KR101181269B1 - Exopolysaccharides with antioxidant and antiaging activity produced by Bacillus sp. strains isolated from kimchi, a fermented korean food and the method for manufacturing the same - Google Patents

Abstract

The present invention Bacillus licheniformis KS-17 ( Bcillus licheniformis KS-17) and B. licheniformis KS-20 ( B. licheniformis KS-20) strain having an extracellular polysaccharide-producing ability from kimchi, antioxidants produced therefrom And an extracellular polysaccharide having an anti-aging ability and a method for producing the same. The extracellular polysaccharides produced under optimal culture conditions from the Bacillus rickenomyces strain of the present invention have excellent antioxidant effects and can be usefully used in the food and cosmetic industries.

Description

Extracellular polysaccharide with antioxidant and anti-aging activity produced by Bacillus sp. Isolated from Kimchi and its production method {Exopolysaccharides with antioxidant and antiaging activity produced by Bacillus sp. strains isolated from kimchi, a fermented korean food and the method for manufacturing the same}

The present invention is isolated from Kimchi Bacillus licheniformis KS-17 ( Bacillus licheniformis KS-17), Bacillus licheniformis KS-20 strain to produce extracellular polysaccharide (exopolysaccharide, EPS) And it relates to a method for producing extracellular polysaccharides having antioxidant activity produced by the strains.

Currently, the antioxidants mainly used in the food industry, such as BHA (Butylated hydroxyanisole), BHT (Butylated hydroxytoluene), PG (Propyl gallate), TBHQ (Tertiary butylhydroquinone) is synthesized using the antioxidant. However, there is a need for a new eco-friendly production method due to problems such as cytotoxicity (Valentao P et al., 2002), carcinogenicity, and hypertrophy between high cities (Lee KH et al., 2006). As such, the development of natural antioxidants is a very important industrial task. Currently, natural plants and microorganisms are used as natural antioxidants. In particular, studies on the antioxidant effect of polysaccharides obtained as metabolites of microorganisms such as fungi and lactic acid bacteria such as mushrooms have been made (Cui Y et al., 2005). Meanwhile, metabolites such as exopolysaccharides (EPS) produced by food-derived microorganisms, such as Lactic acid bacteria (LAB), Propionibacteria, and bifidobacteria, are considered GRAS (generally regarded as safe).

Primary or secondary metabolites of microorganisms, which form part of the microbial cell wall around the cell wall or accumulate during fermentation in the form of a viscous form outside the cell wall, are called extracellular polysaccharides (exopolysaccharides (EPS)) (Kim DJ et al., 2001). Polysaccharides produced from microorganisms are long-chain and high-molecular-mass polymers with various functions such as texturizers, thickeners, emulsifiers, and film-forming ability. It has been used for a long time in the food industry, and has recently been used for blood sugar control effect (Cho EJ et al., 2007), sunscreen effect (Bae JT et al., 2005), antioxidant effect (Asker MMS et al., 2009; Wang J et al., 2007), antimicrobial and immune-enhancing effects (Kolodzieja H et al., 2007) have been reported to be of great importance as a functional material that can be used in a wide range of applications, including the food and pharmaceutical industries. Increasing (Shivakumar S et al., 2006). In addition, it is reported that the extracellular polysaccharides produced by microorganisms can greatly increase the productivity by improving the composition of the medium and culture conditions (Vijayendra SVN et al., 2008).

These functional polysaccharides are known to be mainly produced in fungi, such as mushrooms, and beta glucan (β-glucan) produced by higher mycelial mushrooms, such as chaga ( Inonotus obliquus ), such as Beta glucan is known to have anti-cancer (Cha JY et al., 2004), antioxidant (Cui Y et al., 2005), anti-hyperlipidemia and glycemic control (Kim MA et al., 2009) from recent studies. It is getting great attention. However, fungi, such as mushrooms, have slower growth rates than bacteria and are difficult to cultivate in large quantities due to their culture characteristics.

On the other hand, studies on extracellular polysaccharides produced from strains isolated from food, especially Lactobacillus sp., Streptococcus sp., Lactococcus sp. There are extracellular polysaccharides produced from lactic acid bacteria such as Leuconostoc sp. And Weissella sp. Isolated from kimchi, but the number of extracellular polysaccharides produced by each microorganism is small. It is known to be very small and has a very difficult problem for industrial use (Vuyst LD et al., 2001; Kim BJ et al., 2001; Kim HJ et al., 2006; Kim MJ et al. , 2008). Therefore, it is necessary to find a method for optimizing culture conditions for polysaccharide production so as to discover a strain that produces extracellular polysaccharides having high functionality and to produce a large amount in a simpler way.

Kimchi, a traditional fermented food, has a high salt concentration unlike dairy products (Park SJ ,. 2001), and is affected by subsidiary materials such as red pepper powder, garlic, and ginger (Lee SH et al., 2005). It is difficult to grow microorganisms (Kim JG et al., 2005). From this point of view, the strain isolated from kimchi may have the advantages of being able to withstand in a relatively extreme environment. To isolate the strain producing high.

To date, only a few previous studies have isolated microorganisms from kimchi, and no previous studies have isolated Bacillus sp. In addition, there are no specific results on the antioxidant and anti-aging effects of exopolysaccharides produced from Bacillus rickenomyces isolated from Kimchi and the conditions for mass production thereof. Therefore, if functional and productivity such as antioxidant and anti-aging of polysaccharides having safety are secured, it is expected to have advantages as a functional material that can be directly applied to the food industry.

Therefore, the present inventors have studied intensively to search for microorganisms capable of producing new extracellular polysaccharides different from conventional exopolysaccharides (EPS). Bacillus sp. Strains that produce polysaccharides were found, and in particular, polysaccharides produced from the strains exhibited antioxidant and anti-aging characteristics, and the mass production process using them was established to complete the present invention.

Therefore, an object of the present invention is to provide a Bacillus licheniformis strain isolated from kimchi.

Another object of the present invention is to provide an antioxidant or anti-aging composition.

Another object of the present invention to provide an extracellular polysaccharide production method having an antioxidant function.

In order to solve the above problems, the present invention is separated from kimchi, Bacillus rickenformis KS-17 ( Bacillus) having accession number KACC91570P licheniformis KS-17) strain is provided.

In addition, the present invention is separated from kimchi, Bacillus rickeniformis KS-20 ( Bacillus) having accession number KACC91571P licheniformis KS-20) strain.

The present invention also provides an extracellular polysaccharide produced from the strain of Bacillus rickeniformis KS-17 or Bacillus rickeniformis KS-20. The extracellular polysaccharide is composed of glucose, KS-17 is characterized in that the average molecular weight is 1.40 to 1.50 kDa, KS-20 is preferably characterized in that the average molecular weight is 1.50 to 1.60 kDa. In addition, the extracellular polysaccharide is cultured in a medium containing sucrose (tryuctone), tryptone, yeast extract (yeast extract), dipotassium phosphate (dipotassium phosphate) and diammonium citrate It is preferable to obtain.

The present invention also provides an antioxidant or anti-aging composition comprising an extracellular polysaccharide produced from KS-17 or KS-20 as an active ingredient.

In another aspect, the present invention provides an antioxidant or anti-aging food comprising an extracellular polysaccharide produced from the KS-17 or KS-20 as an active ingredient.

In another aspect, the present invention provides an antioxidant or anti-aging cosmetic composition comprising an extracellular polysaccharide produced from KS-17 or KS-20 as an active ingredient.

In another aspect, the present invention provides a method for producing or recovering the extracellular polysaccharide produced from the KS-17 or KS-20. More specifically, extracellular polysaccharides produced from KS-17 include sucrose, tryptone, yeast extract, dipotassium phosphate, and diammonium citrate. After inoculating Bacillus rickeniformis KS-17 strain to the medium containing the extracellular polysaccharide can be produced or recovered from the culture medium incubated at 36.5 ℃ to 37.5 ℃ conditions at an initial pH of 6.5 to 7.5. In the medium, sucrose is 90 to 110 g / L, tryptone is 8 to 12 g / L, yeast extract is 4 to 6 g / L, dipotassium phosphate is 4 to 6 g / L, diammonium citric acid is 4 to 6 g / It is preferable that it is L.

In addition, extracellular polysaccharides produced from KS-20 were added to medium containing sucrose, tryptone, yeast extract, dipotassium phosphate, and diammonium citrate. After inoculation of Bacillus rickenomyces KS-20 strain, extracellular polysaccharides can be produced or recovered from a culture medium cultured at 36.5 ° C. to 37.5 ° C. at an initial pH of 5.5 to 6.5. In the medium, sucrose 65-85g / L, tryptone 8-12g / L, yeast extract 4-6g / L, dipotassium phosphate 4-6g / L, diammonium citrate 4-6g / L Is preferably.

As described above, Bacillus licheniformis KS-17 and Bacillus licheniformis KS-20 ( Bacillus licheniformis KS-20) produced from the new strain isolated from Kimchi according to the present invention Extracellular polysaccharides having high antioxidant capacity or antiaging capacity are natural high functional polysaccharides that are safe for human body, and are manufactured according to the optimum medium and culture conditions of the present invention. It can be usefully used as a functional material.

Figure 1 is a photograph of Bacillus rickeniformis KS-17 and KS-20 cultured in a medium containing sucrose (Sucrose).
Figure 2 is a molecular biological phylogenetic diagram of Bacillus rickeniformis KS-17 strain.
3 is a molecular biological phylogenetic diagram of Bacillus rickeniformis KS-20 strain.
Figure 4 is a graph showing the molecular weight of the extracellular polysaccharides (EPS KS-17 and KS-20) produced from Bacillus rickeniformis KS-17 and KS-20 strain.
Figure 5 shows the analysis of monosaccharide components by TLC of the extracellular polysaccharides KS-17 and KS-20.
Figure 6 shows the analysis of monosaccharide components by HPLC-ELSD analysis of extracellular polysaccharides KS-17 and KS-20.
Figure 7 shows the structural analysis by FT-IR spectra of the extracellular polysaccharides KS-17 and KS-20.
8 is a graph showing the specific rate of the extracellular polysaccharides per cell according to the sucrose concentration of the extracellular polysaccharides KS-17 and KS-20.
9 is a graph showing the specific rate of extracellular polysaccharides per cell according to the initial pH of the extracellular polysaccharides KS-17 and KS-20.
10 is a graph showing the specific rate of the extracellular polysaccharides per cell according to the culture temperature of the extracellular polysaccharides KS-17 and KS-20.
11 is a graph showing the specific rate of extracellular polysaccharides per cell according to the culture time of the extracellular polysaccharides KS-17 and KS-20.
12 is a graph showing the effect on the electron donating ability of the extracellular polysaccharides KS-17 and KS-20.
Figure 13 is a graph showing the antioxidant capacity against the SOD-like activity of the extracellular polysaccharides KS-17 and KS-20.
14 is a graph showing the antioxidant capacity of tyrosinase inhibitory activity of the extracellular polysaccharides KS-17 and KS-20.

The present invention relates to Bacillus licheniformis KS-17, KS-20 strain isolated from kimchi.

The strain is a strain that produces microbial extracellular polysaccharides from kimchi, purely isolated and identified by 16S rDNA sequencing and biochemical characterization, it was identified as belonging to the genus Bacillus sp. Bacillus licheniformis KS-17 ( Bacillus licheniformis KS-17) and Bacillus licheniformis KS-20 ( B. licheniformis KS-20).

In addition, the strains Bacillus rickenformis KS-17 and Bacillus rickenformis KS-20 are in accordance with the provisions of the Budapest Treaty on the International Approval of Microbial Deposits in Patent Procedures, and on July 28, 2010, Korean Agricultural Culture Collection, KACC91570P and KACC91571P.

In one embodiment of the present invention, the strain is cultured in a medium containing sucrose, tryptone, yeast extract, yeast extract, dipotassium phosphate, and diammonium citrate. To produce an extracellular polysaccharide having antioxidant function. The extracellular polysaccharide produced from Bacillus rickenformis KS-17 consists of glucose, the average molecular weight was 1.45 kDa, and the extracellular polysaccharide produced from Bacillus liqueniformis KS-20 consists of glucose, The average molecular weight was 1.55 kDa.

In another aspect, the present invention provides a composition and culture conditions of the medium having the highest antioxidant or anti-aging function in the method for producing extracellular polysaccharides from the strains.

In the present invention, the medium refers to a solid medium or a liquid medium containing nutrients necessary for growing animal cells, plant cells or bacteria, and the culture medium means cultured by inoculating a strain into a liquid medium.

Inoculated with Bacillus rickenomyces KS-17 strain in a medium containing sucrose, tryptone, yeast extract, dipotassium phosphate and diammonium citrate After that, the extracellular polysaccharide may be produced from the culture medium incubated at 36.5 ° C. to 37.5 ° C. at an initial pH of 6.5 to 7.5. Preferably, the sucrose is 90 to 110g / L, tryptone is 8 to 12g / L, yeast extract is 4 to 6g / L, dipotassium phosphate is 4 to 6g / L, diammonium citric acid is 4 to 6g / L, the initial pH is 7, the temperature is 37 ℃.

In addition, Bacillus rickenomyces KS-20 strain was added to a medium containing sucrose, tryptone, yeast extract, dipotassium phosphate, and diammonium citrate. After inoculation, extracellular polysaccharides can be produced from the culture cultured at 36.5 ° C. to 37.5 ° C. at an initial pH of 5.5 to 6.5. Preferably, the sucrose is 65 to 85 g / L, tryptone is 8 to 12 g / L, yeast extract is 4 to 6 g / L, dipotassium phosphate is 4 to 6 g / L, diammonium citric acid is 4 to 6 g / L, initial pH is 6, temperature is 37 degreeC.

The culture solution may be a culture filtrate, which comprises a strain or cultured by inoculating the strain, and then removing the bacteria. The concentrate of the culture medium refers to the concentration of the culture solution, and the dried product of the culture solution means that the water of the culture solution is removed.

The extracellular polysaccharide of the present invention produced by the above method has an antioxidant or anti-aging function. In one embodiment of the present invention, the electron donating ability, the superradical radical (superoxide radical) scavenging ability and tyrosinase according to the DPPH radical scavenging ability of the extracellular polysaccharides produced from Bacillus rickeniformis KS-17 and Bacillus rickeniformis KS-20 Tyrosinase) inhibitory activity was measured, it was confirmed that it has a higher antioxidant activity than the control group β-glucan.

Accordingly, the present invention provides an antioxidant or anti-aging composition comprising extracellular polysaccharides produced from Bacillus rickeniformis KS-17 or Bacillus rickeniformis KS-20.

The composition may be a pharmaceutical composition and may be used as an antioxidant. The compositions of the present invention can be formulated and used in pharmaceutical unit dosage forms with the addition of pharmaceutically acceptable carriers, excipients or diluents.

The compositions of the present invention may be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral dosage forms, external preparations, suppositories, and sterile injectable solutions, respectively, according to a conventional method. have.

The present invention also provides an antioxidant or anti-aging food comprising extracellular polysaccharides produced from Bacillus rickeniformis KS-17 or Bacillus rickeniformis KS-20.

The food of the present invention means a natural product or processed product containing one or more nutrients, and preferably means a state in which it can be directly eaten through some processing process, for example, fruits, vegetables, and fruits. Dried or cut products of fruits, vegetables, fruit juices, vegetable juices, mixed juices or chips, noodles, livestock processed foods, fish processed foods, dairy foods, fermented foods, legumes foods, cereals, microbial fermented foods, confectionery , Seasonings, processed meats, acidic beverages, licorice, herbs, etc., but is not limited thereto.

The food may be, for example, a health functional food, fermented food or a beverage.

The health functional food means a food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. using raw materials or ingredients having useful functions for the human body. Functionality refers to obtaining useful effects on health use such as nutrient control or physiological action on the structure and function of the human body.

Food containing the extracellular polysaccharide of the present invention has the advantage that there is no side effect that may occur when taking a long-term use of the drug as a raw material, unlike the general medicine, there is an excellent portability.

In the present invention, the drink refers to a generic term for drinking to quench thirst or enjoy the taste, and includes alcoholic beverages, soft drinks, water, syrup, tea, coffee, fruit drinks, and the like, and include lactic acid bacteria drinks. The lactic acid bacteria drink refers to a beverage in which lactic acid bacteria are cultured and lactically fermented to add distilled water and dilute sugar, spices, and the like. The beverage is not particularly limited to other ingredients other than the extracellular polysaccharides produced from KS-17 or KS-20 as essential ingredients in the indicated ratios, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. Can be.

The present invention also provides an antioxidant or anti-aging cosmetic composition comprising an extracellular polysaccharide produced from Bacillus rickeniformis KS-17 or Bacillus rickeniformis KS-20.

The cosmetic composition according to the present invention may be formulated as an external preparation for skin or cosmetics. The external preparation for the skin may include an ointment, a warning agent, a spray, a suspension, an emulsion, a cream, a gel, and the like. Components included in the cosmetic composition of the present invention may include components conventionally used in cosmetic compositions in addition to the extracellular polysaccharides as an active ingredient, for example, such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavorings Phosphorus aids, and carriers. In addition, the cosmetic composition may further include a skin absorption promoting substance to enhance the effect.

Hereinafter, the present invention will be described in detail through examples. The following examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples according to the gist of the present invention to those skilled in the art. Will be self-evident.

Example 1 Isolation of Strains That Produce Extracellular Polysaccharides from Kimchi

In order to separate the EPS-producing strains from kimchi, the entire contents of the collected kimchi were crushed, and then diluted sequentially using a sterilized dilution solution 0.1% peptone water in a decimal method. Take 100 μl of each diluted sample and spread it on MRS (Difco Laboratories, MI, USA) agar medium (1.5% agar, w / v) to which 2% CaCO ₃ is added and incubate for 30 hours at 37 ° C. The strain was separated by. In order to investigate the EPS generation ability of the isolated strains, the colony of each isolate was inoculated in a solid medium adjusted to pH 7.0 by sucrose medium containing 1.5% agar (Table 1) for 30 hours at 37 ° C. Two strains were selected which were cultured and produced high EPS.

Altered Composition of Sucrose Media Component gram / Liter Sucrose 50.00 Tryptone 10.00 Yeast extract  5.00 Dipotassium phosphate  5.00 Diammonium citrate  5.00

Example 2 Strain Identification

Selected EPS producing strains were identified by 16S rDNA sequencing and biochemical characterization. Oligonucleotide primers used for sequencing 16S rDNA gene were universal primers proF (5'-AGA GTT TGA TCC TGG CTC AG-3 ') and 534r (5'-ATT ACC GCG GCT GCT GG-3'), 9Rev (5'-AAG GAG GTG ATC CAG CC-3 ') Three primers were used, PCR was performed using the PCR cycler Primus (MWG-BIOTECH) (Table 2). The sequencing was determined by ABI PRISM 3730 DNA analyzer and the analyzed sequencing showed homology results compared with ribosomal RNA gene sequencing registered in Genbank by DDBJ program.

PCT reaction condition Reaction mixture PCR condition 10 ㅧ Taq reaction buffer 5 μl 95 ℃, 2min
95 ℃, 1min ━━━┑
54 ℃, 30sec 30 cycle
72 ℃, 1min ━━━┙
72 ℃, 5min  dNTP mix (2.5mM each) 4 μl primer proF 0.5 μl primer 534r 0.5 μl template 15 μl Enzyme (2.5U / ul) 0.5 μl D.W up to 50 μl

In addition, the analysis of the biochemical properties of the selected EPS-producing isolates was examined using the VITEK (VITEK 2 System), the results are shown in Table 3.

Biochemical Analysis of Strains Using VITEK Strain Strain Biochemical details KS-17 KS-20 Biochemical details KS-17 KS-20 BXTL - - dMAN + + LysA - - dMNE + + AspA (-) - dMLZ + - LeuA + + NAG - - PheA + + PLE + + ProA - - IRHA - - BGAL + + BGLU + + PyrA + + BMAN - - AGAL - - PHC - - AlaA - - PVATE - - TyrA + + AGLU + + BNAG - - dTAG + + APPA + (-) dTRE + + CDEX + + INU - - dGAL - - dGLU + + GLYG - - dRIB - - INO - - PSCNa - - MdG + + NaCl6.5% + + ELLM + + KAN - - MdX - - OLD - - AMAN - - ESC + + MTE + + TTZ - - GlyA + + POLYB_R - -

Strain identification results from the above method are shown in FIGS. 2 and 3. As shown in FIG. 2, the isolated strain KS-17 showed 99% homology with Bacillus licheniformis , and biochemical analysis using VITEK showed 85% similarity with Bacillus rickeniformis. . As shown in FIG. 3, the isolated strain KS-20 had a homology of 99.1% with Bacillus rickeniformis, and showed 85% similarity with Bacillus rickeniformis using VITEK.

In other words, isolates KS-17 and KS-20, which produce exopolysaccharides isolated from kimchi, were identified as Bacillus licheniformis ( B, licheniformis ) as a result of 16S rDNA sequencing and biochemical analysis. 17 was named Bacillus rickeniformis KS-17 ( B. licheniformis KS-17) and KS-20 was named B. licheniformis KS-20 ( B. licheniformis KS-20).

Example 3 Extracellular Polysaccharide Purification

To produce EPS from the strain, 50 g / L sucrose, 10 g / L tryptone, 5 g / L yeast extract, 5 g / L dipotassium phosphate and diammonium citric acid (diammonium citrate) was incubated for 48 hours at 37 ℃, 150rpm using a medium prepared with 5g / L. After completion of the culture, the cell culture solution was inactivated by boiling water at 100 ° C. for 20 minutes in a water bath, and then trichloroacetic acid (trichloroacetic) was added to a final concentration of 4% in a culture solution at 4 ° C. to remove protein. acid) to remove the cells and precipitated protein by centrifugation (10,000rpm, 25 minutes, 4 ℃). The supernatant was extracted and precipitated overnight at 4 ° C. after adding and stirring 3 times ethanol (EtOH) at 4 ° C., and the precipitated EPS was recovered by centrifugation (10,000 rpm, 20 minutes, 4 ° C.), The remaining ethanol was removed using a vacuum concentrator (MG-2200, EYELA, Japan) and redissolved in tertiary distilled water and dialyzed (Mw cut off 12,000) for 24 hours at 4 ° C to remove free sugar from the culture. After freeze-drying it was used as a sample in the form of a powder.

Example 4 Characterization of Extracellular Polysaccharides

4-1. Molecular Weight Measurement

In order to measure the molecular weight of purified EPS KS-17 and KS-20, 1 ml of an aqueous solution of 1 mg / ml (w / v) dissolved in 0.5 M Phosphate buffer (pH 6.8) containing 0.15 M NaCl was added to sephadex G-. Gel filtration column chromatography using 200 as a standard, blue dextran 2000 (GE Healthcare) and gel filtration standard (thyroglobulin 670kDa, bovine gamma-globulin 158kDa, chicken ovalbumin 44kDa, equine myogolbin 17kDa, vitamin B ₁₂ 1.35 kDa) using the elution time ratio and the molecular weight is shown in Figure 4 the result. The molecular weight of KS-17 was 1.45 kDa and the molecular weight of KS-20 was about 1.55 kDa.

4-2. Confirmation per configuration

In order to investigate the constituent sugars of EPS KS-17 and KS-20, 10mg of EPS samples were hydrolyzed by phenol-sulfuric acid and analyzed by TLC and HPLC-ELSD. TLC analysis was performed using glucose, galactose, fructose and mannose as standard, and the silica gel TLC plate (Merck, TLC aluminum sheet, silica gel 60F ₂₅₄ ) was used. The standard and the sample were instilled in 1μl and developed twice with n-butanol / 2-propanol / water / acetic acid (7: 5: 4: 2, v / v / v / v), followed by drying, followed by ethanol / sulfuric acid / p-anisaladehyde (18: 1: 1, v / v / v) color sprinkling solution was confirmed by coloring for 1 minute at 120 ℃. As a result, as shown in FIG. 5, the main component of the reducing sugars constituting KS-17 and KS-20 was glucose. As a standard, high-performance liquid chromatography (HPLC-ELSD) was used as a standard. When using glucose, galactose, fructose, fructose, mannose and lactose, homopolysaccharides composed of glucose to form polymers were analyzed under the conditions of Table 4 Was determined to be. The other peak in KS-17 showed a different retention time than the monosaccharide used as a standard as an unknown material, but it is finally considered to be a DNA polymer extracted together during the extraction process. EPS produced by KS-20 was found to be a polysaccharide consisting of glucose.

HPLC-ELSD Analysis Conditions Instrument Waters 1515 Binary HPLC Pump Detector ELSD 800 (Alltech) Column Supelcosil ^TM LC-NH2, 5㎛, 4.6 ㅧ 250 mm
(GL Sciences Inc. Japan) Mobile phase water: MeCN = 25: 75 Drift tube Temp. 55 ° C Column Temp. room temp. Flow rate 1.0 mL / min Injection Vol. 10 μl

4-3. FT-IR Spectrum for Structural Analysis

EPS KS-17 and KS-20 from the infrared absorption spectrum for the structural analysis of the polysaccharide from specific absorption band of 3410 cm ^-1 of the polysaccharide in the high OH group, 2936 cm ^-1 in the CH group, 1645 cm ^-1 C = O stretching band (Ha JY et al., 2010 ), CH 2 band at ^{1456cm -1 (Ha JY et al.} , 2010) and 1127 cm ^-1, 1061 cm ^-1 and 1015 cm ^-1 are to exhibit a strong COC stretch band The results of the two polysaccharides were similar (FIG. 7).

Example 5 Optimal Culture Conditions for Extracellular Polysaccharides

To optimize the optimal culture conditions of EPS KS-17 and KS-20, sucrose 50g / L, tryptone 10g / L, yeast extract 5g / L, dipotassium phosphate 5g / L, diammonium citrate 5g / L Incubating for 48 hours at 37 ℃, 150rpm was the basic conditions (Table 1). In addition, the measurement of cell mass and EPS amount was recovered by centrifugation (10,000 rpm, 25 minutes, 4 ° C.) after the completion of culture to recover the precipitated cells, and the supernatant was trichloroacetic acid so that the final concentration was 4%. ), The protein was precipitated and centrifuged (10,000 rpm, 25 minutes, 4 ° C.) to the supernatant removed by 3 times of 4 ° C. ethanol and precipitated overnight at 4 ° C., followed by centrifugation (10,000 rpm, 25 minutes, 4 ° C.). Recovered by precipitation). The cells and EPS thus recovered were dried under reduced pressure and dried.

5-1. Impact of Carbon Sources

In order to investigate the cell mass and EPS amount according to the carbon source and the specific ratio (Specific rate = EPS production / biomass, w / w) under the preparation condition of the culture medium of the isolated strain, Lactose (fructose), galactose (galactose), glucose (glucose), lactose (lactose), sucrose (sucrose) was prepared by the ratio of 50g / L, respectively. As shown in Table 5, both Bacillus rickenformis KS-17 and Bacillus rickenformis KS-20 produced 6.44 ± 0.39 and 7.51 ± 2.03 g / L, respectively, using sucrose as the carbon source. The KS-17 produces 0.99 ± 0.05 g / L of glucose, which is about 6.5 times higher than the lowest production glucose, and the KS-20 produces 0.83 ± 0.63 g / L, which produces about 9 times the EPS. The maximum yield was shown.

Cell mass and EPS production according to carbone source Carbon
source B. licheniformis KS-17 B. licheniformis KS-20 Biomass
(g / L) EPS
(g / L) specific
rate of EPS Biomass
(g / L) EPS
(g / L) specific
rate of EPS fructose 3.39 ± 0.22 1.12 ± 0.11 0.35 ± 0.03 2.79 ± 0.22 1.43 ± 0.11 0.51 ± 0.06 galactose 2.32 ± 0.25 1.45 ± 0.18 0.63 ± 0.12 3.11 ± 0.22 0.83 ± 0.63 0.27 ± 0.21 glucose 2.69 ± 0.24 0.99 ± 0.50 0.36 ± 0.15 2.33 ± 0.29 1.63 ± 0.21 0.70 ± 0.04 lactose 2.23 ± 0.12 1.48 ± 0.40 0.66 ± 0.15 1.96 ± 0.03 1.16 ± 0.37 0.59 ± 0.18 sucrose 5.87 ± 0.76 6.44 ± 0.39 1.10 ± 0.09 5.01 ± 0.19 7.51 ± 2.03 1.51 ± 0.46

5-2. Influence of sucrose concentration

Cell weight and EPS amount according to the concentration of sucrose, which is the carbon source selected in Example 5-1, under the preparation conditions of the culture medium of the isolated strain, and the EPS specific ratio according to it (Specific rate = EPS production amount / w / w) In order to investigate the carbon source in the basic culture medium composition described above was confirmed by the preparation of each concentration of 0g, 25g, 50g, 75g 100g / L. As shown in FIG. 7, Bacillus rickenformis KS-17 exhibited a maximum EPS yield and specific rate of 11.6 ± 0.6 g / L when the sucrose concentration was 100 g / L. -20 was the maximum EPS yield of 10.6 ± 0.9g / L when the sucrose concentration was 75g / L (p <0.05).

5-3. Effect of Initial pH

Basic culture medium composition described above to investigate the cell mass and EPS amount according to the initial pH and the resulting EPS specific ratio (Specific rate = EPS production / cell mass, w / w) in the basic medium preparation conditions of the culture medium of the isolated strain The initial pH was determined by preparing 4.0, 5.0, 6.0, 7.0, and 8.0, respectively. As shown in FIG. 8, Bacillus rickenformis KS-17 exhibited maximum EPS production and specific rate at 10.7 ± 0.3 g / L when the initial pH was 7.0. When the pH was 6.0, the maximum EPS production and specific rate were 12.5 ± 0.8 g / L, while both strains did not grow at pH 4 (p <0.001).

5-4. Effect of incubation temperature

The basic culture medium composition described above was investigated in order to investigate the cell mass and EPS amount according to the culture temperature at the basal medium composition conditions of the isolated strains and the corresponding EPS specific ratio (Specific rate = EPS production / biomass, w / w). Based on the culture temperature was confirmed by setting to 20, 30, 37, 40, 45 ℃. As a result, as shown in Fig. 9, both Bacillus rickenformis KS-17 and Bacillus rickenformis KS-20 were 13.2 ± 0.7g / L and 12.4 ± 0.5g / L when the incubation temperature was 37 ° C, respectively. The maximum EPS yield and specific rate are shown (p <0.001).

5-5. Production amount according to incubation time

Based on the basal medium composition and culture conditions described above to investigate the cell mass and EPS amount according to the culture time of the isolated strain and the resulting EPS specific ratio (Specific rate = EPS production / cell mass, w / w) Incubation time was confirmed the production amount every 0, 12, 24, 36, 48, 60, 72 hours. As a result, as shown in Fig. 10, Bacillus rickeniformis KS-17 shows the maximum EPS production and specific rate of 18.9 ± 0.4g / L at 60 hours after incubation, after which the EPS production and specific rate is rather long This has been reduced. On the other hand, Bacillus liqueniformis KS-20 showed a tendency to increase steadily up to 72 hours after incubation, showed an EPS production of 15.2 ± 0.4 g / L and a high specific rate at 72 hours.

Example 6 Antioxidant Activity of Extracellular Polysaccharides

In order to test the antioxidant capacity of EPS KS-17 and KS-20 obtained from the present invention was compared and analyzed by the same concentration gradient with β-glucan extracted from Chaga ( Inonotus obliquus ). To date, Chaga mushroom (fruiting body) has been researched on the effects of anti-cancer (Cha JY et al., 2004), antioxidant (Cui Y et al., 2005), antihyperlipidemia and glycemic control (Kim MA et al., 2009). It is made from, and the material is attracting attention as a material for functional food development for its functionality.

6-1. Electron donating ability according to DPPH radical scavenging ability

DPPH radical scavenging activity assays use DPPH, which is used to measure electron donating ability (EDA), to have relatively stable radicals, and to have high antioxidant activity and other radicals, including free radicals, if the ability to reduce or offset radicals is high. As it is expected to have high elimination activity, the method was mainly used to measure the antioxidant activity of a specific substance (Lee SL, 2009). 0.4 ml of 0.4 mM DPPH solution and 0.1 ml of sample prepared by dissolving EPS KS-17 and EPS KS-20 in distilled water were stirred well and reacted for 30 minutes at 37 ° C in a dark state and measured at absorbance at 517 nm. It became. The calculation formula was electron donating ability (%) = [1- (sample absorbance / control absorbance)] x 100. At this time, after confirming whether the experiment was performed properly with ascorbic acid, and compared with β-glucan extracted from known chaga mushroom having excellent antioxidant capacity. As a result, as shown in Table 6 and Figure 11, both KS-17 and KS-20 at 1-20mg / ml concentration was increased as the concentration gradient increased the activity and showed higher antioxidant capacity than β-glucan ( P <0.01).

Electron donating ability of EPS KS-17 and KS-20 (%)
(Mean ± Stdev) mg / ml One 2.5 5 7.5 10 15 20 KS-17 26.8 ± 1.7 ^b 33.5 ± 1.5 33.6 ± 0.8 33.9 ± 1.9 ^a 34.9 ± 0.7 37.8 ± 1.2 38.3 ± 1.1 KS-20 30.0 ± 0.6 ^a 32.7 ± 2.9 33.5 ± 2.6 35.5 ± 0.7 ^a 36.1 ± 0.2 37.7 ± 2.3 38.8 ± 1.4 β-glucan 31.1 ± 1.6 ^a 30.5 ± 2.8 30.9 ± 1.9 31.1 ± 1.2 ^b 33.1 ± 2.1 - - * ab: Value with different superscrips within the same colume are significantly different by ANOVA with Dancan's multiple range test at p <0.01.

6-2. Superoxide radical scavenging ability

Superradical scavenging activity is a low-molecular substance that plays a similar role as SOD (superoxide dismutase), and belongs to phytochemicals and is known to protect against oxidative disorders by inhibiting the reactivity of superoxide (Marklund et al., 1974). In addition to the anti-aging, the use of a material is considered to be a way to prevent oxidative damage. This assay was performed by mixing 1 ml of the sample prepared at a concentration of 0-10 mg / ml with 3 ml of 0.5 mM tris-HCL buffer (50 mM tris (hydroxymethyl) amino-methane + 10 mM EDTA) adjusted to pH 8.2, followed by 0.2 ml 7.2 mM pyrogallol. After reacting at 25 ° C. for 10 minutes and stopping the reaction with 0.5 ml of 1N HCl, absorbance was analyzed at 420 nm. The calculation formula was SOD-like activity (%) = 100-[(sample absorbance / control absorbance) x 100]. At this time, after confirming whether the experiment was properly performed with ascorbic acid, and compared with β-glucan extracted from known chaga mushroom having excellent antioxidant capacity. As a result, as shown in Table 7 and Figure 12, both KS-17 and KS-20 increased activity with increasing concentration gradient at the concentration of 1-10mg / ml, and KS-20 was relatively higher than β-glucan. At low concentrations, KS-17 showed higher antioxidant activity at higher concentrations (P <0.001).

SOD-like activity of EPS KS-17 and KS-20 (%)
(Mean ± Stdev) mg / ml EPS17 EPS20 β-glucan One 5.66 ± 3.25 ^c 18.52 ± 3.80 ^b 14.65 ± 0.23 ^b 2 8.42 ± 2.21 ^d 21.59 ± 2.04 ^b 16.76 ± 0.30 ^c 3 11.91 ± 1.75 ^d 21.77 ± 0.68 ^b 17.61 ± 0.33 ^c 4 12.51 ± 3.51 ^d 25.93 ± 2.10 ^b 19.12 ± 0.28 ^c 5 15.11 ± 1.84 ^d 25.55 ± 1.21 ^b 18.97 ± 0.34 ^c 6 17.83 ± 0.69 ^d 27.8 ± 1.30 ^b 21.03 ± 0.15 ^c 7 17.68 ± 2.51 ^d 26.70 ± 0.79 ^b 20.39 ± 0.33 ^c 8 22.79 ± 1.91 ^c 26.25 ±± 2.14 ^b 23.16 ± 0.28 ^c 9 23.66 ± 2.29 ^b 25.82 ±± 3.08 ^b 27.38 ± 0.34 ^b 10 29.79 ± 1.59 ^b 25.18 ± 0.77 ^d 27.23 ± 0.34 ^c * abcd: Value with different superscrips within the same colume are significantly different by ANOVA with Dancan's multiple range test at p <0.001.

6-3. Tyrosinase Inhibitory Activity

Tyrosinase is an enzyme that is involved in the production of dark brown melanin pigment, which may cause dark brown pigmentation of skin in humans, and is known to be a cause of skin blackening phenomenon. In addition to inhibition, it is considered as a way to protect against oxidative damage. This assay was performed by mixing 1 ml of sample prepared at a concentration of 0-10 mg / ml, 0.12 ml of 8.3 mM L-DOPA dissolved in 67 mM phosphate buffer adjusted to pH 6.8, and 0.04 ml of sample, before absorbing at 495 nm. Measured. Thereafter, 0.04 ml of 125 U / ml mushroom tyrosinase was added, followed by enzymatic reaction at 37 ° C. for 20 minutes, and absorbance at 495 nm was used to calculate the amount of DOPA chrome produced by comparing the calculated values. Calculations were made with tyrosinase inhibitory activity (%) = [{(Control _Abs -Blank _Abs )-(Sample _Abs -Blank _Abs )} / (Control _Abs -Blank _Abs )] x 100. At this time, after confirming whether the experiment was properly performed with ascorbic acid, and compared with β-glucan extracted from chaga mushroom known to have excellent antioxidant capacity. As a result, as shown in Table 8 and Figure 13, both KS-17 and KS-20 increased activity with increasing concentration gradient at the concentration of 1-10mg / ml, and KS-20 was relatively higher than β-glucan. At low concentrations, KS-17 showed higher antioxidant activity at higher concentrations (P <0.001).

Tyrosinase Inhibitory Activity of EPS KS-17 and KS-20 (%)
(Mean ± Stdev) mg / ml KS-17 KS-20 β-glucan One 22.66 ± 2.86 ^a 23.09 ± 0.63 ^a 5.62 ± 2.67 ^b 2 22.41 ± 1.80 ^a 22.23 ± 1.37 ^a 9.30 ± 1.30 ^b 3 22.84 ± 2.65 ^b 28.43 ± 0.80 ^a 9.49 ± 1.49 ^c 4 25.21 ± 3.17 ^a 27.63 ± 1.27 ^a 10.79 ± 1.75 ^b 5 25.10 ± 3.42 ^a 26.75 ± 1.85 ^a 9.95 ± 1.95 ^b 6 24.41 ± 1.91 ^a 26.95 ± 0.98 ^a 11.27 ± 2.10 ^b 7 24.58 ± 3.44 ^b 31.53 ± 0.46 ^a 17.94 ± 2.08 ^c 8 26.96 ± 2.53 ^b 33.42 ± 0.96 ^a 16.53 ± 3.70 ^c 9 30.72 ± 2.20 ^a 33.41 ± 0.98 ^a 16.64 ± 3.58 ^b 10 31.13 ± 3.65 ^b 38.00 ± 2.06 ^a 14.96 ± 1.80 ^c abc: Value with different superscrips within the same colume are significantly different by ANOVA with Dancan's multiple range test at p <0.01.

Example 7 Statistical Processing

All experiments were performed in three iterations with mean and standard deviation, and significance test was performed using the Version 12.0 SPSS (Statistical Package for Social Sciences, SPSS Inc., Chicago, IL, USA) software package program. It was done.

Agricultural Biotechnology Research Institute KACC91570 20100714 Agricultural Biotechnology Research Institute KACC91571 20100714

Claims (13)

Bacillus licheniformis KS, an extracellular polysaccharide with antioxidant and tyrosinase inhibitory activity, which is isolated from kimchi and has an average molecular weight of 1.40 to 1.50 kDa and is a homopolysaccharide consisting solely of glucose (glucose) -17 strain (Accession No. KACC91570P).
Bacillus licheniformis KS, an extracellular polysaccharide having antioxidant and tyrosinase inhibitory activity, which is isolated from kimchi and has an average molecular weight of 1.50 to 1.60 kDa and is a homopolysaccharide consisting solely of glucose (glucose) -20 strain (Accession No. KACC91571P).
delete Antioxidant activity and tyrosinase produced from Bacillus licheniformis KS-17 strain (Accession No. KACC91570P) of claim 1, having an average molecular weight of 1.40 to 1.50 kDa and a homopolysaccharide consisting only of glucose (glucose) Extracellular polysaccharides with inhibitory activity.
Antioxidant activity and tyrosinase produced from Bacillus licheniformis KS-20 strain (Accession No. KACC91571P) of claim 2 and having an average molecular weight of 1.50 to 1.60 kDa and a homopolysaccharide consisting only of glucose (glucose) Extracellular polysaccharides with inhibitory activity.
The method according to claim 4 or 5, wherein the extracellular polysaccharide comprises sucrose, tryptone, yeast extract, dipotassium phosphate, and diammonium citrate. An extracellular polysaccharide obtained by culturing a strain in a containing medium.
delete An antioxidant food composition comprising an extracellular polysaccharide having the antioxidant activity and tyrosinase inhibitory activity of any one of claims 4 and 5 as an active ingredient.
Antioxidant cosmetic composition comprising an extracellular polysaccharide having the antioxidant activity and tyrosinase inhibitory activity of any one of claims 4 or 5 as an active ingredient.
The Bacillus liqueniformis KS-17 of claim 1 in a medium containing sucrose, tryptone, yeast extract, dipotassium phosphate and diammonium citrate. After inoculation of the strain, the culture medium incubated at a temperature of 36.5 ℃ to 37.5 ℃ at pH 6.5 to 7.5 has an average molecular weight of 1.40 to 1.50 kDa, it is a homopolysaccharide consisting of glucose, antioxidant activity and tyrosinase inhibition A method for producing or recovering extracellular polysaccharides having activity.
According to claim 10, wherein the sucrose is 90 to 110g / L, tryptone is 8 to 12g / L, yeast extract is 4 to 6g / L, dipotassium phosphate is 4 to 6g / L, diammonium citrate is 4 To 6 g / L.
The Bacillus liqueniformis KS-20 of claim 2 in a medium containing sucrose, tryptone, yeast extract, dipotassium phosphate and diammonium citrate. After inoculation of the strain, the culture medium incubated at a temperature of 36.5 ℃ to 37.5 ℃ at pH 5.5 to 6.5 has an average molecular weight of 1.50 to 1.60 kDa, it is a homopolysaccharide consisting of glucose, antioxidant activity and tyrosinase inhibition A method for producing or recovering extracellular polysaccharides having activity.
The method according to claim 12, wherein the sucrose is 65 to 85g / L, tryptone is 8 to 12g / L, yeast extract is 4 to 6g / L, dipotassium phosphate is 4 to 6g / L, diammonium citrate is 4 to 6 g / L.

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