KR102007421B1 - Composition for anti-influenza virus comprising as active ingredient polysaccharide derived from Lactobacillus plantarum and method for producing the polysaccharide - Google Patents

Abstract

The present invention relates to a composition for an anti-influenza virus containing a polysaccharide derived from Lactobacillus plantarum as an active component and a method for manufacturing the polysaccharide. Since the polysaccharide contains sialic acid to inhibit infection of influenza virus, the composition containing the polysaccharide as the active component has excellent anti-influenza virus activity.

Description

Composition for anti-influenza virus comprising as active ingredient polysaccharide derived from Lactobacillus plantarum and method for producing including polysaccharide derived from Lactobacillus plantarum as an active ingredient the polysaccharide}

The present invention relates to a composition for anti-influenza virus comprising a polysaccharide derived from Lactobacillus plantarum as an active ingredient and a method for producing the polysaccharide.

Probiotics are microorganisms that produce lactic acid using sugars. Lactic acid bacteria are known to promote beneficial physical health by inhibiting harmful bacteria in the intestines of humans or animals, preventing the intestinal diseases and activating the digestive organs. Accordingly, lactic acid bacteria are attracting attention as a health supplement functional material, and are currently being widely used in addition to foods such as feed additives, pharmaceuticals, and cosmetics.

The efficacy of lactic acid bacteria varies greatly, even if it is a medically confirmed function, such as intestinal action, blood cholesterol reduction, immune enhancement, endogenous infection inhibition, anti-cancer effects. Many livestock were killed in Korea as a foot-and-mouth disease between 2011 and 2012, but livestock fed with lactic acid bacteria (probiotics) were not caught in foot-and-mouth disease, and livestock farms are gradually trying to introduce lactic acid bacteria as feed additives.

Lactic acid bacteria produces various exopolysaccharides as immune enhancing materials as well as antimicrobial proteins such as bacteriocin, which makes it possible to develop various bio-products such as health supplements, cosmetics, medical treatment materials, animal treatments, and viral infection prevention aids. It is expected to be used as a promising material.

However, the majority of domestic research and products are mainly product development using live bacteria of lactic acid bacteria itself. In order to enhance the high value added of cosmetics and pharmaceuticals, specific identification and biological evaluation of functional components of polysaccharides and peptides having the efficacy of anti-virus, anti-bacteria, and anti-bacteria are required.

Prior arts relating to strains or substances having anti-influenza virus efficacy derived from lactic acid bacteria are as follows: novel lactic acid bacteria having an avian influenza virus infection inhibitory effect and a composition comprising the same (application number: KR 10-2009-0053845); Novel lactic acid bacteria having an avian influenza virus infection inhibitory effect, natural antiviral agents using the same, and compositions comprising the same (Registration No. KR 10-1269595); Lactobacillus luterii H. Y7501 having anti-influenza virus efficacy and a product containing it as an active ingredient (Registration No. KR 10-1001767).

However, many of the above-mentioned prior arts refer to lactic acid bacteria and lactic acid bacteria cultures having anti-influenza virus activity, and there is no specific reference to the specific substances produced by the lactic acid bacteria, Examples of anti-influenza virus efficacy through comparison with previously validated standards are not clearly described.

An object of the present invention is a polysaccharide recovered from Lactobacillus plantarum culture medium; Or to provide an anti-influenza virus composition comprising a; a culture solution containing the polysaccharide; as an active ingredient.

Another object of the present invention to provide a pharmaceutical composition for the prevention or treatment of infection of influenza virus comprising the composition.

Still another object of the present invention is a) culturing Lactobacillus plantarum ( Lactobacillus plantarum ) in the medium; b) recovering the polysaccharide from the Lactobacillus plantarium cultured in the step a) or its culture solution; to provide a method for producing a polysaccharide.

Each description and embodiment disclosed in the present invention may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. In addition, the scope of the present invention is not to be limited by the specific description described below.

As one aspect for achieving the object of the present invention, the present invention is a polysaccharide recovered from the Lactobacillus plantarium culture medium; Or it provides a composition for an anti-influenza virus comprising; a culture solution containing the polysaccharide; as an active ingredient.

In the present invention, the term " Lactobacillus plantarum " is an aerobic, or anaerobic, gram-positive bacillus genus microorganism widely distributed in nature.

Specifically, the Lactobacillus plantarium may be Lactobacillus plantarium CK401. In an embodiment of the present invention, Lactobacillus plantarium CK401 was cultured.

As used herein, the term "culture liquid" includes various materials which are required for culture of conventional Lactobacillus plantarium or Lactobacillus plantarium discharged into the medium during growth. In addition, the culture medium may further include components that synergistically act on the growth of the Lactobacillus plantarium as well as the medium or material, and the composition thereof may be easily selected by those skilled in the art. Can be.

Specifically, the culture solution may include a polysaccharide (polysaccharide) discharged by the Lactobacillus plantarium, specifically, a polysaccharide added with sialic acid.

In the embodiment of the present invention, Lactobacillus plantarium CK401 was incubated for 72 hours at 37 ℃ using MRS medium, the culture solution obtained through this process was used.

In the present invention, the term “polysaccharide” refers to a saccharide in which three or more monosaccharides make a large molecule through glycosidic bonds, and when hydrolyzed, it becomes a monosaccharide. By hydrolysis, more than 10 monosaccharides are produced in 1 molecule of polysaccharide, and usually 50-100 monosaccharides are condensed.

Specifically, the polysaccharide of the present invention is Glc (Glucose); GlcNAc (N-acetylglucosamine) or GlcN (glucosamine); GalNAc (N-acetylgalactosamine) or GalN (galactosamine); Gal (Galactose); Man (Mannose); N-acetylneuraminic acid (Neu5Ac); And Neu5Gc (N-glycolylneuraminic acid); It may be to include.

In the embodiment of the present invention, the polysaccharide was recovered from the Lactobacillus plantarium culture medium, and the monosaccharide was obtained by acid hydrolysis of the polysaccharide, and then labeled with a fluorescent substance and analyzed by HPLC to determine the type of sugar constituting the polysaccharide. It was confirmed (FIG. 2). As a result, 132 mM GlcNAc or GlcN in the polysaccharide; 44.3 mM GalNAc or GalN; 10.6 mM Gal; 8.5 mM Man; 996 mM Glc was identified and sialic acid was 0.27 mM Neu5Ac; 0.15 mM Neu5Gc was identified (Table 1). That is, it was confirmed that the polysaccharide of the present invention contains sialic acid.

Specifically, the ratio of the polysaccharide and the Glc Neu5Ac is 1: 1x10 ^-5 to 1: 1x10 ^-3 which can be, more specifically 1: 1x10 ^-5 to 1: 1x10 ^-4, and more more particularly 1 1: 1 × 10 ⁻⁴ to 1: 1 × 10 ⁻³ , and more specifically 1: 2.5 × 10 ⁻⁴ to 1: 1 × 10 ⁻³ .

Specifically, the ratio of the polysaccharide and the Neu5Gc is Glc 1: 1x10 ^-5 to 1: 1x10 ^-3 which can be, more specifically 1: 1x10 ^-5 to 1: 1x10 ^-4, and more more particularly 1 : 1 × 10 ⁻⁴ to 1: 1 × 10 ⁻³ , and more specifically 1: 1.0 × 10 ⁻⁴ to 1: 5.0 × 10 ⁻⁴ .

In the embodiment of the present invention, the concentration of each monosaccharide was calculated as the relative concentration ratio when the concentration of Glc or GlcNAc (or GlcN) was set to 1. As a result, the ratio of Glc and Neu5Gc in the polysaccharide was 1: 1.5 × 10 ⁻⁴ , and the ratio of Glc and Neu5Ac was 1: 2.7 × 10 ⁻⁴ . Was 2.0x10 ^-3 (Table 1): In addition, the polysaccharides within GlcNAc (or GlcN), and the ratio of Neu5Gc was 1: 1.1x10 ^-3, GlcNAc (or GlcN), and the ratio of Neu5Ac are first. That is, the polysaccharide of the present invention is characterized by containing sialic acid (Neu5Ac, Neu5Gc).

Specifically, the polysaccharide may comprise one or more residues selected from the group consisting of:

Neu5Acα2-6Gal-R residues;

Neu5Acα2-6GalNAc-R residues;

Neu5Acα2-3Galβ1-4Glc-R residues;

Neu5Acα2-3Galβ1-4GlcNAc-R residues;

Neu5Gcα2-3Galβ1-4Glc-R residues; And

Neu5Gcα2-3Galβ1-4GlcNAc-R residues;

SNA lectins that selectively bind to Neu5Acα2-6Gal-R residues or Neu5Acα2-6GalNAc-R residues in an embodiment of the invention; Or MAA lectins that selectively bind to Neu5Acα2-3Galβ1-4Glc-R residues, Neu5Acα2-3Galβ1-4GlcNAc-R residues, Neu5Gcα2-3Galβ1-4Glc-R residues or Neu5Gcα2-3Galβ1-4GlcNAc-R residues; When the immobilized 96 well plate was treated with the polysaccharide of the present invention, it was confirmed that the polysaccharide of the present invention binds to both the SNA lectin and the MAA lectin (Examples 5 and 6). Therefore, it can be seen that the polysaccharide of the present invention contains at least one of the six residues.

Specifically, the polysaccharide may have a molecular weight of 10 to 200 kDa. In the embodiment of the present invention, the anti-influenza virus activity of two polysaccharides (polysaccharide-L: molecular weight greater than 500 kDa, polysaccharide-S: molecular weight 10-200 kDa) recovered from the culture medium of Lactobacillus plantarium was measured. It was confirmed that only the polysaccharide-S having a molecular weight of 10 to 200 kDa inhibits the influenza virus infection.

Specifically, the polysaccharide may be produced by fermentation of Lactobacillus plantarium. In the embodiment of the present invention for producing polysaccharides derived from the Lactobacillus plantarium strain, the strain was incubated at 37 ℃, 1,000 rpm Batch culture or Fed-batch culture method using a fermentor using MRS medium.

 In the present invention, the term "anti-influenza virus" refers to the property of inhibiting the infection of influenza virus.

Specifically, the influenza virus may be an influenza A virus or an avian influenza virus.

In the embodiment of the present invention, the anti-influenza virus activity of two polysaccharides (polysaccharide-S, polysaccharide-L) recovered from the culture medium of Lactobacillus plantarium was measured, and influenza A (H1N1, H3N2 only in polysaccharide-S) was measured. It was confirmed that the virus inhibits the infection, it was confirmed that the polysaccharide-S has anti-influenza virus activity (Table 2). This suggests that the composition comprising polysaccharide-S can be used as an anti-influenza virus composition. In particular, the polysaccharide-S was confirmed to exhibit a particularly high anti-influenza virus activity against influenza A compared to influenza B.

In addition, in an embodiment of the present invention, the polysaccharide was treated before, concurrent with or after infection with the virus and the anti-influenza virus activity of the polysaccharide was measured. As a result, it was confirmed that the anti-influenza virus activity of the polysaccharide was better when the polysaccharide was treated after the virus infection (Table 2). This suggests that even after treatment with the virus, the inventive polysaccharides will exhibit sufficiently good anti-influenza virus activity.

 In addition, in the embodiment of the present invention, the sialic acid sugar chain (sialoglycoconjugate), which is a key structure for the polysaccharide to have anti-influenza virus activity, is added to the sugar-structure to which sialic acid is added in the sugar-precursor (sugar chain, glycolipid and glycoprotein). As a result of using lectin for selective binding, it was confirmed that the majority of sialic acid constituting the polysaccharide was added to the galactose as α (2,3) bonds instead of α (2,6) (FIG. 6). . This suggests that the polysaccharide may be particularly effective against avian influenza virus that is infected via silyl-α (2,3) -galactose.

As another aspect for achieving the object of the present invention, the present invention provides a pharmaceutical composition for the prevention or treatment of influenza virus infection comprising the composition for anti-influenza virus.

The term "prevention" in the present invention means any action that inhibits or delays the infection of the influenza virus by administration of the composition of the present invention.

The term "treatment" in the present invention means any action that improves or advantageously changes the symptoms caused by the influenza virus infection by administration of the composition of the present invention.

The pharmaceutical composition of the present invention is in the form of tablets, pills, powders, granules, capsules, suspensions, solvents, emulsions, syrups, aerosols, sterile injectable solutions and the like according to conventional methods for preventing and treating influenza virus infection. Formulation is possible.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations are prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose, lactose, gelatin, and the like. Can be. In addition to the simple excipients, lubricants such as magnesium stearate, talc can also be used.

Liquid preparations for oral administration include suspensions, solvents, emulsions, and syrups, and various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to water and liquid paraffin, which are commonly used simple diluents, include Can be used.

Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, suppositories, and the like. As the non-aqueous solvent and the suspension solvent, vegetable oils such as propylene glycol, polyethylene glycol, olive oil, etc., injectable esters such as ethyl oleate, and the like can be used.

In addition, the pharmaceutical compositions of the present invention may further comprise a carrier, excipient or diluent. Carriers, excipients or diluents include lactose, textose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, hydroxy Mineral oils such as propyl methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, flophyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, silicon dioxide and the like can be used. have.

The specific dosage of the pharmaceutical composition according to the present invention depends on factors such as the formulation method, the condition and weight of the patient, the sex of the patient, the age, the extent of the disease, the form of the drug, the route and duration of the administration, the rate of excretion, the sensitivity of the reaction, and the like. It can be variously selected by those skilled in the art, and the dosage and frequency do not limit the scope of the present invention in any aspect.

The pharmaceutical composition of the present invention can be administered to mammals such as mice, mice, livestock, humans, etc. through various routes. All modes of administration can be expected and can be administered, for example, by oral, intravenous, intramuscular or subcutaneous injection.

As another aspect to achieve the object of the present invention, the present invention comprises the steps of: a) culturing Lactobacillus plantarum ( Lactobacillus plantarum ) in the medium; b) recovering the polysaccharide from the Lactobacillus plantarium cultured in the step a) or its culture solution; provides a method for producing a polysaccharide comprising a.

The Lactobacillus plantarium, the culture medium is as described above.

The term "culture" of the present invention means to grow microorganisms under moderately controlled environmental conditions. The method of culturing the microorganism of the present invention can be carried out according to suitable medium and culture conditions well known in the art, and can be easily adjusted and used by those skilled in the art according to the strain selected. Specifically, examples of the culture method include, but are not limited to, batch culture, continuous culture, and fed-batch culture.

As used herein, the term "media" refers to a medium containing a carbon source, a nitrogen source, and an inorganic salt so that the microorganism can produce a polysaccharide as used for culturing bacteria, in particular, Lactobacillus plantarium. The medium may include various substances discharged into the medium during the growth of the microorganism together with the medium component prepared for culturing the strain, and specifically, may include a polysaccharide as a target substance.

The medium used for cultivation must meet the requirements of the particular strain in an appropriate manner. Culture media for Lactobacillus plantarium are known (e.g., Manual of Methods for General Bacteriology. American Society for Bacteriology.Washington D.C., USA, 1981). Carbon sources in the medium include, for example, sugars and carbohydrates such as glucose, sugar, sodium citrate, fructose, lactose or maltose, soybean oil, sunflower oil, Oils such as castor oil, coconut oil and the like, fatty acids such as palmitic acid, stearic acid, linoleic acid, alcohols such as glycerol, ethanol, organic acids such as acetic acid, but are not limited thereto. These materials can be used individually or as a mixture.

As the nitrogen source in the medium, for example, compounds containing peptone, meat extract, yeast extract, dried yeast, corn steep liquor, soybean cake, urea, thiourea, ammonium salt, nitrate and other organic or inorganic nitrogen may be used. It is not limited to this. The inorganic salts included in the medium may include compounds including magnesium, manganese, potassium, calcium, iron, zinc, cobalt, and the like, but are not limited thereto.

Personnel that can be used in the medium may include, but are not limited to, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or a corresponding sodium-containing salt. In addition, the culture medium may include metal salts such as magnesium sulfate or iron sulfate required for growth. The compounds may be used individually or as a mixture, but are not limited thereto.

In addition to the components of the carbon source, nitrogen source and inorganic salts, amino acids, vitamins, nucleic acids and related compounds may be further added to the medium of the present invention. The above-mentioned raw materials may be added batchwise or continuously by a suitable method for the medium in the culture process.

On the other hand, the pH of the medium can be adjusted by using a basic compound such as calcium carbonate, sodium hydroxide, potassium hydroxide, ammonia or an acid compound such as phosphoric acid or sulfuric acid in an appropriate manner. In addition, antifoaming agents such as fatty acid polyglycol esters can be used to inhibit bubble generation. Oxygen or oxygen-containing gas (eg, air) may be injected into the medium to maintain aerobic conditions. The temperature of the culture may be 20 to 45 ℃ but is not limited thereto. Cultivation can be continued until the desired amount of polysaccharide production is achieved, and can be achieved in 10 to 160 hours, but is not limited thereto.

The resulting polysaccharide may be discharged into culture medium (culture medium) or contained in microorganisms (Lactobacillus plantarium). The step of recovering the polysaccharide from the culture medium, the microorganism, or a mixture of the cultured microorganism and the culture medium is a variety of methods well known in the art, for example, cell lysis, centrifugation, filtration, Exclusion chromatography, ion chromatography, sugar fractionation column, crystallization and HPLC may be used, but is not limited thereto. In addition, the recovery step may include a purification process.

In the embodiment of the present invention, L. plantarum CK401 was incubated in a batch culture or Fed-batch culture method at 37 ℃, 1,000 rpm in a 1 L or 5 L fermentor using MRS medium. The culture was then centrifuged and the supernatant concentrated and ultrafiltered with a membrane having a pore size of 3-100 kDa (MWCO). Thereafter, the fraction containing the polysaccharide of the present invention was recovered through size exclusion chromatography, ion chromatography, and continuous column separation purification of the sugar fraction column.

It was confirmed that the polysaccharide prepared by the production method of the present invention has a high sialic acid content, and thus has excellent anti-influenza virus activity (Examples 1, 2 and 4).

The present invention Lactobacillus plantarum ( Lactobacillus plantarum ) Derived polysaccharide inhibits the influenza virus infection, the composition comprising the polysaccharide as an active ingredient is excellent in anti-influenza virus activity.

Figure 1 is a L. plantarum culture (A), obtained by concentrating the culture to the membrane, lactic acid (lactate) removed polysaccharide polysaccharide fraction (B) of at least 10 kDa, the lactate, passed through the membrane ) And a culture solution (C) containing a low molecular weight material of 10 kDa or less containing acetic acid is analyzed by HPLC using an organic acid column.
FIG. 2 shows the types of neutral sugars (GlcNAc or GlcN; GalNAc or GalN; Gal; Man; Glc;) and acidic sugars (Neu5Ac; NeuGc) constituting the polysaccharide of the present invention. Fluorescent monosaccharides obtained by labeling are shown by quantitative analysis using HPLC.
FIG. 3 illustrates chemical functional groups present in the polysaccharide of the present invention using Fourier Transform Infrared spectroscopy (FT-IR) and Fourier Transform Raman spectroscopy (FT-Raman). It shows the result confirmed.
4 is a mass spectrum (MS) profile obtained by analyzing LC-MS of monosaccharides and oligosaccharides obtained by acid hydrolysis of polysaccharides to analyze the kinds of sugars (monosaccharides or oligosaccharides) constituting the polysaccharide of the present invention.
5 is a lectin that selectively binds to alpha-2,6-linkage to confirm the binding structure of sialic acid added to the polysaccharide of the present invention, SNA ( Sambucus nigra) It is a schematic diagram illustrating lectin analysis using MAA ( Maackia amurensis lectin) as a lectin that selectively binds to lectin) and alpha-2,3-linkage.
FIG. 6 shows a fluorescently-labeled polysaccharide with APTS that binds to the SNA lectin that selectively binds to alpha-2,6-binding or MAA lectin that selectively binds to alpha-2,3-binding immobilized on 96 well plates. As a result of measuring the fluorescence sensitivity of, the fluorescence sensitivity of the polysaccharide bound to the lectin before and after the sialidase treatment to hydrolyze the sialic acid is noticeable:
SNA: SNA lectin, which selectively binds to alpha-2,6-binding,
MAA: MAA lectin, which selectively binds to alpha-2,3-binding
(-) sialidase: before sialidase treatment,
(+) α2,3-sialidase: after treatment with Streptococcus pneumoniae sialidase,
(+) α-2,3,6-sialidase: after treatment with Althrobacter urepaciens sialidase,
Control: Negative control without fluorescence polysaccharide treatment.

Hereinafter, the present invention will be described in more detail through Examples and Experimental Examples. However, these Examples and Experimental Examples are for illustrative purposes only and the scope of the present invention is not limited to these Examples and Experimental Examples.

Example 1. Lactobacillus plantarum  Production of Polysaccharides Through Culture

In order to produce polysaccharides from L. plantarum CK401 strain, using either MRS (de Man, Rogosa and Sharpe) medium, 1 L or 5 L fermentor (jar fermentor) at 37 ℃, 1,000 rpm by batch culture or Fed-batch culture method The strain was cultured.

In batch culture, the culture of L. plantarum CK401 was found to convert glucose to lactate rapidly within 48 hours using MRS medium containing 20 g / L glucose. In addition, it was confirmed that about 15.3 ± 2.1 g / L lactate was prepared at a conversion rate of 0.75 g lactate / g glucose after 72 hours of incubation.

At this time, the polysaccharide was confirmed that the amount is prepared in almost mg level in the ratio of 1: 1,000,000 of lactate production rate. After 72 hours the pH of the culture was found to be less than 2.5.

In the fed batch culture, an appropriate amount of solid state calcium carbonate was added to the MRS medium to adjust the pH of the culture. At this time, the initial glucose amount was 40 g / L and incubated at 37 rpm and 1,000 rpm with a 5 L fermenter. When the concentration of glucose dropped below 5 g / L, step-feeding was maintained. It was.

L. plantarum strains consumed MRS medium containing 40 g / L glucose within 36 hours and added 40 g / L glucose at 24 hour intervals. The majority of feeding glucose was found to be converted to lactate. It was observed that the excessively produced lactate was precipitated in the culture medium in the form of lactate salt in combination with free calcium from the solid-state calcium carbonate for pH adjustment in the medium.

The amount of polysaccharide was found to be measurable by HPLC after 72 hours, and it was confirmed that it was continuously produced up to 144 hours. The final polysaccharide produced was about 0.62 ± 0.15 g / L. This was confirmed to be produced in a ratio of 1: 1,000,000, similar to the batch culture, when converted to the concentration ratio with lactate dissolved in an aqueous solution.

Example 2. L. plantarum  Isolation of Polysaccharides from Culture

Cells and particulate matter were removed by centrifugation of the culture solution recovered in Example 1. The supernatant recovered by centrifugation was passed through a 0.2 μm filter to remove particulate matter. The culture solution passed through the filter was concentrated and ultrafiltered into a membrane having a pore size of 3-100 kDa (Molecular Weight Cut Off).

Alternatively, the same amount as that of the culture was added using an organic solvent having ethanol or similar properties to stand at low temperature for 24 hours, and the precipitate was recovered by centrifugation. Thereafter, the supernatant was recovered by dialysis with a cellulose membrane having a pore size of 3-100 kDa to remove the low molecular weight material, followed by centrifugation. The recovered fractions were subjected to continuous column separation purification such as size exclusion chromatography, ion chromatography, and sugar fractionation column to recover the fractions containing polysaccharide.

The general method of separation and purification is as follows:

The culture medium concentrated using the membrane or organic solvent from L.plantarum culture medium was concentrated about 1000 times from the initial culture medium to the final concentration solution, and the lactic acid and low molecular material were completely removed by filtration from the concentrated culture medium. The low molecular weight material is removed and a solution containing polysaccharide is added to an ion-exchange column, and an ionic material such as ammonium sulfate, sodium chloride, potassium chloride, sodium acetate, potassium acetate, sodium phosphate or potassium phosphate is 0 to 0. The polysaccharide material was recovered from the column through a concentration gradient of 1 M to 3 M. The polysaccharide-containing fractions were concentrated again and subjected to size-exclusion chromatography to separate the polysaccharides by size, so that the polysaccharides had a polysaccharide-S ranging from 10 to 200 kDa and polysaccharides of 500 kDa or more. -L was isolated and purified. Each polysaccharide was separated into third polysaccharide using a divalent metal ion immobilized column, and the polysaccharide fraction was separated into a Rezex ROA-Organic acid H + organic acid column and an Aminex HPX-87P. Analysis was carried out by HPLC equipped with sugar column. The analysis conditions are as follows:

Detector: refractive index detector (Agilent);

HPLC (High-performance liquid chromatography) system with autosampler (Agilent, USA);

Analytical column: SecurityGard ^TM guard with Rezex ROA-Organic Acid H ⁺ column (7.8 x 300 mm; Rhenomenex, Torrance, CA, USA) and guard cartridge (KJ0-4282; Rhenomenex, Torrance, CA, USA) column;

Column temperature: 65 ° C .;

Mobile phase: 0.005 mM sulfuric acid solution;

Flow rate, 0.5 mL min ⁻¹ .

The polysaccharide in the organic acid column was 8.2 min; Lactate was 16.1 min; Acetic acid was detected at 18.8 minutes.

Only the fraction containing the polysaccharide was collected and concentrated through a membrane, and after dialysis, the polysaccharide portion was recovered through separation and purification with a sugar analysis column.

The analysis conditions of the analytical column per Aminex ^® HPX-87P (Biorad) are as follows:

Column temperature: 80 ° C .;

Mobile phase: distilled water;

Flow rate: 0.6 mL min ⁻¹ .

After recovering only the fraction containing the polysaccharide prepared as a pool, the sample was prepared in a solid state for further component analysis and anti-influenza efficacy analysis of the polysaccharide through reduced pressure lyophilization.

The concentrated solution of L. plantarum culture, the polysaccharide fraction obtained after ultrafiltration and dialysis as a membrane capable of fractionating polysaccharides of 3 to 100 kDa size, and the culture medium passed through the membrane were analyzed by HPLC using an organic acid column. The results are shown in Figs. 1A, 1B, and 1C, respectively.

Referring to Figure 1, the polysaccharide fraction obtained by concentrating the L. plantarum culture to the membrane was removed the lactic acid and acetic acid, the main fermentation products in the culture, it can be seen that the polysaccharide has a molecular weight of at least 10 kDa (Fig. 1).

Example 3. Analysis of the composition of sugars constituting the polysaccharide and molecular weight analysis of the polysaccharide

Quantitative, qualitative, size analysis of the following polysaccharides describes the results for polysaccharide-S having anti-influenza activity in Example 4 below among the polysaccharides isolated and purified in Example 2 above.

Example 3-1: Quantitative Analysis

For quantitative analysis of sugars and polysaccharides constituting the polysaccharide, neutral sugars and acidic sugars were analyzed by HPLC, and the component sugars were analyzed by HPLC by labeling the monosaccharides obtained through acid hydrolysis with fluorescent substances.

For the analysis of neutral sugars or amino sugars, 2M trifluoroacetic acid (TFA) was added to the polysaccharide sample, and the reaction was performed for 3 hours at 100 ℃ heat block to monosaccharide polysaccharide, and then the acid valence was removed by removing the TFA using the Speed Vac System. Digested monosaccharides were obtained.

Acid hydrolysis for the analysis of acidic sugar was added 0.1 M TFA to the polysaccharide sample and reacted for 1 hour at 80 ℃, the method is the same as the neutral sugar assay.

Isolated neutral sugars or amino sugars were labeled with 2-AA (2-aminobenzoic acid) fluorescent material for HPLC analysis. First, 30 mg 2-AA and 30 mg sodium cyanoborohydride were each prepared in a 1.5 ml tube, followed by 500 μl of a solution of 4% sodium acetate-3H ₂ O (W / V) and 2% boric acid in methanol. Was dissolved to prepare a fluorescent material labeling solution. Thereafter, the resultant was placed in the acid-hydrolyzed sample obtained as described above and reacted at 80 ° C. for 45 minutes to perform fluorescent labeling.

Acidic sugars containing sialic acid were labeled with DMB (1,2-diamino-4,5-methylenedioxybenzene.2HCl) phosphor. A certain amount of DMB was mixed with a solution containing 5 mM trifluoroacetic acid / 1 M 2-mercaptoethanol / 18 mM sodium hydrosulfite to produce 7 mM, and the acid hydrolyzed sample obtained above was added at the same volume ratio. The mixture was reacted at 50 ° C. for 2 hours, and fluorescently labeled.

Solution A (0.2% butylamine (v / v), 0.5% phosphoric acid (v / v), 1% tetrahydrofuran in water) and Solution B (mobile phase A: ACN = 1: 1) were used as mobile phase for HPLC analysis. . 5% of solution B was stabilized by flowing at a rate of 1 ml / min, then flowed in a gradient to 100% for 70 minutes, maintained for 5 minutes, and then solution B was flowed at 5% for 5 minutes.

A Waters Alliance HPLC system (Milford, Mass.) And a Waters 2475 fluorescence detector were used and analyzed at an excitation wavelength of 360 nm and an emission wavelength of 425 nm using a Shodex Asipak NH2P-50 (Showa Denko, Tokyo, Japan) column.

Neutral sugars are N-acetylglucosamine (GlcNAc) or glucosamine (GlcN), N-acetylgalactosamine (GalNAc) or galactosamine (GalN), galactose (Gal), mannose (Man), glucose using a monosaccharide mix reference (Ludger) as a standard. (Glc) and fucose (Fuc) were analyzed. Acidic sugars were analyzed using N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) as standards.

As a result of sample analysis, GlcNAc or GlcN, GalNAc or GalN, Gal, Man and Glc were identified as the neutral sugar constituting the polysaccharide, and the acidic sugars were Neu5Ac and Neu5Gc (FIG. 2). Specifically, 132 mM GlcNAc or GlcN; 44.3 mM GalNAc or GalN; 10.6 mM Gal; 8.5 mM Man; 996 mM Glc was identified, sialic acid being 0.27 mM Neu5Ac; 0.15 mM Neu5Gc was confirmed.

When the concentration of Glc or GlcNAc was set to 1 for quantitative analysis, the relative molar ratio of other sugars was converted, and the results are shown in Table 1.

This analysis confirmed that sialic acid (Neu5Ac), a receptor for influenza virus, was added to the polysaccharide of the present invention.

Composition ratio of neutral sugar and acid sugar constituting polysaccharide Monosaccharide Type
(monosaccharide) density
(mM) Relative concentration ratio Glc (1) GlcNAc (1) Glucose (glc) 996.0 1.00 7.54 N-Acetylglucosamine (GlcNAc)
Or Glucosamine (GlcN) 132.0 0.13 1.00 N-Acetylgalactosamine (GalNAc)
Or Galacotosamine (GalN) 44.3 0.04 0.33 Galactose (gal) 10.6 0.01 0.08 Mannose (man) 8.5 8.5 x 10 ^-3 0.06 N-Glycolylneuraminic acid (Neu5Gc) 0.15 1.5x10 ^-4 1.1 x 10 ^-3 N-Acetylneuraminic acid (Neu5Ac) 0.27 2.7 x 10 ^-4 2.0 x 10 ^-3

Example 3-2: Qualitative Analysis

In order to qualitatively analyze the sugar component constituting the isolated polysaccharide in Example 2, it is present in the polysaccharide through Fourier-transform infrared (FT-IR) (FIG. 3 (A)) and FT Raman spectroscopy (FIG. 3 (B)). Functional groups were analyzed. As a result, the CHO-, CO-, CH _2- , CH _3- , -OH groups identified in the typical carbohydrate structure of the polysaccharide of the present invention were identified, and amide group, sulfur dioxide (SO ₂ ) The sulfonyl group (R-SO ₂ -R) to which was added was confirmed. Amide groups are believed to be present in GlcNAc or GalNAc, and sulfonyl groups are present in sulfated hexosamine or hexose.

In order to identify the sugars in the form of monosaccharides or oligosaccharides constituting the polysaccharide, unlabeled monosaccharides obtained through acid hydrolysis were subjected to mass spectrometry using LC-MS. The mass (MS) of the peaks corresponding to monosaccharides or oligosaccharides liberated from the polysaccharides was analyzed by comparative analysis with the Mass Spectrometer spectrum profile of the buffer solution without polysaccharides in the cationic mode. As a result, [Hex + Na] ⁺ in the form of Na ⁺ ion bound to hexose, [Hex + Hex] ^{+ in the} form of oligosaccharide, [HexNAc + Deoxyhex] ⁺ , [Hex + Neu5Ac] ⁺ , [HexNAc] Molecular weights of 203, 360, 383, 453, 204, 309 and 659 corresponding to ⁺ , [Neu5Ac] ⁺ , [HexNac + Deoxyhex + Neu5Ac] ⁺ , respectively, were confirmed (FIG. 4). In the above, [Hex] is hexose (hexose), [HexNAc] is hexosamine (hexosamine), [deoxyhex] is deoxyhexose, [Neu5Ac] represents sialic acid (Neu5Ac).

Example 3-3 Molecular Weight Analysis of Polysaccharides

Molecular weight determination of the polysaccharide isolated and purified in Example 2 was carried out using a Sephacryl S-300 column, size-exclusion chromatography under non-reducing conditions, to analyze the relative size of the polysaccharide using a standard protein of known molecular weight. It was. The standard protein used was 440 kDa Fertin, 158 kDa Aldose, 44 kDa Ovalbumin, 29 kDa carbonic anhydrase from the Molecular weight calibration kit (GE healthcare). (Carbonic anhydrase), 13.7 kDa Ribonuclease A and 6.5 kDa Aprotinin. The polysaccharide contained in the fraction eluted from the Sephacryl S-300 column was confirmed by HPLC equipped with a Rezex ROA-Organic acid H + organic acid column and an Aminex HPX-87P sugar analysis column in the same manner as in Example 2 above. The chromatogram was compared with the standard protein to confirm the relative size. As a result, it was confirmed that the molecular weight of the isolated polysaccharide is 10 ~ 200 kDa.

Example 4. Identification of anti-influenza virus activity of polysaccharides

L. plantarum Cell experiments were conducted to confirm the anti-influenza virus activity of the polysaccharide isolated from the culture.

The polysaccharides used at this time are polysaccharides (polysaccharide-L) having a molecular weight of greater than 500 kDa and polysaccharides (polysaccharide-S) having a molecular weight of 10 to 200 kDa, separated by size-exclusion chromatography.

After the solid polysaccharide was dissolved in distilled water and permeated through a 0.45 μm filter, a solution having a polysaccharide concentration of 0.015, 0.05, 0.14, 0.4, 1.2, 3.7, 11.1, 33.3, 100, 300 μg / ml was prepared.

Subsequently, H1N1 A / Puerto Rico / 8/34, H3N2 A / Hong Kong / 8/68, or influenza virus B (Flu B), influenza virus A (Flu A) type, were viewed. The activity of the inventive polysaccharide was confirmed. When infecting a virus and processing polysaccharide in a Maddin-Darby Canine Kidney (MDCK) cell line, the polysaccharide is either pre-treatment prior to viral infection, co-treatment with infection, or post-infection. treatment).

Specifically, polysaccharide pre-treatment first treated the polysaccharides with the cells for 30 minutes before infecting the MDCK cells with the influenza virus, and then infected the cells for 1 hour. Polysaccharide co-treatment was a mixture of polysaccharide and influenza virus and then treated with cells for 1 hour. Post-treatment treatment was performed for 3 days after influenza virus infection for 1 hour.

Thereafter, the toxicity of the polysaccharide in the MDCK cell line was confirmed by cell viability (CC ₅₀ , ㎍ / ㎖). In addition, the anti-influenza virus activity of the polysaccharide confirmed the effective concentrations for 50% plaque reduction (EC ₅₀ , ㎍ / ㎖) by the cytophatic effect (CPE) inhibition assay and the results are shown in Table 2.

TABLE 2

In this case, RBV (ribavirin), an inhibitor of viral RNA polymerase, and an anti-influenza agent OSV-C (Oseltamivir carboxylate), which are currently used commercially, were used as positive controls. In addition, influenza A M2 inhibitor AMT (amantadine hydrochloride) was used as a control.

In Table 2, polysaccharide-L of two polysaccharides having different molecular weights did not show an anti-influenza virus effect, and polysaccharide-S was Flu A type H1N1 A / Puerto Rico / 8/34 and H3N2 A / Hong. Only anti-viral activity was shown in Kong / 8/68. In addition, both polysaccharides showed no activity against Flu B type. In addition, no toxicity was observed for both polysaccharides.

The anti-influenza activity of polysaccharide-S (EC ₅₀ ) was found to be 140.2 ug / mL for pretreatment, 127.1 ug / mL for co-treatment and 42.4 ug / mL for post-treatment for H1N1 A / Puerto Rico / 8/34. . In addition, H3N2 A / Hong Kong / 8/68 was found to be ineffective in pretreatment, 181.5 ug / mL in simultaneous treatment, and 67.7 ug / mL in post treatment. Therefore, polysaccharide-S showed better activity against influenza virus of H1N1 type, and acted as an inhibitory factor against H1N1 and H3N2 influenza of Flu A type at the early stage of infection, and the longer the treatment, the better the effect.

Example 5 Identification of Polysaccharides Added with Sialic Acid

To determine whether the polysaccharide of the present invention contains sialic acid addition glycosides having anti-influenza activity, the glycoconjugate residues containing sialic acid added to the polysaccharides were analyzed using lectins. .

N-acetylneuraminic acid (N-acetylneuraminic acid, Neu5Ac) is linked to galactose (galactose, Gal) or N-acetylgalactosamine (N-acetylgalactosamine, GalNAc) in the form of alpha-2,6-bond (alpha- Lectins that selectively bind to 2,6-linkage), such as SNA ( Sambucus nigra lectin); Alpha-2,3-binding of lactose or lactosamine, a disaccharide structure in which N-acetylneuraminic acid or N-glycolylneuraminic acid (Ne5Gc) is added to galactose The chemical binding structure of sialic acid added to the polysaccharide was confirmed using MAA ( Maackia amurensis lectin); as a lectin that selectively binds to (alpha-2,3-linkage).

In addition, as well as confirming the selective binding ability of the lectin to the polysaccharide, the chemical binding structure of the sialic acid added to the polysaccharide by simultaneously performing the lectin analysis of the bisial oxidized polysaccharide after removing sialic acid added to the polysaccharide through enzymatic hydrolysis. It was confirmed. An enzyme used for enzymatic hydrolysis of sialic acid. Both Streptococcus pneumoniae sialidase and alpha-2,3,6-linkage can selectively hydrolyze alpha-2,3-linkage. Hydrolysis of Arthrobacter ureafaciens sialidase was used.

The experimental method is illustrated in FIG. 5, and the specific method is as follows: polysaccharides fluorescently labeled with APTS (9-Aminopyrene-1,4,6-trisulfonic acid) were treated with the sialidase, followed by dialysis and concentration. Recovered through. SNA and MAA lectins were immobilized on a Nunc MaxiSorp ^® flat-bottom 96 well plate (eBIOscience, Cat. No. 44-2404) by methods suggested by those skilled in the art through chemical methods. After reacting the fluorescently labeled polysaccharide with SNA and MAA lectin-immobilized 96 well plates for 2 hours at room temperature, the fluorescently labeled polysaccharide was washed three times or more using PBS buffer solution, and finally the fluorescence remaining on the lectin-immobilized 96 well plate Relative amounts of labeled polysaccharides were quantified at Ex 425 nm / Em 503 nm using fluorospectroscopy.

As a result, the polysaccharide of the present invention was measured in both the SNA and MAA lectin (Fig. 6), the sialic acid is alpha-2,3-linked compared to the SNA lectin that binds to the oligosaccharide to which sialic acid is alpha-2,6-linked Measured with a relatively high fluorescence sensitivity in the MAA lectin binding to the oligosaccharide, it can be seen that the polysaccharide of the present invention is added to the alpha-2,3-linked sialic acid relatively higher than the alpha-2,6-linked (FIG. 6).

In addition, in the case of the group treated with sialidase which hydrolyzes the sialic acid of the polysaccharide, the fluorescence sensitivity was low (FIG. 6). In SNA lectin, there is little change in fluorescence signal after sialidase treatment that recognizes alpha-2,3-sialic acid, but the fluorescence signal remarkably after sialidase treatment that recognizes alpha-2,3,6-sialic acid. It was confirmed that it is measured low. On the other hand, in the MAA lectin, it was confirmed that the fluorescence signal was hardly measured after sialidase treatment (FIG. 6), and the polysaccharide of the present invention was found that sialic acid was mostly added by alpha-2,3-linkage. This suggests that the polysaccharide of the present invention may be particularly effective against avian influenza viruses that are infected via siaryl α (2,3) galactose.

Claims (12)

Polysaccharides having a molecular weight of 10 to 200 kDa, recovered from Lactobacillus plantarum culture; Or a culture solution comprising the polysaccharide; an anti-influenza A virus composition comprising a.
According to claim 1, wherein the polysaccharide is Glc (Glucose); GlcNAc (N-acetylglucosamine) or GlcN (glucosamine); GalNAc (N-acetylgalactosamine) or GalN (galactosamine); Gal (Galactose); Man (Mannose); N-acetylneuraminic acid (Neu5Ac); And Neu5Gc (N-glycolylneuraminic acid); It comprises, composition.
The composition of claim 2, wherein the molar ratio of Glc and Neu5Ac is 1: 1 × 10 ⁻⁵ to 1: 1 × 10 ⁻³ .
The composition of claim 2, wherein the molar ratio of Glc and Neu5Gc is 1: 1 × 10 ⁻⁵ to 1: 1 × 10 ⁻³ .
The composition of claim 1, wherein the polysaccharide comprises one or more residues selected from the group consisting of:
Neu5Acα2-6Gal-R residues;
Neu5Acα2-6GalNAc-R residues;
Neu5Acα2-3Galβ1-4Glc-R residues;
Neu5Acα2-3Galβ1-4GlcNAc-R residues;
Neu5Gcα2-3Galβ1-4Glc-R residues; And
Neu5Gcα2-3Galβ1-4GlcNAc-R residues;
delete The composition of claim 1, wherein the polysaccharide is produced by fermentation of Lactobacillus plantarium.
The composition of claim 1, wherein the Lactobacillus plantarium is Lactobacillus plantarium CK401.
delete A pharmaceutical composition for preventing or treating influenza A virus infection, comprising the composition of any one of claims 1 to 5 and 7 to 8.
a) culturing Lactobacillus plantarum in a medium;
b) recovering the polysaccharide having a molecular weight of 10 to 200 kDa from the Lactobacillus plantarium cultured in step a) or a culture thereof;
Including, polysaccharide production method.
The method of claim 11, wherein the Lactobacillus plantarium is Lactobacillus plantarium CK401.

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