KR101523205B1 - Pediococcus pentosaceus kft-18 and mass production method of exopolysaccharides using the same - Google Patents

Abstract

The present invention relates to a novel strain of Pediococcus pentosaceus that mass-produces EPS and a method for mass-producing EPS using the same. The novel Pediococcus pentosaceus KFT-18 of the present invention has a property of mass-producing EPS, and the EPS produced has excellent immunity-enhancing activity. Therefore, the EPS produced by the method of the present invention can be utilized as various pharmaceuticals or food compositions containing it.

Description

PEDIOCOCCUS PENTUSACEUS KFT-18 AND MASS PRODUCTION METHOD OF EXOPOLYSACCHARIDES USING THE SAME <br> <br> <br> Patents - stay tuned to the technology PEDIOCOCCUS PENTUSACEUS KFT-18 AND MASS PRODUCTION METHOD OF EXOPOLYSACCHARIDES USING THE SAME

The present invention relates to a novel strain of Pediococcus pentosaceus that mass-produces EPS and a method for mass-producing EPS using the same.

The viscous substance has attracted attention as a physiological functional material as well as a physical property of a food. By selecting microorganisms that produce viscous materials using natural food materials and understanding optimal production conditions of the viscous materials, it is expected that highly functional compositions using microorganisms will be developed.

Polysaccharides in the form of slurries are divided into homopolysaccharides and heteropolysaccharides, depending on the constituent sugars.

Homo polysaccharide mainly consists only in one type of sugar such as fructose and glucose, typically, Streptococcus salivarius (Streptococcus salivarius) and streptococcus mutans (Streptococcus mutans) program ruktan (fructan) type of Levan (levan) and inulin (inulin) and, as teroyi glucan type flow Pocono stock mesen teroyi des subspecies mesen des (Leuconostoc by mesenteroides subsp. mesenteroides , Leuconostoc mesenteroides , Leuconostoc &lt; RTI ID = 0.0 &gt; mesenteroides subsp. dextran by dextranicum and alternan by leuconostoc mesenteroides and mutans by Streptococcus sobrinus have been known (see, for example, . Among these, dextran produced by Lukono Stokes has stickiness, hygroscopicity and thermal stability, and can be involved in the control of the physical properties of the food. Thus, it is possible to improve the moisturizing property of sugar, candy and sugar enhancer, emulsifier, , Gelation of gum and jelly, prevention of freezing of ice cream, and control of physical properties of fluid foods.

Heteropolysaccharides are composed of two or more monosaccharides, i.e., glucose, galactose, fructose, and rhamnose, at different ratios. In some cases, the heterosaccharide is not a sugar such as N-acetyl-amino sugar, phosphate, acetyl or glycerol There are also substances. Mainly Leuconostoc lactis &lt; RTI ID = 0.0 &gt; lactis subsp. lactis , Lactobacillus &lt; RTI ID = 0.0 &gt; casei , Lactobacillus sakei ), and Lactobacillus acidophilus ( Lactobacillus acidophilus), Lactobacillus spp delbu rwiki Bulgaria kusu (Lactobacillus delbrueckii subsp. bulgalicus , Streptococcus thermophilus , etc. Generally, it shows low productivity compared with homopolysaccharide, and the production of viscous product is temporary.

Lactic acid bacteria and other microorganisms produce not only the physical properties of foods but also physiological functional materials. Lactobacillus delbrueckii Lactobacillus and Lactobacillus HERBERTICUS strain Lactobacillus ( Lactobacillus sp. helverticus jugurti have been reported to have anticancer effects, and the secretions produced from Streptococcus have immunological effects, anti-ulcer effects and cholesterol-lowering activity.

Some microorganisms are biosynthesizing extracellular polysaccharides (EPS), which also contribute to the texture and the texture of the product. EPS is a microbial polysaccharide that forms part of the cell wall around the cell wall or accumulates as a slime form outside the cell wall during fermentation, and is a primary or secondary metabolite. In particular, the lactic acid bacteria producing EPS, which is a major problem in lactic acid fermented milk products, alleviates the phenomenon of whey separation, increases the viscosity and softens the tissues to give fullness and texture to the mouth during ingestion.

EPS produced by microorganisms is not used as an energy source but acts to protect itself from external environments such as drying, phagocytosis, protozoan wetting, antibiotics, toxic compounds, and osmotic stress. In addition, EPS is a long chain polymer polymer that dissolves or disperses in water, and plays an important role in thickeners and gelation in processed food products.

EPS is located outside the cell wall and is adsorbed on the cell wall in the form of a capillary membrane or secreted into the extracellular environment as a slime form, which increases the viscosity of the milk, inhibits syneresis and graininess, It is widely used as a stabilizer substitute for fermented milk because it plays an important role in fluidization and organization of fermented milk by giving natural turbidity. It is also known that the EPS isolated from the culture broth of lactic acid bacteria has anticancer activity.

EPS is a polysaccharide produced by the largest amount of microorganisms from a practical point of view, and has a very high commercial potential because it is easily recovered from a culture medium and has a low purification cost. It has been previously reported that EPS produced by lactic acid bacteria has different types and ratios of constituent sugars depending on the species.

The production of EPS in microorganisms is important for chemical fermentation conditions (medium conditions, initial lactose content, carbon and nitrogen ratio) and physical fermentation conditions (temperature, pH, oxygen partial pressure).

Korean Patent Laid-Open Publication No. 10-2011-0122048 discloses a Bacillus licheniformis strain having an ability to produce an extracellular polysaccharide from kimchi.

 It is an object of the present invention to find a polysaccharide component derived from a microorganism having an immunity-enhancing activity capable of enhancing a biodefense mechanism of a living entity by utilizing microbial resources derived from traditional foods such as kimchi, In order to develop a technique capable of increasing the number of cells.

In the present invention, the polysaccharide high productivity microorganism strains isolated from kimchi were screened and identified, and their productivity and methods of enhancing their productivity and their constituents were analyzed to examine the optimum culture conditions for polysaccharide production of each strain and their applicability. The characteristics of polysaccharide and polysaccharide production were investigated. As a result, they found a novel Pediococcus pentosaceus strain capable of producing a large amount of EPS and completed the present invention.

The novel Pediococcus pentosaceus KFT-18 of the present invention has a property of mass-producing EPS, and the EPS produced has excellent immunity-enhancing activity. Thus, the mass-produced EPS may be utilized as a variety of pharmaceuticals or food compositions containing it.

FIG. 1 shows colonies of microorganism strains cultured in a solid medium into 18 types.
FIGS. 2 to 8 show strains and utilization sugars in the colonies classified in FIG. 1.
9 to 24 show the results of viscosity measurement for the 16 strains selected.
Figure 25 shows a standard calibration curve for total quantitative analysis.
Figs. 26 to 33 show the sugar content analysis results of the microbial culture broth.
FIG. 34 shows the results of measurement of nitric oxide production amount of EPS produced using 6 kinds of Kimchi isolates selected according to the present invention.
Figure 35 is a flow diagram illustrating a phylogenetic detection approach for the comparison of homology between 16S rRNA sequences.

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 present invention provides a novel strain Pediococcus pentosaceus KFT-18 ( Pediococcus pentosaceus KFT-18, Accession No. KCCM 11309P).

After isolating a number of microbial strains from various kimchi, the present inventors selected optimal microorganisms by comprehensively evaluating the amount of EPS produced and the amount of NO generated by the produced EPS, etc., and identified the selected microorganisms to obtain Pediococcus pen It was confirmed to be Tosa Suse. This strain was named as KFT-18 strain and deposited with KCCM 11309P on Oct. 17, 2012 at the Korean Society for Microbiological Conservation.

In another aspect, the present invention relates to a method for mass-producing extracellular polysaccharides (EPS) using Pediococcus pentosaceus KFT-18 and a composition containing EPS produced thereby.

In one embodiment, the composition may be a pharmaceutical composition for treating or preventing an immune-related disease. The immune related disease may be, but is not limited to, an infection, an inflammation, or an autoimmune disease. In addition, the composition may be a health functional food for enhancing immunity.

NO is known to be a multifunctional mediator that is produced by various cells and acts on these cells and is involved in the destruction of inflammatory and autoimmune mediated tissues. These NOs are produced in vivo by nitric oxide synthase (NOS).

The pharmaceutical composition according to the present invention may be formulated into oral compositions such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, external preparations, suppository syringes, pre-filled syringes ) Solution form, or a lyophilized form, but the present invention is not limited thereto.

In the case of formulation, it may be prepared by using diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, surfactants and the like which are generally used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one or more excipients such as starch, calcium carbonate, sucrose, lactose, gelatin, . In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups, and the like. Various excipients such as wetting agents, sweetening agents, fragrances, preservatives and the like are included in addition to water and liquid paraffin which are conventionally used simple diluents . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, and the like.

The preferred dosage of the pharmaceutical composition of the present invention can be appropriately selected depending on the condition and body weight of the patient, the degree of obesity, the type of disease, the drug form, the administration route and the period.

In one embodiment of the present invention, the health functional food of the present invention may further contain at least one additive that is acceptable for food. The at least one additive that is acceptable for foodstuffs is not particularly limited and may contain conventional ingredients that can be incorporated into foods, such as water, flavor, flavor, colorant, stabilizer, preservative, alcohol, Vitamins, plant extracts, organic acids, minerals, nutrients, and the like.

The form of the health functional food of the present invention is not particularly limited, but may be any form selected from the group consisting of beverage, gum, tea, confection, vitamin complex, and health supplement.

In another embodiment, the composition may be a cosmetic composition. The cosmetic composition of the present invention may further contain at least one cosmetically acceptable additive. The at least one additive that is acceptable for cosmetics is not particularly limited and may be selected from conventional ingredients incorporated in common cosmetics such as oil, water, a surfactant, a moisturizer, an alcohol, a thickener, a colorant, an antiseptic, a flavor, a chelating agent, , Sunscreen agents, fatty acids, and the like.

The cosmetic composition of the present invention may be any one selected from the group consisting of a skin, a lotion, a cream, a gel, an essence, a foundation, a lotion, a patch, a pack, a soap, a cleanser and a detergent without particular limitation.

The present invention will be described in more detail with reference to the following examples. However, the following examples are for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention.

[Example]

Selection of strains and EPS  Production amount measurement

Selection of strains

99 strains were isolated from kimchi and named KFT-01 to KFT-99. Unidentified lactic acid bacteria, Enterococcus nose kusu (Enterococcus), Lactobacillus nose kusu (Lactococcus), flow Pocono stock (Leuconostoc), Phedi OKO kusu (Pediococcus), was the like by Sela (Weissella) (Table 1 to Table 4).

The saccharides added to the solid medium were prepared by mixing the monosaccharides arabinose, fructose, glucose, mannose, rhamnose and ribose and the disaccharides lactose, maltose, sucrose and the like, using the constituent sugar of API kit 50 CHL and the Bergey's manual And trehalose, trisaccharide raffinose were used. A total of 16 sugar alcohols such as erythritol, maltitol, mannitol, sorbitol and xylitol were used. Based on what is specified in the API kit 50 CHL, they are selected based on sugars that use more than 50% sugar as a substrate and sugar used as a substrate more than 90% by the Bergey manual (Table 5 and Table 6).

99 kinds of microorganisms stored at -70 DEG C were subcultured twice to MRS broth (Merck) and used for the experiment. The production of polysaccharides of lactic acid bacteria was investigated by colony forming by inoculation of lactic acid bacteria after solid medium was prepared by substituting sugar components based on the constituents of MRS agar (Merck) as shown in Table 7 below.

First, the components of MRS (de Man Rogosa and Sharpe, complex medium) agar except for sugar were dissolved in distilled water and sterilized by high pressure steam at 121 ° C for 15 minutes. The sugar was dissolved in each sterilized distilled water and sterilized with a 0.45 μm filter Filtered and added to the MRS agar solution to a final concentration of 2%. After the medium solution was stirred well, the medium was stuck on a sterilized agar plate to prepare a medium. Microorganism inoculation was carried out in a sterilized wooden rod having a diameter of 2 mm, soaked in the culture medium, and the medium was incubated at 30 ° C for 48 hours .

Colony morphology was classified into 18 categories according to color, morphology, transparency, ring formation and degree of viscosity (Fig. 1). Microorganisms made by the microorganisms are not only in liquid form but also in dry crystalline form. They distinguish between strains and sugars by distinguishing stickiness, wetness and flow. As a result of the morphological characteristics of the colonies, the dry crystalline form was in the form of 5, 6, 11 and 15, and the colonies in the flowing form were 14, 16 and 17.

In microorganisms, EPS is mainly produced by using low molecular weight sugars such as glucose or lactose, which may vary depending on the components of the growth medium. MRS, which is mainly used for culturing lactic acid bacteria, has been reported to be affected not only by glucose but also by the addition of emulsifiers such as tweens and complex saccharides such as beef extract, peptone and yeast extract Stacy A. Kimmel. Et al, 1998). In this example, the type of sugar produced by the EPS was investigated by first examining the EPS and the cluster type generated according to the sugar group using the MRS component as a control group.

EPS production in the solid medium is divided into three types, non-slime form, dry crystalline form and liquid slime form depending on the colony type, and the colony size Divided by 4 mm or less (+). 4 to 6 mm (++) and more than 6 mm (+++).

The strains and sugars which had a transparent colloidal shape and flowed on the surface were selected. When the maltose, mannose, ribose, raffinose and trehalose were used, coagulated transparent ring-forming EPS was produced. In addition, strains were produced when arabinose, lactose and glucose were used.

FIG. 2 shows the strain showing the generation of the coagulated transparent ring corresponding to No. 5 in the classification of FIG. 1, FIG. 3 shows the strain showing the coagulated transparent ring production corresponding to No. 6 of the classification shown in FIG. 1, FIG. 4 shows the strain showing the production of the coagulated transparent ring corresponding to No. 11 in the classification of FIG. 1, and FIG. 5 shows the strain showing the coagulated transparent ring production corresponding to No. 15 of the classification shown in FIG.

A large amount of EPS was used to select strains and sugars having a size of not less than 6 mm and a colony morphology of not more than 6 mm. In the case of sugar, a liquid form was formed when sucrose was used. ConnoStock, Pediococcus, and Bacela, and some Lactobacillus were selected.

FIG. 6 is a view showing a strain showing a flow-like colony formation corresponding to 14 of the classification shown in FIG. 1, FIG. 7 is a view showing a flow-forming colony corresponding to 16 of the classification shown in FIG. 1, FIG. 8 shows a strain showing a flow-like colony formation corresponding to No. 17 in the classification of FIG. 1. FIG.

Sixteen strains and 12 sugars were selected which produced viscous products in the solid medium or exhibited specific colony morphology (Table 8).

Viscosity of medium

After confirming the shape and size of the colonies in the solid medium, the strains which were more than 4 mm in size and which used EPS as a substrate were selected. MRS broth (manufactured by Merck) was added to all liquids except for glucose to prepare a liquid medium. Then, each sugar solution was added by filtration sterilization so as to be 2% of the total amount (Table 9).

0.5% of the microbial solution was added to the completed liquid medium and cultured at 30 캜. The culture solution was placed in a tube of 30 mL every 0 hours, 12 hours, and 24 hours, and the viscosity (cP) was measured with a viscometer (LVDV-II manufactured by Brookfield). The viscosity was measured by spindle No. 1 at 25 ℃. 1 for 15 seconds at 100 rpm.

Sixteen kinds of microorganisms selected in the prepared liquid medium were inoculated, and the liquid medium was collected every 12 hours while culturing at 30 DEG C for 48 hours, and the viscosity was measured. Each liquid medium was dispensed into a sterile 50 mL Falcon tube (30 mL each), and then measured at a temperature of 26 ± 1 ° C for 15 seconds at 100 rpm. The results of the viscosity analysis of the selected 16 strains are shown in Figs. 9 to 24.

EPS Generation  Measure

The polysaccharide produced in the culture broth was recovered and the amount thereof was measured using 30 mL of each microorganism culture as a sample. Polysaccharide precipitation was done by ethanol precipitation method. After centrifugation (J-251, manufactured by Beckman) for 15 minutes at 12,000 xg, the precipitate was removed, and the supernatant obtained by separation was taken and added with 2 volumes of 95% ethanol (manufactured by Daejung Co.) And then precipitated at 4 ° C for 24 hours. The precipitated polysaccharide was centrifuged at 12,000 xg for 15 minutes to remove the supernatant, and the recovered polysaccharide was recovered and diluted with distilled water (DW) of the same volume as the initial culture solution.

The yield of polysaccharides recovered was determined by phenol-sulfuric acid method (Dubois et al., 1956). First, 150 μl of the test solution was diluted, 150 μl of the diluted solution was added, and an equal amount of 5% phenol solution was added. 750 μl of concentrated sulfuric acid (96% concentration) was added to the solution and reacted at room temperature for 30 minutes and absorbance was measured at 470 nm . The standard calibration curve for total quantitative analysis is shown in Fig. 25, and the sugar content analysis of the culture solution of microorganism is shown in Fig. 26 to Fig.

As a result of measuring the amount of glutathione produced in the culture, it was confirmed that the KFT-09 strain culture medium had a high EPS-derived sugar content after culturing when lactose, rhamnose and sucrose were used as substrates. KFT-15 strains were found to be superior in EPS-derived sugar production when sucrose was used as a substrate (Fig. 26).

In the case of KFT-16 strain, the amount of sugar production was relatively low as compared with that of other strains, and it was confirmed that the production amount of EPS-derived sugar was active when rhamnose and sucrose were used as substrates. In case of KFT-18, EPS production was highest when sucrose was used as a substrate (FIG. 27).

In the case of KFT-19 culture, the production of EPS-derived sugars was highest in the case of rhamnose and sucrose as a substrate. In the case of KFT-22, the content of lactose, rhamnose and sucrose was higher than that of EPS, (Fig. 28).

KFT-30 and KFT-51 showed no significant increase in EPS-derived glucose production. In KFT-30, mannitol, raffinose and sorbitol, KFT-51 was found to have a high EPS-derived sugar content in rhamnose (FIG. 29).

KFT-63 also did not have a significantly higher EPS-derived glucose-containing culture. However, in the case of KFT-69, it was confirmed that the EPS-producing sugar-producing ability was very high in sucrose (FIG. 30).

In the case of KFT-10, the EPS-derived saccharide content was high when raffinose, rhamnose, ribose and sucrose were used as substrates, and KFT-84 had low EPS-derived saccharide content in all culture media (FIG.

In the case of KFT-87 and KFT-88, the EPS-derived saccharide content was not highly measured, but the EPS-derived saccharide content was measured to be high when Ramanose was used as a substrate (FIG. 32).

In KFT-90, EPS-derived saccharide content was measured to be high when lactose, rhamnose and sucrose were used as substrates. KFT-95 showed low EPS-derived saccharide content without significant difference according to sugar type (FIG.

KFT-09, KFT-15, KFT-16, KFT-18, KFT-19 and KFT-22 strains were isolated from kimchi. These strains were inoculated to 100% MRS broth at 1% in the activated state, cultured at 30 ° C for 24 hours, and centrifuged to obtain a viscous substance. Lactose, maltose, rhamnose and sucrose were used as the substrates, and the amount of the lactose, maltose, rhamnose and sucrose was determined by phenol-sulfuric acid analysis.

Specifically, the strain was activated and each strain was cultured in 5 mL of sterilized MRS broth (Merck) in 100 mL portions for 24 hours. In addition, sugar was replaced with 2% (w / w) of lactose, maltose, rhamnose and sucrose instead of glucose based on the constituents of MRS. MRS broth and 0.5% (v / v) of the strain solution cultured in the substituted MRS medium were inoculated and cultured at 30 ° C for 24 hours. After centrifugation at 10,000 rpm for 15 minutes, the cells were removed, and the same amount of ethanol was added to the culture solution, followed by precipitation at 4 ° C for 24 hours. The precipitate was collected by centrifugation at 11,000 rpm for 20 minutes, dissolved in 1 mL of distilled water (DW), diluted to 2 mL and stored at -20 ° C. The cultures were also diluted to 2 mL and stored in vials. Quantitative analysis of EPS was carried out by the phenol-sulfuric acid method. After measuring the OD value at 470 nm, the EPS content was calculated using a calibration curve.

The polysaccharide production (mg / mL) of the six Kimchi-derived strains is shown in Table 10 below.

Most of kimchi-derived microorganisms were found to produce a large amount of slime-type slime when added with sucrose, and polysaccharide production was mainly confirmed in maltose, raffinose, trehalose, rhamnose and lactose addition medium in addition to sucrose .

The content (mg / mL) of 6 kinds of strains per EPS is shown in Table 11 below. Polysaccharide production was highest in sucrose and sugar alcohol content was relatively low. Maltose, rhamnose, sucrose, KFT-15, and KFT-16 strains were selected for the strains that produced more than 30 mg / mL, and KFT-16 strain was sucrose, The strains KFT-18 and KFT-19 were identified as lactose, rhamnose and sucrose, respectively, and KFT-22 as mannitol, raffinose and sorbitol. Based on the constituents of MRS, it was confirmed that KFT-18 was cultured in the presence of 2% (w / w) sucrose instead of glucose to produce the largest amount of EPS at 117.39 mg / mL.

NO (Nitric oxide) production measurement

NO production in the culture was evaluated by measuring nitrite, which is an oxide of NO. Raw 264.7 cells were seeded at a concentration of 2 x 10 ⁵ cells / mL in a 96-well plate and cultured for 24 hours. Then, 500 μg / mL of Raw 264.7 cells were seeded at 50 μg / mL And then cultured for 20 hours. After the incubation, 100 μL of the supernatant was transferred to a new 96-well plate, mixed with Griess reagent (Sigma-Aldrich) at a ratio of 1: 1, reacted for 10 minutes in a shaker, Absorbance was measured at 540 nm. The standard curve was measured using sodium nitrite. The results are shown in Table 12 and FIG. Interferon-γ (IFN-γ) and lipopolysaccharide (LPS) were used as control.

As a result of observing the NO production ability of Raw 264.7 cells by lyophilization of the EPS of strains cultured with maltose, sucrose, rhamnose, and lactose, which were active in EPS production ability, 4 strains of 6 strains produced maltose as a carbon source EPS showed a NO production ability of about 70% compared with the control (LPS) level. Among them, EPS of KFT-18 was 67.3 ± 5.3%, which showed the highest NO production inducing ability among samples.

Identification of the strain

Eight species of Kimchi isolates stored at -70 DEG C were subcultured twice in TS broth (Merck), fully activated, and cultured at 30 DEG C for 24 hours. The results obtained are shown in Table 13 below.

The positions of the 8 strains were identified through a phylogenetic detection approach for comparison of homology between 16S rRNA sequences (Fig. 35).

Korea Microorganism Conservation Center KCCM11309P 20121017

Claims (6)

As a strain of Pediococcus pentosaceus KFT-18, which has the ability to produce extracellular polysaccharide (EPS), KFTM11309P, KFT-18,
A strain that forms colonies that produce extracellular polysaccharides only in solid medium containing rhamnose or sucrose per substrate. Culturing the Pediococcus pentosaceus KFT-18 of claim 1 at 30 占 폚 in a medium wherein glucose is replaced with sucrose in the MRS medium to prepare a strain solution;
Inoculating 0.5% (v / v) of the cultured strain solution to a culture medium containing 2% (w / w) sucrose and culturing at 30 ° C; And
A method for producing an exopolysaccharide (EPS) using Pediococcus pentosaceus KFT-18 of Accession No. KCCM 11309P, comprising the step of removing the strain from the culture and recovering and purifying the polysaccharide. The method according to claim 2, wherein the extracellular polysaccharide is a polysaccharide accumulated around the cell wall of Pediococcus pentosaceus KFT-18 of claim 1 during fermentation. delete An immunocompromising health functional food comprising an extracellular polysaccharide produced by the method of claim 2. A cosmetic composition comprising an extracellular polysaccharide produced according to the method of claim 2.

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