KR20180072225A - Antibacterial composition containing exopolysaccharides derived from lactic acid bacteria - Google Patents

Abstract

The present invention relates to an antibacterial activity of polysaccharide derived from Lactobacillus kefiranofaciens DN1 (KCCM11869P). In particular, a condition for producing EPS at optimal efficiency from Lactobacillus kefiranofaciens DN1 (KCCM11869P) having the most excellent EPS production ability among 22 lactic acid bacteria isolated from kefir is established. The produced EPS is purified, and a structure thereof is confirmed. The present invention has excellent antibacterial activities to Listeria monocytogenes and Salmonella Enteritidis strains which are EPS food pointing causative bacteria, and thus can be used as an antibacterial pharmaceutical composition, a food poisoning preventing or treating pharmaceutical composition, a food composition, and a fodder composition.

Description

TECHNICAL FIELD [0001] The present invention relates to an antimicrobial composition containing an extracellular polysaccharide derived from a lactic acid bacterium,

The present invention relates to the antimicrobial activity of polysaccharides derived from Lactobacillus kefiranofaciens DN1 (KCCM11869P).

Various polysaccharides are produced by plants, algae and bacteria, among which polysaccharides of plant origin are cellulose, pectin, starch, alginate polysaccharides are agar, carrageenan and alginate, bacterial polysaccharides are dextran , Gellan, flunan, and xanthan (Sutherland, 1998). Recently, Lactic Acid Bacteria (LAB) not only produce substances such as aromas, spices and enzymes that can affect the sensory properties of fermented foods, but also some fermented food products such as rheology, an exopolysaccharide that can affect texture, viscosity, viscofying, sweetening, stability, water-binding agents, gelling, thickening properties, (2006), and the results of this study are summarized as follows.

Lactobacillus kefiranofaciens is the first lactic acid bacterium isolated from kefir fermented milk, a longevity food from the former Soviet Caucasus region. Kefir is a dairy product produced by the combined fermentation of more than 50 kinds of lactic acid bacteria, yeast and acetic acid bacteria in the Caucasus region of Soviet Union. The beneficial effects of fermented foods are known to be caused by various metabolites such as polysaccharides, proteins, and lipids that they produce, as well as microorganisms that cause fermentation. The extracellular polysaccharide (EPS), a typical metabolite, is a viscous polysaccharide that accumulates around or outside the cells during fermentation of the microorganism. It is a primary or secondary metabolite produced by bacteria. These are the most abundant of the saccharide metabolites produced by microorganisms, and their structure is stable and viscous, so they are applied for the purpose of improving emulsifying agent and texture of fermented food. Recently, however, EPS has been attracting attention as a physiologically active function such as immunoregulation, anticancer effect, and blood cholesterol lowering effect, not merely for improving the physical properties of the past. In addition, EPS produced by microorganisms is recognized to have greater potential as physiological performance and functional new material than commercially available plant-derived EPS. However, to date, there has been little research on the isolation and purification of EPS produced by KPI fermented milk and its physiological activity.

Listeria Monocytogenes and Salmonella Enteritidis are representative pathogenic bacteria and belong to the cause of Top 5 food poisoning in Korea. These can be propagated by foods such as meat, vegetables, etc., and are used as a method to control them because they are the main microorganisms that are managed by defining the detection standard according to the ingredient standard of the livestock product and the food revolution. Although antibiotics have been used most actively as a representative product, the recent restrictions on antibiotic use and regulations have been strengthened at home and abroad due to the recent development of antibiotic-resistant and antibiotic resistant bacteria. Accordingly, studies for developing an antibiotic substitute have been actively carried out, and organic acid and plant extracts have been developed as substitutes. However, they are limited in their use in the food industry due to poor water solubility and environmental pollution. Therefore, there is a steady increase in interest in substances that have no side effects and are not harmful to the environment.

It is an object of the present invention to provide an extracellular polysaccharide (EPS) derived from Lactobacillus kefiranofaciens DN1 (KCCM11869P).

It is an object of the present invention to provide a pharmaceutical composition for antibacterial use containing an extracellular polysaccharide derived from Lactobacillus kefiranopathies DN1 (KCCM11869P) as an effective ingredient.

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating food poisoning which contains an extracellular polysaccharide derived from Lactobacillus kefiranopathies DN1 (KCCM11869P) as an active ingredient.

It is an object of the present invention to provide a food composition containing an extracellular polysaccharide derived from Lactobacillus kefiranopathies DN1 (KCCM11869P) as an active ingredient.

It is an object of the present invention to provide a feed composition comprising an extracellular polysaccharide derived from Lactobacillus kefiranopathies DN1 (KCCM11869P) as an active ingredient.

It is an object of the present invention to provide a method for producing an extracellular polysaccharide from Lactobacillus kefiranopathies DN1 (KCCM11869P).

In order to achieve the above object, the present inventors selected Lactobacillus kefiranopathies DN1 (KCCM11869P) having the highest ability to produce EPS among 22 lactic acid bacteria isolated from kefir, , And the produced EPS was purified and its composition was confirmed and its antibacterial effect was confirmed.

EPS consisting of monosaccharides of mannose, arabinose, glucose, galactose and rhamnose, produced by Lactobacillus kefiranopathies DN1 (KCCM11869P) of the present invention, Has excellent antimicrobial activity against Listeria monocytogenes and Salmonella Enteritidis, which are food poisoning bacteria, and thus it can be used as a pharmaceutical composition for antimicrobial use, a pharmaceutical composition for preventing or treating food poisoning, a food composition and a feed Can be used as a composition.

FIG. 1 is a photograph showing the EPS production by negative staining of Lactobacillus kefiranofaciens strain DN1.
FIG. 2 is a graph showing the amount of crude EPS produced according to the concentration of glucose, which is a carbon source.
3 is a graph showing the growth curve analysis results for pathogenic bacteria according to the addition amount of EPS_DN1:
A: Listeria monocytogenes Growth curve analysis results for ATCC 51776;
B: Growth curve analysis results for Salmonella Enteritidis 108;
NB: control group without EPS_DN1 added (nutrient medium);
NB + 0.3% EPS DN1: group with EPS_DN1 added to nutrient medium at a concentration of 0.3%;
NB + 1% EPS DN1: 1% concentration of EPS_DN1 in nutrient medium; And
NB + 2.5% EPS DN1: A nutrient medium supplemented with EPS_DN1 at a concentration of 2.5%.

Hereinafter, the present invention will be described in detail. However, it should be understood that the present invention may be embodied in many other specific forms without departing from the spirit or essential characteristics thereof.

In one aspect, the invention relates to a polysaccharide derived from Lactobacillus kefiranofaciens DN1 (KCCM11869P).

In one embodiment, the polysaccharide from Lactobacillus kefiranopathiae DN1 (KCCM11869P) of the present invention is a polysaccharide derived from monosaccharide of mannose, arabinose, glucose, galactose and rhamnose, , And in one embodiment, the constituent ratio of the monosaccharide is shown in Table 4. < tb > < TABLE >

In one aspect, the present invention relates to a pharmaceutical composition for antimicrobial use containing an exopolysaccharide (EPS) derived from Lactobacillus kefiranopathies DN1 (KCCM11869P) as an active ingredient.

In one embodiment, the Lactobacillus kefiranopathies DN1 (KCCM11869P) can be isolated from kefir.

In one embodiment, the extracellular polysaccharide may have an antimicrobial activity against Listeria monocytogenes and / or Salmonella Enteritidis strains.

The antimicrobial pharmaceutical composition according to the present invention contains the extracellular polysaccharide of the present invention as an active ingredient, thereby achieving the pharmacological effect of the antimicrobial activity.

The term "antibacterial" or "antibacterial activity" of the present invention means a property of resisting microorganisms such as bacteria or fungi, more specifically, a property of inhibiting growth or proliferation of bacteria or killing bacteria themselves . For the purpose of the present invention, the antimicrobial or antimicrobial activity is preferably a pathogenic microorganism, more preferably E. coli , Staphylococcus aureus , Shigella flexneri , Listeria monocytogenes monocytogenes), Salmonella Entebbe utility tooth (Salmonella Enteritidis) and Salmonella typhimurium (selected from the group consisting of micro-organisms Salmonella typhimurium), and most preferably from Zenith L. monocytogenes (Listeria monocytogenes ATCC 51776 and Salmonella Enteritidis 108, but are not particularly limited thereto.

In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating food poisoning, comprising an extracellular polysaccharide derived from Lactobacillus kefiranopathies DN1 (KCCM11869P) as an active ingredient.

In one embodiment, the extracellular polysaccharide may have an antimicrobial activity against Listeria monocytogenes and / or Salmonella Enteritidis strains.

In one embodiment, since the extracellular polysaccharide derived from Lactobacillus kefiranopathies DN1 (KCCM11869P) exhibits antimicrobial activity against Listeria monocytogenes and / or Salmonella enteritidis, it is possible to prevent the disease Can be prevented or treated.

In one embodiment, the pharmaceutical composition may be one or more formulations selected from the group comprising oral formulations, external preparations, suppositories, sterile injectable solutions and sprays.

In the pharmaceutical composition for preventing or treating food poisoning according to the present invention, the prevention and treatment of food poisoning can be carried out by an antibacterial action against any causative bacteria causing food poisoning, but preferably, E. coli , Staphylococcus aureus , Shigella flexneri), L. monocytogenes Zenith (Listeria monocytogenes), Salmonella Entebbe utility tooth (Salmonella Enteritidis) and Salmonella typhimurium (Salmonella typhimurium) selected food poisoning bacteria from the group consisting of and most preferably L. monocytogenes Zenith (Listeria monocytogenes) ATCC 51776 or Salmonella Enteritidis 108, which is known in the art.

The pharmaceutically effective amount of the composition of the present invention may vary depending on various factors, such as the method of administration, the site of administration, the condition of the patient, and the like. Therefore, when used in the human body, the dosage should be determined in consideration of safety and efficacy. It is also possible to estimate the amount used in humans from the effective amount determined through animal experiments. Such considerations in determining the effective amount are described, for example, in Hardman and Limbird, eds., Goodman and Gilman ' s Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; And E.W. Martin ed., Remington ' s Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.

Compositions of the present invention may also include carriers, diluents, excipients, or a combination of two or more thereof commonly used in biological formulations. The pharmaceutically acceptable carrier is not particularly limited as long as the composition is suitable for in vivo delivery, for example, Merck Index, 13th ed., Merck & Inc. A buffered saline solution, a buffer solution, a dextrose solution, a maltodextrin solution, glycerol, ethanol, and one or more of these components may be mixed and used, and if necessary, an antioxidant, a buffer, Conventional additives may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into main dosage forms such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules or tablets. Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990) in a suitable manner in the art.

The composition of the present invention may further contain one or more active ingredients showing the same or similar functions. The composition of the present invention contains 0.0001 to 10% by weight, preferably 0.001 to 1% by weight, of the oligosaccharide, based on the total weight of the composition.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable additive, wherein the pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, Wherein the starch is selected from the group consisting of lactose, mannitol, sugar, arabic gum, pregelatinized starch, cornstarch, powdered cellulose, hydroxypropyl cellulose, opaques, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, Calcium, white sugar, dextrose, sorbitol and talc may be used. The pharmaceutically acceptable additives according to the present invention are preferably included in the composition in an amount of 0.1 to 90 parts by weight, but are not limited thereto.

The composition of the present invention may be administered orally or non-orally (for example, intravenously, subcutaneously, intraperitoneally or topically) or orally administered in accordance with a desired method, and the dose may be appropriately determined depending on the patient's body weight, The range varies depending on diet, time of administration, method of administration, excretion rate, and severity of the disease. The daily dose of the composition according to the present invention is 0.0001 to 10 mg / ml, preferably 0.0001 to 5 mg / ml, more preferably administered once to several times a day.

Examples of the liquid preparation for oral administration of the composition of the present invention include suspensions, solutions, emulsions, syrups, and the like. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, Etc. may be included together. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, and the like.

In one aspect, the present invention relates to a food composition comprising as an active ingredient an extracellular polysaccharide derived from Lactobacillus kefiranopathies DN1 (KCCM11869P).

The term "food" as used in the present invention means a natural product or a processed product containing one or more nutrients, preferably a state of being able to be eaten directly through a certain degree of processing, It is intended to include food, food additives, functional foods and beverages as a meaning.

The food composition of the present invention can be manufactured in the form of, but not limited to, various drinks, gums, tea, confectionery, vitamin complexes, health supplements and the like.

The preferred intake amount of the food composition of the present invention varies depending on the condition and the weight of the recipient, the degree of symptoms, the type of food, the period of consumption, and can be appropriately selected. It is preferable for the food composition of the present invention that the active ingredient is ingested at 0.2 mg / kg to 200 mg / kg per day for optimal effect.

In one aspect, the present invention relates to a feed composition comprising an extracellular polysaccharide derived from Lactobacillus kefiranopathies DN1 (KCCM11869P) as an active ingredient.

In one aspect, the present invention provides a method for producing lactobacillus kefiranopathies DN1 (KCCM11869P); Centrifuging the cultured medium and recovering the supernatant; Adding ethanol to the supernatant; Centrifuging to obtain pellets; Removing protein and bacterial debris from the pellet; And adding ethanol to the mixture, followed by centrifugation to obtain pellets. The present invention relates to a method for producing an extracellular polysaccharide from Lactobacillus kefiranopathies DN1 (KCCM11869P).

In one embodiment, the concentration of the glycogen in the culture of the strain may be less than or equal to 60 g / L of glucose.

In one embodiment, the step of removing protein and bacterial debris from the pellet may be a step of adding trichloroacetic acid (TCA) to the pellet followed by filtering.

The present invention will be described in more detail with reference to the following examples. However, the following examples are only for the purpose of illustrating the present invention, and thus the present invention is not limited thereto.

Example One. Kefir  Derived lactic acid bacteria Extracellular polysaccharide Generation  compare

1-1. Kefir  Isolation and Identification of Derived Lactic Acid Bacteria

For the production of kefir fermented milk, 50 g of kefir grains were added to 1 L of sterilized milk (5% w / v) and cultured at 25 캜 for 24 hours. Fermented milk was collected in a sterile loop to separate the lactic acid bacteria from the fermented kefir and cultured under anaerobic conditions for 48 hours at 37 ° C after staining with MRS (de Man, Rogosa, and Sharpe) agar. After the culture, morphological characteristics of the colonies were firstly screened, and 3% hydrogen peroxide was brought into contact with a drop, followed by a catalase test to confirm the occurrence of bubbles. As a result, Respectively. Twenty - two kinds of lactic acid bacteria selected by Lactobacillus were streaked on MRS agar medium and cultured for 2 hours under anaerobic condition at 30 ℃ for 48 hours. After that, DNA was extracted from each strain using a NucliSENS easyMAG (bioMerieux, Marcy l'Etoile, France) machine, and the base sequence of the 16S ribosomal RNA gene was analyzed. Universal PCR primer 27F / 1492R was used as a primer. The species was identified by confirming the similarity of 16S rRNA sequences of each strain using BLAST of GeneBank.

Sequencing of the 16s rRNA gene of Lactobacillus strain 22 isolated from Kefir was analyzed and identified as the following lactic acid bacteria with a similarity of 99.4-99.9%.

Strain Species 16S rRNA  gene sequence identity, % DN1 Lactobacillus kefiranofaciens 99.8 DN2 Lactobacillus kefiranofaciens 99.7 DN3 Lactobacillus kefiranofaciens 99.6 DN4 Lactobacillus kefiranofaciens 99.9 DN5 Lactobacillus kefiranofaciens 99.5 DN6 Lactobacillus kefiranofaciens 99.6 DN7 Lactobacillus kefiranofaciens 99.6 DN8 Lactobacillus kefiranofaciens 99.9 DN9 Lactobacillus kefiri 99.8 DN10 Lactobacillus kefiri 99.8 DN11 Lactococcus lactis 99.4 DN12 Lactococcus lactis 99.9 DN13 Lactococcus lactis 99.6 DN14 Lactococcus lactis 99.6 DN15 Lactococcus lactis 99.4 DN16 Lactococcus lactis 99.9 DN17 Lactococcus lactis 99.5 DN18 Leuconostoc mesenteroides 99.7 DN19 Leuconostoc mesenteroides 99.9 DN20 Leuconostoc mesenteroides 99.5 DN21 Leuconostoc mesenteroides 99.4 DN22 Leuconostoc mesenteroides 99.8

1-2. Selection of EPS-forming lactic acid bacteria

Negative staining was performed in order to first select the lactic acid bacteria forming the EPS among the 22 lactic acid bacteria. Specifically, 22 isolated strains were plated on MRS agar medium and cultured for 48 hours at 37 ° C in an anaerobic tank to produce EPS. After that, the colony of each strain was looped onto a glass slide. To stain the cells, the cells were immersed in a crystal violet for 30 seconds, rinsed, immersed in a 20% CuSO ₄ aqueous solution for 30 seconds, and washed with water. Then, the strains DN1, DN3, DN4, DN5, DN9, DN11, DN6, DN7, DN6, DN7, DN6, DN12, DN15, DN18 and DN21 were selected as EPS-forming strains (Table 2).

Strain Species
(16S rRNA gene sequence identity%) EPS formation
(negative staining) DN1 Lactobacillus kefiranofaciens (99.8) + DN2 Lactobacillus kefiranofaciens (99.7) - DN3 Lactobacillus kefiranofaciens (99.6) + DN4 Lactobacillus kefiranofaciens (99.9) + DN5 Lactobacillus kefiranofaciens (99.5) + DN6 Lactobacillus kefiranofaciens (99.6) - DN7 Lactobacillus kefiranofaciens (99.6) - DN8 Lactobacillus kefiranofaciens (99.9) - DN9 Lactobacillus kefiri (99.8) + DN10 Lactobacillus kefiri (99.8) - DN11 Lactococcus lactis (99.4) + DN12 Lactococcus lactis (99.9) + DN13 Lactococcus lactis (99.6) - DN14 Lactococcus lactis (99.6) - DN15 Lactococcus lactis (99.4) + DN16 Lactococcus lactis (99.9) - DN17 Lactococcus lactis (99.5) - DN18 Leuconostoc mesenteroides (99.7) + DN19 Leuconostoc mesenteroides (99.9) - DN20 Leuconostoc mesenteroides (99.5) - DN21 Leuconostoc mesenteroides (99.4) + DN22 Leuconostoc mesenteroides (99.8) -

1-3. Kefir  Identification of EPS yield of derived lactic acid bacteria

To confirm the EPS production yield of EPS-producing lactic acid bacteria derived from Kepir, 10 strains selected in Example 1-2 were added to 200 mL of MRS broth (pH 5.5) so as to have a concentration of 1.0 × 10 ⁸ CFU / mL Inoculated and cultured at 37 ° C for 72 hours under anaerobic conditions to produce EPS. Each culture was centrifuged at 10,000 x g for 25 minutes at 4 ° C to remove the cells and recover the supernatant. 99% chilled ethanol was added to the supernatant twice its volume and then kept at 4 ° C overnight. After that, the pellet was obtained by centrifuging again (10,000 × g, 25 minutes, 4 ° C.), and the remaining ethanol was air-dried in a dry-oven and then freeze-dried. The obtained EPS was named crude EPS and the yields (yields) of the selected strains were compared.

As a result, the crude EPS yields of the ten lactic acid bacteria DN1, DN3, DN4, DN5, DN9, DN11, DN12, DN15, DN18 and DN21 selected in Example 1-2 were compared, Lactobacillus kefiranofaciens The DN1 strain showed the highest EPS production with a yield of 1.38 + - 0.21 g per liter.

Strain Species
(16S rRNA  gene sequence identity%) Yield of crude EPS
(g / L in MRS broth, mean + - SD) DN1 Lactobacillus kefiranofaciens (99.8) 1.38 ± 0.21 DN2 Lactobacillus kefiranofaciens (99.7) - DN3 Lactobacillus kefiranofaciens (99.6) 0.83 + - 0.25 DN4 Lactobacillus kefiranofaciens (99.9) 0.60 + 0.38 DN5 Lactobacillus kefiranofaciens (99.5) 0.02 ± 0.01 DN6 Lactobacillus kefiranofaciens (99.6) - DN7 Lactobacillus kefiranofaciens (99.6) - DN8 Lactobacillus kefiranofaciens (99.9) - DN9 Lactobacillus kefiri (99.8) 0.94 0.25 DN10 Lactobacillus kefiri (99.8) - DN11 Lactococcus lactis (99.4) 0.72 + 0.15 DN12 Lactococcus lactis (99.9) 0.68 ± 0.22 DN13 Lactococcus lactis (99.6) - DN14 Lactococcus lactis (99.6) - DN15 Lactococcus lactis (99.4) 0.03 ± 0.01 DN16 Lactococcus lactis (99.9) - DN17 Lactococcus lactis (99.5) - DN18 Leuconostoc mesenteroides (99.7) 0.30 ± 0.05 DN19 Leuconostoc mesenteroides (99.9) - DN20 Leuconostoc mesenteroides (99.5) - DN21 Leuconostoc mesenteroides (99.4) 0.08 0.02 DN22 Leuconostoc mesenteroides (99.8) -

Example  2. L kefiranofaciens  EPS purification of strain DN1

Additional purification of crude EPS of L. kefiranofaciens strain DN1, which produces the most EPS among lactic acid bacteria derived from kefir, was performed. Specifically, 10% (w / v) trichloroacetic acid (TCA) was added to the crude EPS produced by L kefiranofaciens strain DN1 to remove the residual bacterial debris and proteins, followed by reaction at 4 ° C for 2 hours to induce protein precipitation Respectively. The remaining residue was filtered using a syringe filter of 0.20-μm pore size, and 99% chilled ethanol was added at the same amount as the supernatant, followed by overnight incubation at 4 ° C. After that, the pellet was obtained by centrifugation (10,000 × g, 25 minutes, 4 ° C.), and the remaining ethanol was air-dried in a dry-oven and lyophilized. Finally, the purified EPS was obtained and named EPS_DN1.

Example  3. L kefiranofaciens  Optimization of EPS yield of strain DN1

To optimize the EPS yield of L kefiranofaciens strain DN1 In order to derive the carbon source concentration, EPS production by glucose concentration was compared. Specifically, Lactobacillus kefiranofaciens strain DN1 was inoculated into 200 mL of MRS broth (pH 5.5) so as to have a concentration of 1.0 × 10 ⁸ CFU / mL, and the glucose in the medium was added to each of 20, 40, 60 and 80 g / L Respectively. This was cultured at 37 ° C for 72 hours under anaerobic conditions to produce EPS. Then, the amount of crude EPS produced by glucose concentration was compared using the method of Example 1 above.

As a result, when the glucose was added at 20 g / L, 1.38 g was formed. When the glucose was added at 40 g / L, the yield was 1.78 g, and when glucose was added at 60 g / L, the yield of EPS was 2.26 g Whereas when glucose was added at 80 g / L, the amount of glucose was not significantly different from that of glucose at 60 g / L (FIG. 2). Through this, Lactobacillus kefiranofaciens The amount of EPS produced by the DN1 strain was increased depending on the glucose concentration, but it was found that 60 g / L was the saturation point. Thus, in order to optimize the EPS yield of L kefiranofaciens strain DN1 It can be seen that the concentration of the carbon source is 60 g / L.

Example  4. Statistical analysis

All experimental results of the present invention were presented as mean ± standard deviation using SPSS (version 18.0, SPSS Inc.). Statistical significance between the quantitative values was analyzed by analysis of variance (ANOVA; Tukey's method). P value <0.05 was considered statistically significant.

Experimental Example  One. Lactobacillus kefiranofaciens  Identification of antimicrobial activity of DN1-derived EPS

Purified EPS_DN1 pathogenic bacteria Listeria Growth curve analysis was performed for 24 hours to confirm the antimicrobial effect against monocytogenes ATCC 51776 and Salmonella Enteritidis 108. Specifically, 3, 10 and 25 mg of EPS_DN1 were added to 1 ml nutrient broth (Oxoid, Basingstoke, Hampshire, UK) to give final concentrations of EPS_DN1 of 0.3, 1 and 2.5% (w / v) . Listeria, which was subcultured twice in nutrient agar for 48 hours at 37 占 폚 in the EPS_DN1 mixed nutrient medium monocytogenes ATCC 51776 and Salmonella Enteritidis 108 (1, 9 and 12 g, m: 1.7) at a concentration of 10 ⁵ -10 ⁶ CFU, respectively. After mixing well, 200 .mu.l of each was dispensed into a 96-well plate and incubated at 37.degree. C., and the absorbance was measured at 450 nm for 24 hours at 1 hour intervals to confirm the growth of the bacteria.

As a result, it was confirmed that the NB group to which EPS_DN1 was not added increased the absorbance of the NB group with increasing the absorbance, but when EPS_DN1 was added at 0.3%, the absorbance of Listeria monocytogenes was restricted to 0.2 or less, , And when the EPS_DN1 was added in an amount of 1% or more, growth of both food-borne bacteria was not confirmed and both bacteria were killed (FIG. 2). Thus, EPS_DN1 isolated and purified according to the present invention exhibited antimicrobial activity against two pathogenic bacteria at a concentration of 10 ⁵ -10 ⁶ CFU when added at 1% (w / v) or more.

Experimental Example  2. Analysis of constituents of EPS_DN1

To confirm the chemical composition of EPS_DN1 produced by Lactobacillus kefiranofaciens strain DN1, the composition ratio of monosaccharides was confirmed. Specifically, the polysaccharide EPS_DN1 purified from the strain of the present invention was decomposed into monosaccharides through high-temperature hydrolysis and then subjected to High-Performance Size Exclusion Chromatography equipped with a CarboPac PA-1 column (250 x 4 mm, Dionex Co., USA) (HPSEC) equipment. The peaks obtained from the pulsed amperometric detector (PAD) were compared with the standard monosaccharide material, and the quantities were compared according to their width.

As a result, the single-party configuration of EPS_DN1 can be confirmed as shown in Table 4 below. Specifically, EPS_DN1 was composed of at least five monosaccharides: mannose, arabinose, glucose, galactose and rhamnose. In the conventional literature, the "polysaccharide", a polysaccharide reported to be produced by Lactobacillus kefiranofaciens, is a substance consisting of glucose and galactose in a ratio of 1: 1. Therefore, EPS_DN1 produced by Lactobacillus kefiranofaciens DN1 isolated from kefir Is a new EPS.

A total of 22 Lactobacillus isolates were isolated from Kepir, and the results were compared with those of Lactobacillus kefiranofaciens The DN1 strain was found to form EPS around the cells, and the crude EPS yielded 1.38 ± 0.21g per liter, showing the highest yield among the lactic acid bacteria isolated from the kefir. In addition, when the glucose was added at 60g per liter, the best EPS production was obtained. The analysis of the constituent components of the sugar showed that EPS_DN1 was different from the previously found EPSs. It was confirmed that EPS_DN1 of the present invention significantly inhibited growth of pathogenic bacterium Listeria monocytogenes and Salmonella enteritidis causing food poisoning, and killed both strains upon addition of 1% (w / v) or more, Thus, the Lactobacillus kefiranofaciens of the present invention The EPS produced by DN1 strain is expected to be used as an antibiotic substitute for improving food hygiene.

Korea Microorganism Conservation Center (overseas) KCCM11869P 20160720

Claims (11)

Exopolysaccharide (EPS) derived from Lactobacillus kefiranofaciens DN1 (KCCM11869P). The method of claim 1, wherein the extracellular polysaccharide comprises monosaccharides of mannose, arabinose, glucose, galactose and rhamnose. Polysaccharides derived from Piranopathies DN1 (KCCM11869P). An antimicrobial pharmaceutical composition comprising the extracellular polysaccharide derived from Lactobacillus kefiranopathies DN1 (KCCM11869P) of claim 1 as an active ingredient. 4. The antimicrobial pharmaceutical composition according to claim 3, wherein the lactobacillus kefiranopathies DN1 (KCCM11869P) is isolated from kefir. A pharmaceutical composition for preventing or treating food poisoning comprising the extracellular polysaccharide derived from Lactobacillus kefiranopathies DN1 (KCCM11869P) of claim 1 as an active ingredient. The pharmaceutical composition for preventing or treating food poisoning according to claim 5, wherein the extracellular polysaccharide has antimicrobial activity against Listeria monocytogenes and Salmonella Enteritidis. A food composition comprising the extracellular polysaccharide derived from Lactobacillus kefiranopathies DN1 (KCCM11869P) of claim 1 as an active ingredient. An animal feed composition comprising the extracellular polysaccharide derived from Lactobacillus kefiranopathies DN1 (KCCM11869P) of claim 1 as an active ingredient. (1) culturing Lactobacillus kefiranopathies DN1 (KCCM11869P) strain under anaerobic conditions;
(2) centrifuging the cultured culture and recovering the supernatant;
(3) adding ethanol to the supernatant;
(4) centrifuging to obtain pellets;
(5) removing protein and bacterial debris from the pellet; And
(6) A method for producing an extracellular polysaccharide from Lactobacillus kefiranopathies DN1 (KCCM11869P), comprising adding ethanol and then centrifuging to obtain pellets. 10. The method of producing an extracellular polysaccharide from Lactobacillus kefiranopathies DN1 (KCCM11869P) according to claim 9, wherein the concentration of the glycogen in the strain is not more than 60 g / L of glucose. The method of producing an extracellular polysaccharide according to claim 9, wherein the step (5) is performed by adding trichloroacetic acid (TCA) to the pellet and then filtering the Lactobacillus kefiranopathies DN1 (KCCM11869P).

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