KR101153771B1 - Novel pediococcus pentosaceus k-51 and extracts from ginseng-fermented products using pediococcus pentosaceus k-51 for stimulating insulin secretion and manufacturing method thereof - Google Patents

Abstract

The present invention is a novel Pediococcus pentosaceus K-51 strain (Pediococcus pentosaceus K-51) (KFCC11465P) (hereinafter abbreviated as K-51), ginseng fermented extract for insulin secretion using the strain and a method for preparing the same It relates to a new K-51 capable of fermenting ginseng in more detail, and relates to a ginseng fermentation extract and fermentation extract that can be applied to enhance insulin secretion by fermenting ginseng with the strain.

      The fermented extract obtained by fermenting ginseng of the present invention with Pediococcus pentosassius K-51 strain has an excellent effect on enhancing insulin secretion and is effective in treating fasting glucose disorders, impaired glucose tolerance, persons diagnosed with prediabetes, and diabetic patients. have.

Description

Novel Pediococcus pentosaceus K-51 and extracts from ginseng-fermented products using Pediococcus pentosaceus K-51 for stimulating insulin secretion and manufacturing method

The present invention is a novel Pediococcus pentosaceus K-51 strain (Pediococcus pentosaceus K-51) (KFCC11465P) (hereinafter abbreviated as K-51), ginseng fermented extract for insulin secretion using the strain and a method for preparing the same It relates to a new K-51 capable of fermenting ginseng in more detail, and relates to a ginseng fermentation extract and fermentation extract that can be applied to enhance insulin secretion by fermenting ginseng with the strain.

Panax ginseng is a perennial root of the genus Panax in the Araliaceae family, and about 11 species of ginseng are known on the planet. Grow wild)

Ginseng is known to improve glucose homeostasis in type 1 and type 2 diabetic animals (Kimura et al., J. Pharmacobio-Dyn. 1981, 4, 410-417; Kimura et al., J. Pharmacobio Dyn. 1981, 4, 402-409; Yokozawa et al., Chem. Pharm. Bull., 1985, 33, 869-872; Kimura et al., Phytotherpy Res. 1999, 13, 484-488 Kimura et al. , Phytotherpy Res. 1999, 13, 484-488; Kimura et al., J. Pharmacobio-Dyn. 1981, 4, 907-915; Chung et al., Arch. Pharmacal.Res. 2001, 24, 214-218; Dey et al., Phytomedicine 2003, 10, 600-605). In addition, ginseng is known to improve the sugar always in human experiments (Sievenpiper et al., Diabetes 2003, 52 (Suppl. 1), A387; Vuksan et al., Diabetes 2003, 52 (Suppl. 1), A137) .

Korean white ginseng extract secretes insulin from pancreatic islets (Kimura M et al., 1981, Journal of Pharmacobiodynamics 4, 410-417), and Korean red ginseng extract has the effect of secreting insulin from pancreatic islets (Kim K and Kim HY). , 2008, Journal of Ethnopharmacology 120: 190-195).

There are various components of ginseng, and among them, saponin, phenolic component, polyacexylene component, alkaloid component, polysaccharide and the like are known as useful components for the human body. In particular, saponin is a specific ingredient contained in ginseng, called ginsenosides (ginsenosides), more than 30 species are known, and is known to play the most important role in ginseng medicinal efficacy (Park Jong-dae, 1997. Chemical composition of Korean ginseng (Review of "20 Years of Korean Ginseng Research" Korean Ginseng Society p. 69-112).

Ginsenosides, which represent saponins in ginseng, account for about 5% of ginseng dry weight and contain glucose, arabinose, and xylose on the triterpenoid-type dammarane skeleton. xylose), rhamnose (rhamnose) is a neutral glycoside combined. Protopanaxadiol (PPD) family saponins for two (3, 12) depending on the number of -OH attached to the non-sugar portion, protopanaxatriol for three (3, 6, 12) (protopanaxatriol; PPT) family is called saponin. Glucose, arabinose, xylose, rhamnose, and the like are combined with -OH at the positions R1, R2 and R3 of the PPD and PPT. More than 30 species of ginsenosides have been reported from ginseng, but a significant amount of ginseng was detected when extracting and analyzing ginseng. Six species of Rb1, Rb2, Rc, Rd, Re, and Rg1 accounted for more than 90% of the total saponin. Saponin components are low in content.

Ginsenosides are not absorbed in the human body as they are, but are degraded by microorganisms such as Bacteriodes, Fusobactrium, Eubacterium, and Provetella species present in the human intestine to absorb their metabolites (Chi et al., 2005). In the case of ginsenoside Rb1, it is sequentially converted to ginsenoside Rd, compound K (hereinafter abbreviated as C-K) and protopanaxadiol by intestinal microorganisms. PPD ginsenosides Rb1, Rb2, Rc, Rd are metabolized and absorbed by 20-O-β-D-glucopyranosyl-20 (S) -protopanaxadiol (C-K) in the intestine (Akao et al., 1998). PPT-based ginsenosides Re, Rf, and Rg1 are metabolized into Rh1, F1 and protopanaxatriol in the gut (Hasegawa 2004; Tawab et al., 2003). Rb1, Rb2, Rc, Rd, etc. are converted to 20 (R)-/ 20 (S) -ginsenoside Rg3 by enteric microorganisms (Cheng et al., Phytochemistry 2008), and Rg3 is converted to Rh2 by enteric microorganisms ( Su et al., J Mol Cat B: Enzymatic 2006).

Intestinal microorganisms that break down ginsenosides differ in their presence and retention according to human constitution and dietary habits, and there is a difference in the conversion rate to metabolites. have. Due to different intestinal microflora, ginsenoside conversion and bioavailability are inevitably different (J. Pharm. Pharmacol., 50, pp. 1155-1160, 1998). Therefore, in order to eliminate the difference in efficacy and absorption rate due to the difference between the intestinal microorganisms, it is necessary to find a microorganism that metabolizes ginsenosides.

On the other hand, ginsenosides are due to the ginseng side effect, but the view that the ginsenoside is a complex action rather than a single ginsenoside is predominant. Therefore, microorganisms that metabolize ginsenosides may be more useful than microorganisms that convert ginsenosides in combination.

Blood glucose in the body is always maintained at 80-120 mg / dL, which is called glucose homeostasis (abbreviated as glucose homeostasis). In the process of maintaining the human body glycemic insulin (insulin) and glucagon (glucagon) secreted from the beta (β) cells and alpha (α) cells of the pancreas, respectively. Immediately after a meal, blood sugar rises, and insulin secreted at this time supplies glucose to the peripheral tissues such as liver, muscle, and fat cells to be used as an energy source, and glucose is stored in the liver and muscle as glycogen (glycogen). Normalize Fasting lowers blood sugar and glucagon is secreted at this time to break down glycogen into glucose in the liver to normalize blood sugar (Miyazaki Y, Glass L, Triplitt C, Wajcberg E, Mandarino LJ, DeFronzo RA.2002. Am J Physiol Endocrinol Metab 283 , E1135-43).

Type 2 diabetes is a condition in which blood sugar levels are higher than normal, and a disease occurs when the balance of insulin secretion and the secretion of insulin from pancreatic beta cells is broken in peripheral tissues such as liver, muscle, and fat cells. Reduction of insulin action in peripheral tissues lowers glucose utilization in most tissue cells, and in particular, despite high blood sugar levels in the liver, decreases glycogen synthesis and increases glucose synthesis through your biosynthesis, resulting in higher blood sugar levels. Play a role. As a result, if insulin secretion is sufficiently increased to compensate for insulin resistance, it does not progress to diabetes, but when insulin secretion is insufficient, it progresses to diabetes.

Therefore, the ability of the human body to detect glucose by secreting glucose in the blood is important for maintaining the glucose always. When insulin secretion is reduced, insulin secretagogue should be taken. Insulin secreting agents include insulin secretion initiators that can secrete insulin by themselves and insulin secreting agents that enhance insulin secretion secreted by the insulin secretion initiator.

Accordingly, the present inventors describe a novel Pediococcus pentosaceus K-51 strain (KFCC11465P) (hereinafter abbreviated as K-51), which is a lactic acid bacterium that complexly converts ginsenosides contained in ginseng. After selection from human feces, the strains were identified by 16S rRNA partial sequencing, which was identified in terms of molecular genetics.

The g-51 is treated with ginseng, and more specifically, ginseng is fermented with the K-51, and ginsenosides Rg1 and Rb1 having low bioavailability in ginseng are ginsenosides Rh1, Rg3, Rh2 and CK having high bioavailability. Or ginsenosides Rh1, Rg3, Rh2, CK and the like which have high bioavailability in ginseng.

Accordingly, an object of the present invention is to provide a lactic acid bacterium K-51 capable of generating and / or converting ginsenosides Rg1, Rb1, Rg1, Re and the like to ginsenosides Rh1, Rg3, Rh2, CK and the like with low bioavailability in ginseng. To provide a method for producing a ginseng fermented product using the K-51, a ginseng fermented product prepared by the method, a pharmaceutical preparation comprising the ginseng fermented product and / or a health functional food comprising the ginseng fermented product. .

       In addition, the present inventors have been reported to increase insulin secretion action of ginseng as mentioned in the background, but since the activity was not high, it was determined that it is necessary to enhance the activity. The present inventors found that insulin secretion activity is increased by fermenting ginseng with K-51, thus completing the present invention. Therefore, another object of the present invention is to provide a ginseng fermentation extract for enhancing insulin secretion by extracting after fermenting ginseng with K-51.

In order to achieve the above object, the present invention provides K-51, and also provides a ginseng fermentation extract for improving insulin secretion prepared by fermenting ginseng with K-51.

In addition, the present invention (1) the step of fermenting ginseng K-51 to obtain a fermentation product; And

(2) to provide a method for preparing the ginseng fermentation extract for insulin secretion to include the step of extracting the fermented product and then filtering the fermentation.

The method of preparing the ginseng fermented extract may further include a step of obtaining the extract by concentration or freeze drying under reduced pressure after mixing or mixing the filtrate after step (2) alone.

The present invention can provide a novel K-51 capable of fermenting ginseng.

The present invention ginseng fermentation extract that can convert and / or generate ginsenosides Rh1, Rg3, Rh2, CK and the like with low bioavailability in ginseng using the novel K-51 It can provide a manufacturing method of.

The ginseng fermentation extract for insulin secretion enhancement according to the present invention increases the insulin secretion in pancreatic islets, showing an increased insulin secretion effect than the effect that ginseng itself exhibits.

The present invention represents a novel Pediococcus pentosaceus K-51 strain (Pediococcus pentosaceus K-51) (KFCC11465P) (hereinafter abbreviated as K-51).

The present invention represents a novel K-51 capable of fermenting ginseng.

The present invention provides a ginseng fermentation extract for enhancing insulin secretion prepared by fermenting ginseng with K-51.

The present invention (1) the step of fermenting ginseng with K-51 to obtain a fermentation product; (2) it provides a method for producing a fermented ginseng extract for insulin secretion comprising the step of extracting the fermented product as a solvent and filtering.

The method of preparing the ginseng fermented extract may further include a step of obtaining the extract by concentration or freeze drying under reduced pressure after mixing or mixing the filtrate after step (2) alone.

The novel K-51 of the present invention receives the feces of the human body and separates a single colony (colon) by counting dilution method in BL blood medium, and then takes a single colony showing the characteristics and properties of lactic acid bacteria again, liquid of MRS broth After inoculating the medium and cultured to obtain a strain. DNA was extracted from the strain, 16S rRNA was performed, and the homology of the strain was searched using the BLAST program. The strain was found to have 100% homology with 16S rRNA gene of strain CTSPL1 of Pediococcus pentosassius. It was named Pediococcus pentosaceus K-51 strain and was deposited on September 17, 2009 at the Korea Microorganism Conservation Center and received the strain number of KFCC11465P.

The novel K-51 of the present invention can grow in high concentration ginseng medium, while metabolizing the highly polar ginsenosides Rg1, Rb1 into the less polar ginsenosides Rh1, Rg3, C-K and Rh2. In the present invention, the ginseng was not fermented simply to increase the contents of Rh1, Rg3, CK, and Rh2, and Rh1 as an indicator component of K-51 fermentation process monitoring to enhance sugar content by fermentation by K-51. , Rg3, CK and Rh2 were set.

In the following examples of the present invention, dried ginseng, which is a kind of ginseng, was prepared to produce white ginseng, and then fermented by separating into main roots (MR) and hairy roots (HR). However, the method of fermenting the white ginseng itself without separating into the main root and the ginseng may also be included in the scope of the present invention. In addition, the method of steaming white ginseng and processing them into red ginseng and ginseng may also fall within the scope of the present invention. In the present invention, ginseng is a solid ginseng cut to a predetermined size, ginseng powdered and suspended in purified water, ginseng suspension, ginseng extracted in purified water and / or an organic solvent to be used in any one selected form of ginseng extract Can be. At this time, the organic solvent may be used ethanol or alcohol having a concentration of 30 to 95%.

As an example of the preparation of ginseng fermentation extract for promoting insulin secretion of the present invention (1) fermenting ginseng suspension with K-51 to obtain a fermentation product, (2) extracting the fermentation product with a solvent of ethanol, alcohol or butanol It may provide a method for preparing a ginseng fermentation extract for promoting insulin secretion comprising the step of filtering after.

The ginseng fermentation extract according to the present invention is filtered by extracting the ginseng fermentation product obtained by inoculating fermentation by inoculating K-51 in the medium using the ginseng powder or liquid medium using the ginseng extract with a solvent of ethanol, alcohol or butanol You can get it.

Insulin secretion promoting extract of the present invention is characterized in that it contains a lot of ginsenoside Rh1 (protopanaxatriol) and Rh2 (protopanaxadiol) at the same time compared to the conventional ginseng fermentation, which This is a further step in the ginseng fermentation patent.

The ginseng fermented extract of the present invention enhances insulin secretion in pancreatic islets isolated from rats.

In addition, the ginseng fermented extract of the present invention can prevent and / or treat insulin secretion abnormalities, glucose intolerance, fasting glucose disorders, prediabetes, and diabetes by improving glycemic condition.

The present invention shows a method for producing a ginseng fermented extract using the novel K-51.

The present invention can convert and / or generate ginsenosides Rg1, Rb1, Rg1, Re and the like to ginsenosides Rh1, Rg3, Rh2, CK and the like in ginseng using the novel K-51. It shows the preparation method of ginseng fermented extract.

The present invention shows a method for producing a ginseng fermented extract which can increase insulin secretion using the novel K-51.

The present invention (1) fermenting the ginseng suspension with K-51 to obtain a fermentation product; And (2) extracting the fermented product as a solvent and then filtering the ginseng fermented extract.

The present invention comprises the steps of (1) fermenting ginseng powder suspension obtained by adding white ginseng root powder, white ginseng samsam powder and ginseng powder to purified water for 3-10 days at 35-37 ° C. with K-51; And (2) extracting the fermented product from 60 ° C. to 80 ° C. for 2 to 4 hours with a solvent of ethanol, alcohol or butanol, followed by filtration.

Ethanol in 30-99% concentration can be used for ethanol in said solvent.

As the alcohol in the solvent, alcohol having a concentration of 30 to 99% can be used.

Investigation on the preparation method of the ginseng fermentation extract using the novel K-51 of the present invention, in order to achieve the object of the present invention, the method for producing a ginseng fermentation extract using the novel K-51 under the above-mentioned conditions It is desirable to provide.

The present invention includes the ginseng fermented extract using the novel K-51 by the above-mentioned method.

The present invention represents a pharmaceutical formulation comprising a ginseng fermented extract prepared using the novel K-51 mentioned above.

The present invention represents a pharmaceutical preparation for insulin secretion enhancement comprising a ginseng fermented extract prepared using the novel K-51 mentioned above.

The ginseng fermented extract prepared using K-51 in the pharmaceutical preparation for insulin secretion enhancement comprising the ginseng fermented extract prepared using K-51 is used to be 10 to 30% by weight relative to the total weight of the pharmaceutical preparation. Can be.

The present invention represents a health functional food comprising a ginseng fermented extract prepared using the novel K-51 mentioned above.

The present invention represents a dietary supplement for insulin secretion containing ginseng fermented extract prepared using the novel K-51 mentioned above.

The ginseng fermented extract prepared using K-51 in the insulin-enhanced dietary supplement containing ginseng fermented extract prepared using K-51 is used to be 10 to 30% by weight relative to the total weight of the dietary supplement. Can be.

Hereinafter, the present invention will be described in detail with reference to Examples and Test Examples. However, these are intended to explain the present invention in more detail, and the scope of the present invention is not limited thereto.

Example 1 Isolation of Pediococcus pentosaceus K-51 Strain (Pediococcus pentosaceus K-51) (KFCC11465P)

Human feces were isolated to separate single colonies by counting dilution in BL blood medium. Among them, a single colony showing characteristics and properties corresponding to lactic acid bacteria was taken and inoculated into a liquid medium of MRS broth, and then cultured in a 37 ° C incubator for 24 hours, and then strains were separated from the culture solution.

DNA was extracted from the strain, 16S rRNA was produced, and homology was searched using the BLAST program.

DNA was extracted from the strain using a DNA purification kit, and the PCR primer [27f (5′-agagtttgatcctggctcag-3 ′) was used to target a highly conserved sequence region at both ends of the 16S rRNA molecule. 1492r (5'-ggctaccttgttacgactt-3 '] was prepared by PCR to obtain a PCR product having a size corresponding to about 67% of the total area.

PCR product detection was performed using agarose gel electrophoresis. The target band was cut out and eluted, and then sequencing was performed using newly prepared sequencing primers [r4L (5'-ACGGGCGGTGTGTACAAG-3 ') and f2L (5'-CCAGCAGCCGCGGTAATAG-3']. Homology was searched using the BLAST program, a program that analyzes sequence data on the bed.

A blast search of the product of the strain revealed that it had 100% identity with the 16S rRNA gene of Pediococcus pentosaceus strain CTSPL1, and the strain was identified as Pediococcus pentosassius K-51. It was named Jude (Pediococcus pentosaceus K-51) and it was deposited with KFCC11465P on September 17, 2009.

Meanwhile, the 16s DNA sequence of K-51 is shown in SEQ ID NO: 5.

Example 2 Mycological Properties of K-51

The bacteriological properties of K-51 are characterized by the classification of microorganisms by Hammes et al. The prokaryotes, 1563-1578, 2nd Edition, Springer-Verlag Co. (1992)], and the results are shown in Table 1. The strain was Gram positive, cocci, well grown with or without oxygen, and catalase positive.

Table 1. Physiological and biochemical properties of K-51

K-51 Shape Coccus Gram Dyeing Positive Indole - Methyl red + Nitrate reduction - Catalase + D-Glucose Availability + D-Fructose Availability + L-Arabinose Availability - D-Xylose Availability - Lactose Availability + Starch Availability - Amygdalin Degradable - Arbutin degradable + Aerobic growth + Anaerobic growth +

Preparation Example 1 Preparation of Ginseng Powder

The ginseng was dried 5 years old root of Anseong, separated into white ginseng root, white ginseng root (misam), and then pulverized to produce white ginseng root powder and white ginseng root (misam) powder.

Ginseng was dried for 5 years, Anseong acid and steamed for 4 hours at 98 ℃ to obtain red ginseng and milled to prepare a red ginseng powder.

Ginseng was dried for 5 years, Anseong acid, and then dried at 120 ° C. for 2.5 hours to obtain ginseng, which was then ground to prepare ginseng powder.

Preparation Example 2 Preparation of Ginseng Distilled Water Extract Powder

10 liters of distilled water was added per 1 kg of ginseng powder of white ginseng root powder and ginseng root powder obtained in Preparation Example 1, extracted at 90 ° C. for 3 hours to obtain an extract, and then lyophilized to prepare a powder.

Preparation Example 3 Preparation of Ginseng Ethanol Extract Powder

10 liters of ethanol per 1 kg of ginseng powder of white ginseng fructus powder and rice ginseng fructus powder obtained in Preparation Example 1 were added thereto, extracted at 70 ° C. for 3 hours, dried under reduced pressure, and lyophilized to prepare a powder.

Example 3-1 Preparation of White Ginseng Root Ferment

2,000 g of distilled water was added to 20 g of white ginseng fructus powder obtained in Preparation Example 1, and sterilized. Then, 2 g of K-51 was added thereto, followed by fermentation at 37 ° C. for 7 days to obtain a fermented product.

The fermented product was extracted with 2,000 ml of ethanol at 70 ° C. for 3 hours and filtered to prepare white ginseng root fermentation extract.

Example 3-2 Preparation of White Ginseng Root Ferment

Distilled water 2,000ml was added to 20g white ginseng root (misam) powder obtained in Preparation Example 1, and sterilized. Then, 2 g of K-51 was added thereto and fermented at 37 ° C. for 7 days to obtain a fermented product.

The fermented product was extracted with 2,000 ml of butanol at 70 ° C. for 3 hours and filtered to prepare a white ginseng root fermentation extract.

Example 3-3 Preparation of White Ginseng Root Ferment

Distilled water 2,000ml was added to 20g white ginseng root (misam) powder obtained in Preparation Example 1, and sterilized. Then, 2 g of K-51 was added thereto and fermented at 37 ° C. for 7 days to obtain a fermented product.

The fermented product was extracted with 2,000 ml of alcohol at 90% concentration for 3 hours at 70 ° C. and filtered to prepare a white ginseng root fermentation extract.

Example 4-1 Preparation of White Ginseng Roots

2,000 g of distilled water was added to 20 g of white ginseng fructus powder obtained in Preparation Example 1, and sterilized. Then, 2 g of K-51 was added thereto, followed by fermentation at 37 ° C. for 7 days to obtain a fermented product.

The fermented product was extracted with 2,000 ml of ethanol at 70 ° C. for 3 hours and filtered to prepare a white ginseng root (misam) fermented extract.

Example 4-2 Preparation of White Ginseng Roots

Distilled water 2,000ml was added to 20g white ginseng root (misam) powder obtained in Preparation Example 1, and sterilized. Then, 2 g of K-51 was added thereto and fermented at 37 ° C. for 7 days to obtain a fermented product.

The fermented product was extracted with 2,000 mL of butanol at 70 ° C. for 3 hours and filtered to prepare a white ginseng root (misam) fermented extract.

Example 4-3 Preparation of White Ginseng Roots

Distilled water 2,000ml was added to 20g white ginseng root (misam) powder obtained in Preparation Example 1, and sterilized. Then, 2 g of K-51 was added thereto and fermented at 37 ° C. for 7 days to obtain a fermented product.

The fermented product was extracted with 2,000 ml of alcohol at 90% concentration for 3 hours at 70 ° C., and filtered to prepare a white ginseng root (misam) fermented extract.

Example 5-1 Preparation of Red Ginseng Fermented Products

2,000 g of distilled water was added to 20 g of red ginseng powder obtained in Preparation Example 1, and sterilized. Then, 2 g of K-51 was added and fermented at 37 ° C. for 7 days to obtain a fermented product.

The fermented product was extracted with 2,000 ml of ethanol at 70 ° C. for 3 hours and filtered to prepare a red ginseng fermented extract.

Example 5-2 Preparation of Red Ginseng Fermented Products

2,000 g of distilled water was added to 20 g of red ginseng powder obtained in Preparation Example 1, and sterilized. Then, 2 g of K-51 was added thereto, followed by fermentation at 37 ° C. for 7 days to obtain a fermented product.

The fermented product was extracted with 2,000 ml of butanol at 70 ° C. for 3 hours and filtered to prepare a red ginseng fermented extract.

Example 5-3 Preparation of Fermented Red Ginseng

2,000 g of distilled water was added to 20 g of red ginseng powder obtained in Preparation Example 1, and sterilized. Then, 2 g of K-51 was added and fermented at 37 ° C. for 7 days to obtain a fermented product.

The fermented product was extracted with 2,000 ml of alcohol at 90% concentration for 3 hours at 70 ° C. and filtered to prepare a red ginseng fermented extract.

<Example 6-1> Preparation of fermented ginseng

2,000 g of distilled water was added to 20 g of the ginseng powder obtained in Preparation Example 1, followed by sterilization, and 2 g of K-51 was added thereto, followed by fermentation at 37 ° C. for 7 days to obtain a fermented product.

The fermented product was extracted with 2,000 ml of ethanol at 70 ° C. for 3 hours and filtered to prepare a ginseng fermented extract.

<Example 6-2> Preparation of fermented ginseng

2,000 g of distilled water was added to 20 g of the ginseng powder obtained in Preparation Example 1, followed by sterilization, and 2 g of K-51 was added thereto, followed by fermentation at 37 ° C. for 7 days to obtain a fermented product.

The fermented product was extracted with 2,000 ml of butanol at 70 ° C. for 3 hours and filtered to prepare a ginseng fermented extract.

<Example 6-3> Preparation of fermented ginseng

2,000 g of distilled water was added to 20 g of the ginseng powder obtained in Preparation Example 1, followed by sterilization, and 2 g of K-51 was added thereto, followed by fermentation at 37 ° C. for 7 days to obtain a fermented product.

The fermented product was extracted with 2,000 ml of alcohol at 90% concentration for 3 hours at 70 ° C. and filtered to prepare a ginseng fermented extract.

Comparative Example 1 Preparation of White Ginseng Extract

2000 g of white ginseng fructus powder obtained in Preparation Example 1 was added to sterilize 2,000 ml of distilled water, extracted with 2,000 ml of ethanol at 70 ° C. for 3 hours, and filtered to prepare white ginseng fructus extract.

Comparative Example 2 Preparation of White Ginseng Misam Extract

2,000 g of distilled water was added to 20 g of the white ginseng rice ginseng powder obtained in Preparation Example 1 and sterilized, followed by extraction with 2,000 ml of ethanol at 70 ° C. for 3 hours, followed by filtration.

Test Example 1 Content Analysis Experiment

Each extract obtained in Example 3-1, Example 4-1, Comparative Example 1, and Comparative Example 2 was added with the same amount of methanol (methanol) and centrifuged to obtain a supernatant.

100 ml of each of the supernatants obtained above was concentrated under reduced pressure, 0.2 g of each extract was extracted, and 100 ml of methanol were extracted three times. The extract was concentrated under reduced pressure, suspended in 100 ml of water, extracted three times with 100 ml of ether, and the remaining water fraction was extracted three times with 100 ml each and concentrated under reduced pressure. Subsequently, it was dissolved in 1 ml of MeOH and analyzed by HPLC to obtain a saponin content analysis result as shown in Table 2.

HPLC analysis conditions: Younglin HPLC system: Younglin HPLC system-[Younglin SP930D quaternary pump (Seoul, Korea) and ELSD (Attech Co., USA) detector]: Column, Develosil ODS-UG-5 Batch # 10054 (NOMURA CHEMICAL, Japan); Solvent, distilled water-acetonitrile [0-35 min (90:10), 35-30 min (5:95)]

As a result of the experiment, Rh1, Rg3, CK, Rh2, which were not contained in white ginseng root, contained 1.6%, 1.4%, 0.6%, 4.5% in fermented white ginseng root of K-51 fermentation of Example 3-1, respectively. Rh1, Rg3, CK, and Rh2, which were not contained in rice ginseng, were contained 4.2%, 3.9%, 2.0%, and 3.0% in fermented rice ginseng by K-51 fermentation of Example 4-1, respectively (Table 2). That is, the components not contained in ginseng were produced in fermented white ginseng and / or fermented rice ginseng by K-51 fermentation.

That is, from the results of Table 2, the fermentation product of the ginseng using K-51 produces components not contained in simple ginseng, and the K-51 produces ginsenosides Rh1, Rg3, CK, and Rh2 in ginseng. And / or can be converted.

Table 2. Effect of Ginseng Fermentation by K-51 on Ginsenoside Content

sample
Ginsenoside content (%) Rg1 Rb1 Rh1 Rg3 C-K Rh2 White ginseng root
(Comparative Example 1) 6.5 ± 0.2 4.5 ± 0.2 ^-1) - - - K-51 Fermented White Ginseng Root
(Example 3-1) 2.2 ± 0.2 0.8 ± 0.1 1.6 ± 0.3 1.4 ± 0.2 0.6 ± 0.0 4.5 ± 0.1 Misam
(Comparative Example 2) 11.5 ± 1.1 9.0 ± 0.3 - - - - K-51 Fermented Misam
(Example 4-1) 9.4 ± 0.5 1.6 ± 0.1 4.2 ± 0.9 3.9 ± 0.1 2.0 ± 0.2 3.0 ± 0.5

¹⁾ -not detected.

Test Example 2 Effect on Insulin Secretion in Pancreatic Islets

-Insulin secretion was verified by primary culture of rat pancreatic islets, a laboratory system that responds to glucose.

Isolation and Culture of Pancreatic Islets

Isolation of pancreatic islets from Sprague-Dawley rats was performed by injecting collagenase directly into the bile duct. The detached pancreas was isolated by using a ficoll gradient. Sodium was contained in Krebs-Ringer bicarbonate buffer (KRB, pH 7.2) and used after incubation in 37 ° C., 5% CO ₂ incubator for 1 day.

Examination of insulin secretion

Pancreatic islets incubated for 24 hours were washed twice with KRB, and then three of them were fixed in size per well. The secretion ability according to the glucose concentration, the secretion ability according to the culture time, and the insulin secretion ability by the bioconversion were measured. The insulin concentration was measured using a kit using enzyme immunoassay (EIA).

From the above results, the ethanol extract of the K-51 fermentation of white ginseng root of the present invention showed insulin secretion at the media concentration of 3.3 mM (baseline) and insulin secretion at the media concentration of 16.7 mM (glucose stimulation) compared to the ginseng extract alone. Was increased compared to the ginseng extract alone. In other words, the effect of the insulin secretion initiator and enhancer was increased compared to the white ginseng root. This is the ideal effect for those who do not always control the sugar, which is shown in Table 3.

The ethanol extract of K-51 fermented ginseng of the present invention had no difference in the (base) insulin secretion at the medium concentration of 3.3 mM, but the insulin secretion (glucose stimulation) at the medium concentration of 16.7 mM compared to that used alone. Was increased compared with that used alone. In other words, the effect as an insulin secretion enhancer is improved compared to ginseng. This is an effect that enhances insulin secretion by glucose is an ideal effect for those who do not always control the glucose, it is shown in Table 4.

Butanol extract of K-51 lactic acid bacterium fermentation of ginseng of the present invention showed no difference in basal insulin secretion at 3.3mM medium compared to that of ginseng extract alone, but insulin secretion at glucose concentration of 16.7mM (glucose stimulation) ) Was increased compared to that used alone. In other words, the effect of insulin secretion enhancer was increased compared to ginseng. This is an effect that enhances insulin secretion by glucose is an ideal effect for those who do not control glucose homeostasis, it is shown in Table 5.

Table 3. Insulin Secretion Effects of Ethanol Extracts from K-51 Fermented White Ginseng Root in Pancreatic Islets

Item
Concentration (㎍ / mL)
Insulin secretion (ratio to control) 3.3mM glucose medium 16.7mM Glucose Medium Control group - 1.000 ± 0.103 8.825 ± 0.729 White Ginseng Freckle Extract
(Comparative Example 1) 200 6.950 ± 0.269 *** 14.278 ± 4.798 K-51 Fermentation
(Example 3-1) 200 24.53 ± 6.76 *** 29.28 ± 7.09 ***

-Values are mean ± SEM (n = 6).

-Significantly different from control group at * P <0.05, *** P <0.001.

-The control group in Table 3 represents white ginseng freckles powder.

-White ginseng freckle extract in Table 3 shows the extract obtained in Comparative Example 1.

-K-51 fermented product in Table 3 represents the ethanol extract of K-51 fermented white ginseng roots obtained in Example 3-1.

Table 4. Effect of insulin secretion on pancreatic islets of ethanol extract of K-51 fermented rice ginseng

Item
Concentration (㎍ / mL)
Insulin secretion (ratio to control) 3.3mM glucose medium 16.7mM Glucose Medium Control group - 1.000 ± 0.103 8.825 ± 0.729 Misam Extract
(Comparative Example 2) 200 12.934 ± 4.991 *** 13.075 ± 4.694 K-51 Fermentation
(Example 4-1) 200 16.51 ± 4.26 *** 34.90 ± 12.09 **

-Values are mean ± SEM (n = 6).

-Significantly different from control group at ** P <0.01, *** P <0.001.

-In Table 4, the control represents the ginseng powder.

-In Table 4, the extract of Samsam represents the extract obtained in Comparative Example 2.

-K-51 fermented product in Table 4 represents the ethanol extract of K-51 fermented rice ginseng obtained in Example 4-1.

Table 5. Insulin Secretion Effects of Butanol Extract of K-51 Fermented Ginseng on Pancreatic Islets

Item
Concentration (㎍ / mL)
Insulin secretion (ratio to control) 3.3mM glucose medium 16.7mM Glucose Medium Control group - 1.000 ± 0.103 8.825 ± 0.729 Misam Extract
(Comparative Example 2) 200 1.007 ± 0.028 17.981 ± 7.024 ^* K-51 Fermentation
(Example 4-2) 200 2.06 ± 1.35 35.71 ± 12.58 ***

-Values are mean ± SEM (n = 6).

Significantly different from control group at * P <0.05, ** P <0.01, *** P <0.001.

-In Table 5, the control represents the ginseng powder.

-In Table 5, the extract of Samsam represents the extract obtained in Comparative Example 2.

K-51 fermented product in Table 5 shows the butanol extract of K-51 fermented rice ginseng obtained in Example 4-2.

<Example 7 to Example 9> pharmaceutical preparations for enhancing insulin secretion

White ginseng fructus fermentation ethanol extract, calcium, arginine, lysine, carnitine, magnesium stearate, lactose, starch and agar according to K-51 of Example 3-1 were mixed and granulated, respectively, in the composition ratios shown in Table 6 below. The following tablet machine was used to prepare a pharmaceutical formulation for enhancing insulin secretion in a tablet form.

Table 6. Composition of pharmaceutical preparations for enhancing insulin secretion (unit: weight%)

Item Example 7 Example 8 Example 9 K-51 Fermentation 10 wt% 20 wt% 30 wt% calcium 5 wt% 5 wt% 5 wt% Argimin 5 wt% 5 wt% 5 wt% Lysine 5 wt% 5 wt% 5 wt% Carnitine 5 wt% 5 wt% 5 wt% Magnesium stearate 1 wt% 1 wt% 1 wt% Lactose 1 wt% 1 wt% 1 wt% Starch 1 wt% 1 wt% 1 wt% Agar 67 wt% 57 wt% 47 wt% Sum 100 wt% 100 wt% 100 wt%

* K-51 fermented product in Table 6 is ethanol extract of white ginseng root fermented by K-51 of Example 3-1.

<Examples 10 to 12> Health functional food for insulin secretion enhancement

White ginseng fructus fermentation ethanol extract, calcium, chlorella powder, xylitol, mulberry leaf powder, dongle green tea powder, green tea powder, agar were mixed and granulated respectively in the composition ratios shown in Table 7 below according to K-51 of Example 3-1. Then, the health functional food for enhancing insulin secretion in the form of a ring was prepared by using a depilator.

Table 7. Composition of dietary supplements for improving insulin secretion (unit: weight%)

Item Example 10 Example 11 Example 12 K-51 Fermentation 10 wt% 20 wt% 30 wt% calcium 5 wt% 5 wt% 5 wt% Chlorella Powder 5 wt% 5 wt% 5 wt% Xylitol 5 wt% 5 wt% 5 wt% Mulberry Leaf Powder 10 wt% 10 wt% 10 wt% Roundle powder 5 wt% 5 wt% 5 wt% Green tea powder 5 wt% 5 wt% 5 wt% Agar 55 wt% 45 wt% 35 wt% Sum 100 wt% 100 wt% 100 wt%

* K-51 fermented product in Table 7 is the ethanol extract of white ginseng roots by K-51 of Example 3-1.

The novel Pediococcus pentosaceus K-51 strain (Pediococcus pentosaceus K-51) (KFCC11465P) of the present invention can ferment ginseng or ginseng extract to obtain a ginseng fermented extract.

The Pediococcus pentosassius K-51 strain of the present invention converts and / or produces low ginsenosides Rg1, Rb1, Rg1, Re and the like into ginsenosides Rh1, Rg3, Rh2, CK, etc. in ginseng. Can be applied to pharmaceutical preparations, health functional foods containing the ginseng fermented extract according to the present invention.

The ginseng fermentation extract for enhancing insulin secretion of the present invention can restore the function of maintaining the sugar always when used as a pharmaceutical composition, fasting glucose impaired, impaired glucose tolerance, those diagnosed with prediabetes, pancreatic beta cells of diabetic patients There is an effect that can be used as a protective agent.

<110> KOREA FOOD RESEARCH INSTITUTE <120> Novel Pediococcus pentosaceus K-51 and extracts from          ginseng-fermented products using Pediococcus pentosaceus K-51 for          stimulating insulin secretion and manufacturing method <160> 5 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> A primer for sequencing of 27f <400> 1 agagtttgat cctggctcag 20 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> A primer for sequencing of 1492r <400> 2 ggctaccttg ttacgactt 19 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> A primer for sequencing of r4L <400> 3 acgggcggtg tgtacaag 18 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> A primer for sequencing of f2L <400> 4 ccagcagccg cggtaatag 19 <210> 5 <211> 1455 <212> DNA <213> Pediococcus pentosaceus K-51 <400> 5 atacatgcaa gtcgaacgaa cttccgttaa ttgattatga cgtacttgta ctgattgaga 60 ttttaacacg aagtgagtgg cgaacgggtg agtaacacgt gggtaacctg cccagaagta 120 ggggataaca cctggaaaca gatgctaata ccgtataaca gagaaaaccg catggttttc 180 ttttaaaaga tggctctgct atcacttctg gatggacccg cggcgtatta gctagttggt 240 gaggtaaagg ctcaccaagg cagtgatacg tagccgacct gagagggtaa tcggccacat 300 tgggactgag acacggccca gactcctacg ggaggcagca gtagggaatc ttccacaatg 360 gacgcaagtc tgatggagca acgccgcgtg agtgaagaag ggtttcggct cgtaaagctc 420 tgttgttaaa gaagaacgtg ggtaagagta actgtttacc cagtgacggt atttaaccag 480 aaagccacgg ctaactacgt gccagcagcc gcggtaatac gtaggtggca agcgttatcc 540 ggatttattg ggcgtaaagc gagcgcaggc ggtcttttaa gtctaatgtg aaagccttcg 600 gctcaaccga agaagtgcat tggaaactgg gagacttgag tgcagaagag gacagtggaa 660 ctccatgtgt agcggtgaaa tgcgtagata tatggaagaa caccagtggc gaaggcggct 720 gtctggtctg caactgacgc tgaggctcga aagcatgggt agcgaacagg attagatacc 780 ctggtagtcc atgccgtaaa cgatgattac taagtgttgg agggtttccg cccttcagtg 840 ctgcagctaa cgcattaagt aatccgcctg gggagtacga ccgcaaggtt gaaactcaaa 900 agaattgacg ggggcccgca caagcggtgg agcatgtggt ttaattcgaa gctacgcgaa 960 gaaccttacc aggtcttgac atcttctgac agtctaagag attagaggtt cccttcgggg 1020 acagaatgac aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt 1080 cccgcaacga gcgcaaccct tattactagt tgccagcatt aagttgggca ctctagtgag 1140 actgccggtg acaaaccgga ggaaggtggg gacgacgtca aatcatcatg ccccttatga 1200 cctgggctac acacgtgcta caatggatgg tacaacgagt cgcgagaccg cgaggttaag 1260 ctaatctctt aaaaccattc tcagttcgga ctgtaggctg caactcgcct acacgaagtc 1320 ggaatcgcta gtaatcgcgg atcagcatgc cgcggtgaat acgttcccgg gccttgtaca 1380 caccgcccgt cacaccatga gagtttgtaa cacccaaagc cggtggggta accttttagg 1440 agctagccgt ctaag 1455  

Claims (7)

A novel Pediococcus pentosaceus K-51 strain (KFCC11465P) having a ginseng fermentation product with improved glucose homeostasis control effect. delete (1) fermenting ginseng to Pediococcus pentosaceus K-51 strain (Pediococcus pentosaceus K-51) (KFCC11465P) to obtain a fermentation product; And (2) Method of producing a ginseng fermentation extract improved glucose homeostasis control effect, characterized in that it comprises the step of extracting the fermented product as a solvent and filtering. The ginseng powder suspension of (1) white ginseng root powder, white ginseng powder, red ginseng powder or ginseng powder is added to purified water. ) (KFCC11465P) to ferment for 3-10 days at 35 ~ 37 ℃ to obtain a fermentation product, (2) the production of ginseng fermentation extracts with improved glucose homeostasis control effect, comprising the step of extracting the fermented product above at 60 ° C. to 80 ° C. for 2 to 4 hours with a solvent of ethanol, alcohol or butanol, followed by filtration. Way. Ginseng fermented extract with improved glucose homeostasis control effect prepared by the method of claim 3 or 4. A pharmaceutical preparation for enhancing insulin secretion comprising a ginseng fermented extract having an improved glucose homeostasis control effect prepared by the method of claim 3 or 4. Claims 3 or 4 health functional foods for improving insulin secretion comprising a ginseng fermented extract with improved glucose homeostasis control effect.