS extract extracted by the T-sol method was more sensitive to macrophage differentiation of RAW264.7 cells compared to the LPS extract extracted by the hot phenol-water method.

E. coli
W3110 S. Typhimurium
14028S A. baumannii
ATCC 19606 ED50 Emax ED50 Emax ED50 Emax ① Hot phenol-water 5.8±2.0 370±28 7.6±6.3 227±69 10.0±9.2 477±90 ⑦ T-sol (pH 8, 60℃) 5.0±3.0 396±48 1.2±1.1 340±60 3.1±3.8 401±97 *The effective dose of LPS (ED50, ng/mL) and the maximum level (Emax, ng/mL) of TNF-α (pg/mL) measured using RAW264.7 cell culture medium were determined by the logistic regression analysis of the minimum threshold. Was calculated by, and the value obtained by repeating three experiments for each strain is expressed as the mean ± standard deviation.

As a result, it can be seen that the T-sol extraction method affects the composition of the LPS extract, thus affecting the activity of immune cells by the LPS extract.

Claims (6)

As a solvent for separating lipopolysaccharide from Gram-negative bacteria,
PH consisting of 30 to 50% phenol, 0.5 to 2 M guanidine thiocyanate, 0.1 to 0.8 M ammonium thiocyanate and 3 to 10% glycerol A solvent for extraction of lipopolysaccharide comprising a sodium acetate solution of 4.5 to 5.5 or a Tris/HCl solution of pH 7.5 to 8.5.
The method according to claim 1,
The sodium acetate solution is a lipopolysaccharide extraction solvent containing 0.05 to 0.5 M sodium acetate.
The method according to claim 1,
The Tris / HCl solvent is a lipopolysaccharide extraction solvent containing 0.05 to 0.5 M of Tris / HCl.
a) preparing a first sample solution by contacting a sample containing Gram-negative bacteria with the solvent for extracting LPS of any one of claims 1 to 3; And.
b) heat-treating the first sample solution at 20 to 70°C for 5 to 50 minutes
Lipopolysaccharide extraction method comprising a.
The method of claim 4,
After step b),
c) preparing a second sample solution by adding chloroform to the heat-treated first sample solution;
d) separating the aqueous phase by centrifuging the second sample solution; And
e) adding ethanol to the aqueous phase to precipitate lipopolysaccharide
Lipopolysaccharide extraction method further comprising a.
The method of claim 4,
In the step b), the first sample solution is heat-treated at 20 to 40°C, or 50 to 70°C.

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