KR101420355B1 - Novel strain of Pediococcus acidilactici M76 and exopolysaccharide produced from the same - Google Patents

Abstract

The present invention relates to a novel Pediococcus acidilactici M76 strain and an extracellular polysaccharide produced therefrom. The Pediococcus acidilactici M76 strain according to the present invention and the extracellular polysaccharide produced from the strain are excellent in the proliferation of pancreatic beta cells and the activity of inhibiting the death of pancreatic cells and thus are useful for the prevention or treatment of diabetes mellitus Lt; / RTI >

Description

[0001] The present invention relates to a novel Pediococcus strain Sedilactii M76 and an extracellular polysaccharide produced therefrom. More particularly, the present invention relates to a novel strain Pediococcus acidilactici M76 and an exopolysaccharide produced from the same,

The present invention relates to novel Pediococcus isolated from rice wine and to Pediococcus acidilactici ) M76 strain, an extracellular polysaccharide produced therefrom, a composition for preventing or treating diabetes mellitus comprising the same as an active ingredient, and a method for producing an extracellular polysaccharide from a cidulacyti strain of Pediococcus.

The microbial polysaccharide is a part of the cell wall of the microorganism and forms a thin membrane around the cell wall or accumulates as a viscous form outside the cell wall during fermentation. The primary or secondary metabolites of these microorganisms are called exopolysaccharides (EPS) (Kim DJ et al., 2001). Polysaccharides produced from microorganisms are polymers with long-chain and high-molecular-mass properties, and have various functions such as texturizers, viscosifiers, emulsifiers, (Wang J et al., 2007), antioxidative effects (Asker MMS et al., 2009), and antioxidant effects (Bae JT et al., 2005), which have recently been used in the food industry. (Kolodzieja H et al., 2007), the importance of the use of functional materials in food and pharmaceutical industries has increased as a result of research on the health benefits (Shivakumar S et al, 2006). In addition, the extracellular polysaccharides produced by these microorganisms have been reported to be able to significantly increase their productivity as they improve the constituents of the medium and the culture conditions (Vijayendra SVN et al., 2008). However, most of the functional polysaccharides currently commercialized in the industry are known to be produced mainly from fungi such as mushrooms, and representative examples include Inonotus obliquus , and the like, which are produced by higher mycelia of mushrooms. These beta-glucans are known to have anti-cancer (Cha JY et al., 2004), antioxidant (Cui Y et al., 2005), anti-hyperlipemia and blood glucose control (Kim MA et al., 2009) It is receiving great attention. However, fungi such as mushroom have a disadvantage in that they are slower in growth rate than bacteria, are relatively difficult to cultivate in a large amount, and produce less extracellular polysaccharides.

On the other hand, studies on extracellular polysaccharides produced from strains isolated from foods have been conducted on Lactobacillus sp., Streptococcus sp., Lactococcus sp. Leuconostoc species isolated from kimchi ( Leuconostoc There is also a small number of extracellular polysaccharides produced from lactic acid bacteria, such as Weissella sp., and Weissella sp., but there are also few studies on the functional demonstration of extracellular polysaccharides produced by each microorganism.

Therefore, there is a need to find a strain capable of producing a highly functional extracellular polysaccharide with high efficiency, and to prove the functionality of the extracellular polysaccharide produced from the strain and the strain, and to find an applicable strain for a fermented food having a future function.

Recently, chronic degenerative diseases have been increasing in Korea due to changes in lifestyle and lifestyle, such as westernized dietary habits and lack of exercise. In particular, the incidence of diabetes mellitus and its complications is rapidly increasing. The incidence of diabetes mellitus was less than 1% of the population in the 1970s, and it has been one of the ten leading causes of death since the 1990s, with 17.2 deaths per 100,000 population. Diabetes mellitus and mortality from domestic diabetes mellitus . About 10% of the population in Korea currently suffers from the disease and the incidence is rapidly increasing, and around 3% of the world is suffering from diabetes. Diabetes is characterized by insulin secretion disorder in the beta (.beta.) Cells in the pancreas and insulin resistance to peripheral tissues by environmental, genetic and metabolic factors and is characterized by hyperglycemia. That is, an endocrine system disease in which insulin action abnormally acts to cause a large amount of glucose in the blood to lead to a hyperglycemia state. If such a hyperglycemic state persists, a cardiovascular disease or a kidney disease, a nervous system disease, a foot ulcer, a diabetic retinopathy It is known as a disease that can cause many complications such as. Treatment of diabetes is divided into dietary therapy, exercise therapy and drug therapy. Insulin medicines and various hypoglycemic agents are used depending on the patient's symptoms. However, the problem of toxicity due to the use of drugs and the problem of tolerance of patients are emerging, and the hypoglycemic drugs currently used in clinical practice are commonly known to cause side effects such as gastrointestinal disorders such as abdominal distension and diarrhea. In addition, diabetes is a complex disease characterized by excessive glucose production in the liver, insulin resistance, and decreased glucose disposal in muscle and adipocytes. Therefore, specific treatment alone can not prevent the occurrence of various side effects. Therefore, in order to overcome the problems of existing drugs, attempts have been recently made to search for pharmacological substances in natural materials, and many prototypes have been introduced in the market. However, And search for ingredients.

Unlike other fermented foods, Makkolli, a traditional fermented food, contains a variety of microorganisms containing various nutrients due to the action of microorganisms derived from rice. When alcohol fermentation proceeds, it causes environmental stress to the lactic acid bacteria To produce polysaccharides strategically. Unlike other alcoholic beverages, Makgeolli is a Korean liquor that is rich in nutrients such as protein, amino acid, vitamin group, and organic acid. It is also known as Takju because it is a turbid liquor made by filtering without removing clear liquor. Traditionally, makgeolli is made by fermenting glutinous rice or rice with raw yeast. In other words, makgeolli is a carbohydrate starch that is decomposed by microorganisms such as leukocyte and formed by fermentation process by various components such as alcohol and sugar components. Although it varies depending on fermentation conditions, 15%. When water is added to this solution, the concentration of alcohol is maintained at 6 ~ 8%. Especially, it is fermented by various microorganisms. It contains valine, leucine, serine which is a decomposition product of yeast proteinase and contains protein, saccharide and vitamin B group unlike general liquor. , Proline, glycine and many other amino acids. Unlike other fermented and distilled spirits, makgeolli has very different characteristics because it contains a large amount of yeast and abundant lactic acid bacteria. However, there are very few studies on the characteristics of lactic acid bacteria derived from such rice wine.

Lactic acid bacteria and yeast are most often used as a probiotic microorganism for, in the case of lactic acid bacteria strains belonging to such a high availability of Lactobacillus (Lactobacillus) genus Bifidobacterium (Bifidobacterium) and Enterococcus genus (Enterococcus) genus. Lactic acid bacteria of various characteristics are separated from fermented food such as host and kimchi, and are developed as a strain for prophylactic antibiotics such as disease prevention and immunity enhancement of the host. Pediococcus acidilactici is a lactic acid bacteria used for fermentation such as kimchi or salted fish, and is widely known as a production strain of pediocin among bacteriocin, which is an antimicrobial peptide. Especially, strong lactic acid productivity, It has been reported that various lactic acid bacteria products have been reported as inhibiting harmful microorganisms, maintaining the quality of liquid feed, decreasing ammonia concentration, improving feed nutritional value, and strengthening immune system.

Prior research by separating the microorganisms from the rice wine until now only a few pieces, especially in Peddie Oh Caucus (Pediococcus sp.) is almost insignificant. In addition, there have been few studies on the functionality of extracellular polysaccharides produced from lactic acid bacteria isolated from makgeolli until now, and there have been no studies on them.

Since it is possible to obtain and manufacture a functional substance from such a safe lactic acid bacterium and it is possible to produce it, it is necessary to study a lactic acid bacterium having safety to produce such a substance and a functional substance derived therefrom.

The inventors of the present invention confirmed that the extracellular polysaccharide produced from lactic acid bacteria isolated from makgeolli was superior to the pancreatic beta cell proliferation and pancreatic cell death suppression activity while studying the functional material produced from the lactic acid bacteria having safety, Thus completing the present invention.

Accordingly, an object of the present invention is to provide a novel Pediococcus isolated from rice wine, a traditional fermented wine of Korea, with Pediococcus acidilactici ) M76 strain (Accession No .: KACC91683P) and an extracellular polysaccharide produced therefrom.

It is also an object of the present invention to provide a method for treating Pediococcus The present invention also provides a pharmaceutical composition and a food composition for preventing or treating diabetes mellitus comprising an acidilactici M76 strain (accession number: KACC91683P) as an active ingredient.

It is also an object of the present invention to provide a pharmaceutical composition and a food composition for preventing or treating diabetes mellitus comprising the extracellular polysaccharide as an active ingredient.

It is also an object of the present invention to provide a method for producing an extracellular polysaccharide from the S. licheniformis strain M76 in the Pediococcus.

In order to achieve the above object, the present invention provides Pediococcus acidilactici M76 (Accession No .: KACC91683P) strain in a novel Pediococcus having the activity of proliferating pancreatic beta cells and inhibiting the death of pancreatic cells .

The present invention also provides an extracellular polysaccharide having pancreatic beta cell proliferation activity and pancreatic cell death inhibitory activity produced from Pediococcus acidilactici M76 (accession number: KACC91683P) strain.

The present invention also relates to a Pediococcus acidilactici M76 (accession number: KACC91683P) strain, a strain of the strain, a concentrate of the culture, a culture of the strain, A pharmaceutical composition for preventing or treating diabetes mellitus, and a food composition for preventing or ameliorating diabetes mellitus, which comprises as an active ingredient any one selected from the group consisting of the dried product of the above culture and a combination thereof.

The present invention also provides a method for producing a cell culture, comprising: 1) culturing a Pediococcus acidilactici strain M76 (accession number: KACC91683P) in Pediococcus isolated from rice wine to obtain a culture medium; 2) treating the cell culture broth with hot water; 3) removing protein from the hot culture treated cell culture medium, adding ethanol, and centrifuging to obtain a precipitate; And 4) removing the remaining ethanol from the precipitate and removing free sugars to obtain purified extracellular polysaccharides. The pediatric beta cell proliferation and pancreatic cell death suppression activity, , Which is composed of glucose and has an average molecular weight of 50 kDa to 70 kDa.

Since the Pediococcus acidilactici M76 strain isolated from rice wine according to the present invention and the extracellular polysaccharide produced from the strain are excellent in the activity of inhibiting pancreatic beta cell proliferation and pancreatic cell death inhibition, Or may be useful for treatment.

Brief Description of the Drawings Fig. 1 is a diagram showing the effect of the pediatric beta cell (RIN-m5F) proliferation of Pediococcus strain S14 Lacticus M76 (Accession No .: KACC91683P) (A: 48 hr.
Fig. 2 is a diagram showing a phylogenetic classification of a Pediococcus strain Sdacticus M76 (Accession No .: KACC91683P).
Fig. 3 is a diagram showing the biochemical characteristics of the Pediococcus Sedilactii M76 strain (Accession No .: KACC91683P).
4 is a graph showing the effect of the extracellular polysaccharide pancreatic beta cells (RIN-m5F) produced from the strain Sidi Lactici M76 (Accession No .: KACC91683P) in Pediococcus.
5 is a graph showing the effect of the addition of the extracellular polysaccharide pancreatic beta cells (RIN-m5F) produced from the strain Pediococcus strain M76 (Accession No .: KACC91683P) to the alloxan (10 mM (A), 15 mM (B) FIG.
6 is a diagram showing the molecular weight of an extracellular polysaccharide produced from a strain Pediococcus Sedilactii M76 (Accession No .: KACC91683P).
FIG. 7 is an analysis of monosaccharide components by TLC and HPLC of extracellular polysaccharides produced from Pediococcus strain S76 (SEQ ID NO: KACC91683P) (A: (a) As the control, an extracellular polysaccharide (B) Mannose, Ara, arabinose, Fru, fructose, Glu, clucose, Gal, galactose, Lac, lactose, EPS, extracellular polysaccharide Sample treatment).
Fig. 8 is a diagram showing the result of analysis of the structure of extracellular polysaccharide produced from Pediococcus Sedilactii M76 strain (Accession No .: KACC91683P) using FT-IR spectrum.

The present invention provides Pediococcus acidilactici M76 strain (Accession No .: KACC91683P) to Pediococcus producing an extracellular polysaccharide having pancreatic beta cell proliferation and pancreatic cell death inhibitory activity.

The strain produced microbial extracellular polysaccharide from rice wine and had insulin secretory cell proliferating ability. The strain was purified and purified by 16S rDNA sequencing and biochemical characterization. As a result, Pediococcus sp. ), Which was named Pediococcus acidilactici M76 in Pediococcus .

In addition, in accordance with the provisions of the Budapest Treaty on the International Recognition of Microorganism Deposit under the Patent Procedure, the above-mentioned strain Pediococcus sydacticis M76 was deposited with the Korean Agricultural Culture Collection (KACC) on October 12, No. KACC91683P.

The present invention also relates to the use of Pediococcus acidic lactic acid M76 (Accession No .: KACC91683P).

The extracellular polysaccharide is excellent in pancreatic beta cell proliferation and pancreatic cell death inhibition activity, and is composed of glucose-type glycoprotein, and has an average molecular weight of 50 kDa to 70 kDa, preferably 67 kDa.

The present invention also relates to a Pediococcus acidilactici strain M76 (accession number: KACC91683P) having a pancreatic beta cell proliferation and pancreatic cell death inhibitory activity, a culture of the strain, a concentrate of the culture , A dried product of the culture, and a combination thereof. The present invention also provides a pharmaceutical composition for preventing or treating diabetes mellitus.

The composition of the present invention may be formulated and used as a pharmaceutical unit dosage form by adding a pharmaceutically acceptable carrier, excipient or diluent.

The pharmaceutical composition of the present invention can be formulated in the form of powders, granules, tablets, capsules, oral preparations such as suspensions, emulsions, syrups and aerosols, external preparations, suppositories, and sterilized injection solutions according to conventional methods Can be used.

The daily dose of Pediococcus acidilactici M76 strain (accession number: KACC91683P) is preferably 0.1 g or more of the strain. The daily dose of extracellular polysaccharide is about 10 to 50 mg / kg It is preferable to administer them once or several times a day. The dosage can be adjusted according to the diabetic condition.

The present invention also relates to a Pediococcus acidilactici strain M76 (accession number: KACC91683P) having a pancreatic beta cell proliferation and pancreatic cell death inhibitory activity, a culture of the strain, a concentrate of the culture , A dried product of the culture, and a combination thereof. The present invention also provides a food composition for preventing or ameliorating diabetes.

The food of the present invention may include, without limitation, supplementary foods processed in the form of tablets, capsules, powders, granules, liquids, rings, etc., using raw materials having functions useful in the human body.

The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). In general, when preparing food, the Pediococcus of the present invention is added with Pediococcus The extracellular polysaccharide produced from the acidilactici ) M76 (Accession No .: KACC91683P) strain is added in an amount of 15% by weight or less, preferably 10% by weight or less based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

There is no particular limitation on the kind of the food. Examples of the food include dairy products such as meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums and ice cream, various soups, drinks, tea, drinks, alcoholic beverages, And includes all health foods in a conventional sense.

In the case of beverages, various flavors or natural carbohydrates may be added as an additional ingredient such as ordinary beverages. Such natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, natural sweeteners such as dextrin and cyclodextrin, synthetic sweeteners such as saccharin and aspartame, and the like . The ratio of the natural carbohydrate is generally about 0.001 to 0.4 g, preferably about 0.002 to 0.03 g per 100 ml of the food composition of the present invention.

In addition to the above, the food of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acids and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, A carbonating agent used in a carbonated beverage, and the like. In addition, the food of the present invention may contain flesh for the production of natural fruit juice, fruit juice drink and vegetable drink. These components may be used independently or in combination. Although the ratio of these additives is not critical, it is generally selected in the range of 0.001 to 0.1 parts by weight per 100 parts by weight of the food composition of the present invention.

The food composition comprising the extracellular polysaccharide having the activity of inhibiting the proliferation of pancreatic beta cells and the activity of suppressing the death of pancreatic cells according to the present invention as an active ingredient may be used as a raw material for food, There is no advantage, and there is an advantage of excellent portability.

The present invention also provides a method for producing a microorganism which comprises: 1) culturing a Pediococcus acidilactici strain M76 (accession number: KACC91683P) in Pediococcus isolated from rice wine to obtain a culture medium; 2) treating the cell culture broth with hot water; 3) removing protein from the hot culture treated cell culture medium, adding ethanol, and centrifuging to obtain a precipitate; And 4) removing the remaining ethanol from the precipitate and removing free sugars to obtain purified extracellular polysaccharides. The pediatric beta cell proliferation and pancreatic cell death suppression activity, , Which is composed of glucose and has an average molecular weight of 50 kDa to 70 kDa.

The extracellular polysaccharide can be produced from a culture obtained by inoculating a strain of MRS in glucose or a medium of agar in a pediococcus strain and culturing at an initial pH of 6.5 to 7.5 at a temperature of 36.5 ° C to 37.5 ° C.

The culture broth can be obtained by culturing a novel strain of Pediococcus Sd lacticus M76 (accession number: KACC91683P) in a culture medium by a method known in the art, and a culture supernatant containing a substance having the function of the present invention, The pure culture medium can be included without limitation. In the present invention, the term "medium" means a solid medium or a liquid medium containing nutrients necessary for growing animal cells, plant cells or bacteria, and the term "medium" means a medium inoculated with a strain in a liquid medium.

The culture solution may be a culture solution containing a strain or a culture obtained by inoculating and inoculating a strain, followed by removal of the bacteria, i.e., strain. The concentrate of the culture liquid means the concentrate of the culture liquid, and the dry matter of the culture liquid means that the water of the culture liquid is removed.

Hereinafter, the present invention will be described in detail with reference to Examples and Preparation Examples. However, the following examples and preparative examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following examples and preparative examples.

Example  1. From makgeolli Lactic acid bacteria  detach

In order to separate lactic acid bacteria from makgeolli, makgeolli which was traditionally produced using yeast was used as a sample. The makgeolli samples were sequentially diluted by decimal method using sterilized diluted solution 0.85% physiological saline. MRS liquids (1.5% agar, w / v), Lamva (0.05 g cystein-HCl, 0.005 g bromocresol green, 15 g agar) containing three agar plates for the isolation of lactic acid bacteria, MRS (Difco Laboratories, (Diluted in 1.5% agar, w / v) medium, supplemented with 2 mg / L vancomycin as the antibiotic in the medium and the pH adjusted to 5.0) 100 mu L of the sample was taken out and smoothed, followed by static incubation at 37 DEG C for 48 hours.

Example  2: pancreatic beta cells RIN Culture conditions of -m5F

The RIN-m5F cell line derived from the insulin secreting rat insulinoma cell line was used from ATCC (American Type Culture Collection). RIN-m5F cell lines were grown in RPMI-1640 medium (Hyclone, Logan, Utah, USA) containing 10% FBS and 1% penicillin-streptomycin solution 10000 units / ml (Hyclone, Logan, Utah, USA) USA) and fed with 5% carbon dioxide Cells and tissue culture media (humidified CO ₂ The cell viability was measured with an EZ-Cytox Enhanced Cell Viability Assay kit at an absorbance of 420 to 480 nm at 1 hour intervals. At this time, a cell culture solution not treated with the culture medium was used as a control group, and the viability of the culture medium was compared with 100%.

Example  3: pancreatic beta cells ( RIN -m5F) Proliferative ability Lactic acid bacteria  detach

(RIN-m5F) proliferating ability from rice wine.

To investigate the RIN-m5F cell proliferative capacity, 54 strains of lactic acid bacteria isolated from makgeolli were randomly selected and each strain was inoculated on MRS liquid medium and cultured at 37 ° C for 48 hours with stirring at 150 rpm. Each culture was centrifuged at 10,000 rpm for 20 minutes, and then the precipitated cells were collected, washed twice with sterilized 50 mM PBS buffer (pH 7.0), and then re-precipitated by centrifugation. The strains washed with 50 mM PBS buffer were resuspended in the same buffer, and the pulverized culture broth was used as a culture medium for investigating pancreatic beta cell proliferative capacity using an ultrasonic disperser (Sonic Dismembrator 550, Fisher).

Methods for testing the ability of pancreatic beta cells (RIN-m5F) to proliferate with a culture broth sample are as follows. The cultured pancreatic beta cell line RIN-m5F was cultured at a concentration of 1 × 10 ⁴ cells per 100 μL at 37 ° C. for 24 hours, and the pulverized culture broth as a sample solution was mixed with sterilized 50 mM PBS buffer at 10 μL / mL And cultured for 48 hours. The survival rate of pancreatic beta cells was measured at an interval of 1 hour at an absorbance of 420 to 480 nm using the EZ-Cytox Enhanced Cell Viability Assay kit. At this time, a cell culture solution not treated with the culture medium was used as a control group, and the viability of the culture medium was compared with 100%.

Among the 54 strains tested, strain M76 was selected as the strain with the highest pancreatic beta cell proliferative activity, showing the highest cell proliferative activity at a concentration of 10 μL / mL compared with the control group.

In order to confirm the pancreatic beta cell proliferative activity of the selected strain M76, the ability of the strain M76 to cultivate the RIN-m5F cell line at various concentrations was confirmed. The bacterial culture broth, which had been pulverized into the pancreatic beta cell line RIN-m5F at a concentration of 1 X 10 ⁴ cells per 100 μl pre-incubated overnight, was adjusted to different concentrations of 1, 5, 10, 50, 100 μL / mL in sterile 50 mM PBS buffer And cultured for 48 hours or 72 hours after being dispensed. Cell viability was measured using the EZ-Cytox Enhanced Cell Viability Assay kit. The results were shown in FIG. 1 (A: 48 hours B: 72 hours).

As shown in FIG. 1, the RIN-m5F cell proliferating ability tended to increase with increasing concentration gradient of the M76 strain culture medium. In particular, when cultured at a concentration of 50 μL / mL for 48 hours, 191% ( P <0.001), respectively.

Example  4: Identification of strain

The isolated M76 strain was identified by 16S rDNA sequencing and biochemical characterization. The oligonucleotide primers used for the sequencing of the 16S rDNA gene were the universal primer 27F (5'-AGT TTG ATC CTG GCT CAG-3 ') and 1490R (5'-GTT ACC TTG TTA CGA CTT C-3') PCR was performed using PCR cycler Primus (Primus 25, MWG Biotech, UK) (Table 1). The nucleotide sequence was determined by ABI PRISM 3730 DNA analyzer. The analyzed nucleotide sequence was compared with the nucleotide sequence of the ribosomal RNA gene registered in Genbank with the DDBJ program, and the homology was shown.

PCR reaction conditions Reaction mixture PCR conditions 10 x Taq reaction buffer 5 μL 95 ° C, 2 minutes
95 ℃, 1 minute ━━━┑
54 ℃, 30 seconds 30 times
72 ℃, 1 minute ━━━┙
72 ° C, 5 min dNTP mix (2.5 mM each) 4 μL Primer 27F 0.5 μL Primer 1490R 0.5 μL template 15 μL Enzyme (2.5 U / μl) 0.5 μL Distilled water ~ 50 μL

For analysis of the biochemical characteristics of the selected strains, API 50CHL reagent kit (BioMerieus, France) was used according to the manufacturer's test method. Single colonies were homogenously homogenized in 5 mL of 0.85% NaCl suspension medium and then 2.5 mL of CHL medium was added. After aseptically dispensing into 49 strips of API 50 CHL kit containing various carbon sources, cultures were incubated at 37 ° C for 48 hours to determine whether the color of the culture medium had changed and whether each of the sugars was used as a carbon source. Identification results were confirmed using apiweb identification software, version (V5.1).

The results of the identification of the strains performed in the above manner are shown in FIG. 2 and FIG.

As shown in Fig. 2, the isolate strain M76 was a strain of Pediococcus acidylactici ) was 99% homologous. Also, as shown in FIG. 3, biochemical analysis using an API 50CHL reagent kit showed that Pediococcus acidilactici and Pediococcus pentosaceus 1 showed the highest similarity to Pediococcus However, they showed different glucose metabolism such as glycerol and erythritol, and they were found to be a novel strain different from the existing strains.

That is, the isolated strain M76, which produces extracellular polysaccharide isolated from makgeolli, was identified as Pediococcus acidilactici in 16S rDNA sequence and biochemical analysis of Pediococcus . Therefore, strain M76 was named Pediococcus acidilactici M76 in Pediococcus . In addition, in accordance with the provisions of the Budapest Treaty on the International Recognition of Microorganism Deposit, it was deposited with the Korean Agricultural Culture Collection (KACC) on October 12, 2011 as a deposit number KACC 91683P.

Example  5: Pedio Caucus Sydy Laktishi M76 ( KACC91683P )of Extracellular polysaccharide Generation

The pediococcus Sedilecticus M76 (KACC91683P), which has been shown to exhibit excellent proliferative activity against pancreatic beta cells, exhibits a mucoid type of mucoid different from that of a strain having a different colony shape during culturing on a solid medium Respectively. Since this mucoid-type colony is one of the characteristics of a strain producing an extracellular polysaccharide, it was tried to test the extracellular polysaccharide production ability of Sedilactii M76 (KACC91683P) in Pediococcus.

In order to investigate the extracellular production ability of Sedilactii M76 (KACC91683P) in Pediococcus, an extracellular polysaccharide was purified from the culture solution of Cedaractii M76 (KACC91683P) in Pediococcus. Pediococcus was inoculated with the Sydactyti M76 (KACC91683P) in the MRS liquid medium and cultured at 37 ° C and 150 rpm for 48 hours. The cell culture broth obtained was treated with hot water treatment at 100 ° C for 20 minutes in a constant-temperature water bath To inactivate the enzyme. Then, to remove the protein from the culture solution, the culture was incubated at 4 ° C with trichloroacetic acid to a final concentration of 4%, and then centrifuged (10,000 rpm, 25 minutes, 4 ° C) Lt; / RTI &gt; To extract the water-soluble polysaccharide from the supernatant, 3 times (v / v) of cold ethanol was added to the supernatant and stirred, followed by extraction and precipitation at 4 ° C overnight. The extracellular polysaccharide as a precipitate was recovered by centrifugation (10,000 rpm, 20 minutes, 4 ° C) and the remaining ethanol was removed using a vacuum concentrator (MG-2200, EYELA, Japan) and then subjected to a dinitrosalicylic acid To determine the extracellular polysaccharide production ability of the strain. In addition, the extracellular polysaccharide from which the ethanol was removed was redissolved in the third distilled water to purify and acquire the extracellular polysaccharide from Sedilexi M76 (KACC91683P) in the finally selected Pediococcus. In order to remove the free sugars from the culture solution (Mw cut off 12,000) at 4 ° C for 24 hours, and then lyophilized to obtain powder form. This was used as an extracellular polysaccharide sample produced by the strain.

As a result of confirming the extracellular polysaccharide production ability of Sidi Lactici M76 (KACC91683P) in Pediococcus, it was confirmed that water-soluble extracellular polysaccharide was produced. Its production ability was at a level capable of producing 2 g or more per 1 L of the culture medium, and 42.7 mg / L / h of extracellular polysaccharide productivity (producticity).

Example  6: Extracellular polysaccharide  Pancreatic beta cells RIN -m5F proliferation test

The cultured pancreatic beta cell line RIN-m5F was cultured at a concentration of 1 × 10 ⁴ cells per 100 μL at 37 ° C. for 24 hours and then diluted with various concentrations of 0.1, 1, 2, 5, 10 and 20 μg / And cultured for 72 hours. Cell viability was measured at an absorbance of 420 to 480 nm at 1 hour intervals using the EZ-Cytox Enhanced Cell Viability Assay kit. At this time, a cell culture solution not treated with extracellular polysaccharide was used as a control, and the viability was compared with 100%.

The results are shown in Fig.

As shown in FIG. 4, the extracellular polysaccharide derived from cedaractic acid M76 in Pediococcus increased cell proliferation activity with increasing concentration gradient at a concentration of 0.1 μg / ml to 20 μg / ml, especially 20 μg / ml in the control group (124%) compared to the control group ( p <0.001).

Example 7 Effect of Extracellular Polysaccharides on Alloxan-Induced Cytotoxicity to Pancreatic Beta Cell Death Inhibition

In order to confirm RIN-m5F-killing activity of extracellular polysaccharide pancreatic beta cell (insulin secreting rat insulinoma cell line) against alloxan-induced cytotoxicity, the following experiment was performed. In other words, alloxan is a substance that stimulates and potentially induces diabetes in experimental animals and is treated with insulin secreting cells to examine the extracellular activity of extracellular polysaccharide to kill insulin.

The cultured pancreatic beta cell line RIN-m5F was cultured at a concentration of 1 × 10 ⁴ cells per 100 μL at 37 ° C. for 24 hours, and the extracellular polysaccharide samples diluted at various concentrations of 1, 2, 5, 10 and 20 μg / And then pretreated for 24 hours. After replacing with fresh medium, the cell line was incubated for an additional 1 hour. The cultured cells were treated with 10 mM or 15 mM alooxane and cultured for 1 h in a 5% CO ₂ incubator. The cultured cells were washed twice with fresh culture medium, and then stained with EZ-Cytox Enhanced Cell Viability Assay kit And the cell viability was measured at an interval of 1 hour at an absorbance of 420 to 480 nm. At this time, a cell culture solution not treated with extracellular polysaccharide was used as a control group, and the viability was compared with 100%.

 The results are shown in Fig.

As shown in FIG. 5, the extracellular polysaccharide derived from Pediococcus sydylaticis M76 (KACC91683P) showed a concentration gradient of 1 to 20 占 퐂 / ml in both 10 mM (A) and 15 mM (B) The cell survival rate of the control group treated with 15 mM alooxane was decreased to 21.9%, while that of the extracellular polysaccharide was decreased to 10 μg / (45.4%) compared to the control group ( p <0.001).

The above examples demonstrate that the sydylaclassi M76 (KACC91683P) in the lactic acid bacterium Pediococcus of the present invention is excellent in insulin-secreting pancreatic beta cell proliferative ability and the extracellular polysaccharide effectively inhibits the death of pancreatic beta cells from diabetic inducers And it can be seen that diabetes can be effectively prevented or treated through this.

Example 8 : Extracellular polysaccharide  Characterization

8.1. Molecular weight measurement

To determine the molecular weight of the extracellular polysaccharide produced and purified from Pediococcus Sydacticus M76 (KACC91683P), the sample was loaded onto a Sephadex G-200 (GE Healthcare) solution containing 0.15 M NaCl at a flow rate of 1.0 mL / To perform gel filtration column chromatography. The elution time ratio was determined using Blue dextran 2000 (GE Healthcare) and gel filtration standard (thyroglobulin 670 kDa, bovine gamma-globulin 158 kDa, chicken ovalbumin 44 kDa, equine myoglobin 17 kDa, vitamin B ₁₂ 1.35 kDa) The results according to the relationship with the molecular weight are shown in Fig. At this time, fractions of 2 minutes intervals were confirmed by analyzing the absorbance at 490 nm using the phenol-sulfuric acid method.

As shown in FIG. 6, the molecular weight analysis showed that the molecular weight of the extracellular polysaccharide (EPS of FIG. 6) having pancreatic cell death suppression and proliferation promoting function produced from Pediococcus sydilactii M76 (KACC91683) was 67 kDa .

8.2. Confirmation per configuration

To examine the constituent sugar of the extracellular polysaccharide produced and purified from Pediococcus Sydiractii M76 (KACC91683P), 10 mg of extracellular polysaccharide sample was added to 1 ml of 2N trifluoroacetic acid, And analyzed by TLC and high-performance liquid chromatography (HPLC-ELSD) using a hydrolyzed sample after heating for 6 hours.

TLC analysis was performed using glucose, galactose, fructose, mannose, maltose, lactose and arabinose as standard materials. 5 μL of the standard material and 10 μL of the sample were added to a silica gel TLC plate (Merck, TLC aluminum sheet, silica gel 60F ₂₅₄ ) (18: 1: 1, v / v / v), followed by color development at 120 ° C for 1 minute. Respectively.

High Performance Liquid Chromatography (HPLC-ELSD) analysis was performed with the conditions of Table 2 using glucose, galactose, fructose, lactose, maltose and arabinose as standards.

HPLC - ELSD  Analysis condition device Waters 1515 Binary HPLC Pump detector ELSD 800 (Alltech) column Supelcosil ^TM LC-NH2, 5 [mu] m, 4.6 x 250 mm
(GL Sciences Inc. Japan) Mobile phase Water: MeCN = 25: 75 Drift tube temperature 55 ° C Column temperature Room temperature speed 1.0 mL / min Injection volume 10 μL

The results of the analysis are shown in FIG.

As shown in Fig. 7, the main component of the reducing sugar that constitutes the extracellular polysaccharide was glucose (B), and the liquid chromatography also confirmed that the extracellular polysaccharide was composed of glucose (A) The extracellular polysaccharide produced by Sicilactii M76 (KACC91683P) in the oocyte was found to be a polysaccharide composed of glucose, in particular, as a saccharide.

8.3. FT-IR spectrum for structural analysis

The results of the infrared absorption spectrum for the structural analysis of the extracellular polysaccharide produced from Sedilexi M76 (KACC91683P) in Pediococcus are shown in FIG.

8, the extracellular polysaccharide-specific absorption band of a strong OH group at 3492 cm ^-1, 2932 cm polysaccharide ^- O in CH group, 1670 cm ^-1 and 1459 cm ^-1 and 2892 cm ^-1 in the ^first = CO group, 1230cm ^{- 1,} it was confirmed that in the 1028 cm ^-1 CO stretch showing a strong band. Also, it was confirmed that NH stretching band at 1547 cm ^-1 had a protein structure.

All test runs of the present invention were repeated three times and expressed as an average value and a standard deviation. The significance test was performed using a Duncan ' s software package program using Version 12.0 SPSS (SPSS Inc., Chicago, multiple range test was performed.

Formulation example  One  : Pharmaceutical preparations

1.1 Manufacture of Powder

Pediococcus Sedilexi M76 (KACC91683P) strain or an extracellular polysaccharide produced therefrom 2g

Lactose 1g

The above components were mixed and packed in airtight bags to prepare powders.

1.2 Preparation of tablets

Pediococcus strain Cydacticys M76 (KACC91683P) or an extracellular polysaccharide produced therefrom

100 mg of corn starch

100 mg of milk

Magnesium stearate 2 mg

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

1.3 Preparation of capsules

Pediococcus strain Cydacticys M76 (KACC91683P) or an extracellular polysaccharide produced therefrom

100 mg of corn starch

100 mg of milk

Magnesium stearate 2 mg

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

Formulation example  2  : Manufacturing of food

Foods comprising the Pediococcus strain Sedilactii M76 (KACC91683P) or the extracellular polysaccharides produced therefrom of the present invention were prepared as follows.

2.1. Manufacture of cooking seasonings

20 to 95% by weight of Sidi Lactici M76 (KACC91683P) strain or an extracellular polysaccharide produced therefrom was added to Pediococcus to prepare a cooking sauce for health promotion.

2.2 Manufacture of tomato ketchup and sauce

0.2-1.0% by weight of Sidi Lactici M76 (KACC91683P) strain or an extracellular polysaccharide produced therefrom was added to Pediococcus to tomato ketchup or sauce to prepare health-promoting tomato ketchup or sauce.

2.3. Manufacture of flour food products

Bread, cake, cookies, crackers and noodles were prepared by adding 0.5 to 5.0% by weight of the seedy lactis M76 (KACC91683P) strain or an extracellular polysaccharide produced therefrom to Pediococcus to wheat flour, Enhancing food was prepared.

2.4. Manufacture of soups and gravies

0.1 to 5.0% by weight of Sidi Lactici M76 (KACC91683P) strain or an extracellular polysaccharide produced therefrom was added to soup and broth to produce poultry meat product, noodle soup and juice of noodle.

2.5. Manufacture of ground beef

10% by weight of Sedilexi M76 (KACC91683P) strain or extracellular polysaccharide produced therefrom was added to ground beef to prepare ground beef for health promotion.

2.6. Manufacture of dairy products

5 to 10% by weight of Sidi Lactici M76 (KACC91683P) strain or an extracellular polysaccharide produced therefrom was added to Pediococcus, and milk was used to produce various dairy products such as butter and ice cream.

Formulation example  3  : Manufacture of beverages

3.1. Manufacture of carbonated drinks

Pediococcus contains 10 to 15% of Sydactyti M76 (KACC91683P) strain or an extracellular polysaccharide produced therefrom, 5 to 10% of sugar, 0.05 to 0.3% of citric acid, 0.005 to 0.02% of caramel, 0.1 to 1% of vitamin C, Were mixed, and 75 ~ 80% of purified water was mixed therein to make a syrup. The syrup was sterilized at 85 to 98 ° C for 20 to 180 seconds and mixed with cooling water at a ratio of 1: 4. 0.5 to 0.82% of carbon dioxide gas was then injected into the Pediococcus to obtain the Sydactytis M76 (KACC91683P) A carbonated drink containing the produced extracellular polysaccharide was prepared.

3.2. Manufacture of health drinks

(2.5%, 97.16%), jujube extract (65 brix, 2.67%), over-complex extract (solid content 70%, 0.12%) and the extracellular polysaccharides produced from the strain Pediococcus M76 (KACC91683P) ), Vitamin C (0.02%), calcium pantothenate (0.02%) and licorice extract (65%, 0.01% solids) were uniformly blended and sterilized instantaneously and packaged in glass bottles, .

3.3. Manufacture of vegetable juice

Healthy vegetable juice was prepared by adding 0.5 g of Sidi Lactici M76 (KACC91683P) strain or an extracellular polysaccharide produced therefrom to Pediococcus to 1,000 ml of tomato or carrot juice.

3.4. Manufacture of fruit juices

Healthy fruit juice was prepared by adding 0.1 g of Sidi Lactici M76 (KACC91683P) strain or an extracellular polysaccharide produced therefrom to 1,000 mg of apple or grape juice to Pediococcus.

Agricultural Biotechnology Research Institute KACC91683p 20111012

Claims (9)

Pediococcus acidilactici M76 strain (Accession No .: KACC91683P) which has pancreatic beta cell proliferation and pancreatic cell death inhibitory activity.
The Pediococcus epidermalis M76 strain according to claim 1, wherein the Pediococcus strain Sedilactii M76 (Accession No .: KACC91683P) is isolated from rice wine.
delete delete A strain of Pediococcus sydilactii M76 (accession number: KACC91683P) having the activity of inhibiting the growth of pancreatic cells and the death of pancreatic cells according to claim 1, a culture of the strain, a concentrate of the culture, a dried product of the culture, Or a pharmaceutically acceptable salt or solvate thereof. The present invention also provides a pharmaceutical composition for preventing or treating diabetes.
A strain of Pediococcus sydilactii M76 (accession number: KACC91683P) having the activity of inhibiting the growth of pancreatic cells and the death of pancreatic cells according to claim 1, a culture of the strain, a concentrate of the culture, a dried product of the culture, Wherein the active ingredient is at least one selected from the group consisting of a combination of the active ingredient and the active ingredient.
An extracellular polysaccharide, which is produced from a strain of Pediococcus sydilactii M76 and has an average molecular weight of 50 kDa to 70 kDa and is composed of glucose, having the activity of inhibiting proliferation of pancreatic cells and death of pancreatic cells according to claim 1 or 2, A pharmaceutical composition for preventing or treating diabetes mellitus as an active ingredient.
An extracellular polysaccharide, which is produced from a strain of Pediococcus sydilactii M76 and has an average molecular weight of 50 kDa to 70 kDa and is composed of glucose, having the activity of inhibiting proliferation of pancreatic cells and death of pancreatic cells according to claim 1 or 2, A food composition for prevention or improvement of diabetes mellitus as an active ingredient.
1) culturing a Pediococcus acidilactici strain M76 (accession number: KACC91683P) in Pediococcus isolated from rice wine to obtain a culture medium;
2) treating the cell culture broth with hot water;
3) removing protein from the hot culture treated cell culture medium, adding ethanol, and centrifuging to obtain a precipitate; And
4) removing the remaining ethanol from the precipitate and removing the free sugars to obtain purified extracellular polysaccharides; and isolating the pediatric beta cell proliferation and pancreatic cell death-suppressing activity, A method for producing an extracellular polysaccharide, which is produced and composed of glucose and has an average molecular weight of 50 kDa to 70 kDa.

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