KR20180090594A - Novel Bacillus subtilis 4-1-1 and method for fermented ginseng using the same - Google Patents

Abstract

The present invention relates to a novel Bacillus subtilis strain and uses of the same. More specifically, the present invention relates to a novel Bacillus subtilis strain 4-1-1 (KCCM: 11953P); fermented ginseng fermented using the same; a method for preparing fermented ginseng; and a functional food composition comprising fermented ginseng. The fermented ginseng fermented using the Bacillus subtilis strain 4-1-1 (KCCM: 11953P) according to the present invention has dramatically increased panaxadiol (PPD) content with respect to panaxatriol (PPT) content; in particular, has an increased content of ginsenoside Rd having various physiological activities; and shows excellent thrombolytic activity. Thus, the fermented ginseng of the present invention has further enhanced functionalities compared to conventional fermented ginseng, which can enhance the health of consumers, and thus can be used as a raw material for functional health foods.

Description

A novel Bacillus subtilis strain and a method for producing fermented ginseng using the same,

The present invention relates to a novel strain of Bacillus subtilis and a use thereof. More specifically, the present invention relates to a novel strain of Bacillus subtilis 4-1-1 (KCCM: 11953P) and a process for producing fermented ginseng using the same.

Ginseng has been used for many diseases such as psychiatric diseases, nervous system diseases and diabetes in the form of herbal medicines in Asian countries such as Korea, China and Japan. Saponin, which is the main ingredient of ginseng, Hypertension, and the like. Currently, the use of ginseng can be classified into red ginseng prepared by heat treatment of white ginseng and fresh ginseng dried at room temperature at a temperature of 98 to 100 ° C, ginseng prepared by heat treatment at 120 to 180 ° C, In the form of heat-treated ginseng.

Saponin, which represents the major efficacy of ginseng, is composed of more than 30 kinds of ginsenosides. Ginsenosides are used to enhance immunity, antiinflammatory action, antiallergic action, anticancer effect, effect on erectile dysfunction, Action, anticholesterol action, antithrombotic action, adult disease and aging.

Saponin of ginseng is a neutral glycoside in which glucose, arabinose, xylose, rhamnose and the like are bonded to a triterpenoid-based dammarane skeleton . When ginseng is extracted with water and alcohol, it is known that a large amount of Rb1, Rb2, Rc, Rg1, etc. is present among the ginsenosides of ginseng. However, these ginsenosides are not well absorbed in the body and are not suitable for use as pharmaceutical compositions and food compositions. The ginsenosides in the ginseng are converted into ginsenosides which are not present in the natural world through the process of being processed with red ginseng or black gins, so that the ginsenosides are easily absorbed in the body and are suitably used as pharmaceutical compositions or food compositions of various diseases . That is, the glycosyl residues are separated from Rb1, Rb2, Rc, Rg1 and the like among the ginsenosides of ginseng to produce ginsenosides Rg2, Rg3, Rh2, Rh1, Rk5, Rk1, Rg6, Rk3, F4, and Rh4 are produced through the dehydration process of the water.

In addition, the ginsenosides are hydrolyzed by the addition of organic acid, and the sugar is removed and the side chain portion is cyclized to form panaxatriol (PPT) or panaxadiol (PPD) . When ginsenoside enters the body, ginsenosides such as Rb1, Rb2, Rc, and Rg1 are degraded by intestinal microorganisms in the body and can be absorbed. The ginsenosides are sequentially degraded by intestinal microorganisms, and Rb1 is sequentially converted to Rd, Compound K and protopanaxadiol. However, depending on the constitution and eating habits of humans, the presence or absence of microorganisms and the degree of retention Are virtually different, absorption by the conversion of metabolites is considerably different, and the effect may also vary considerably.

Therefore, in order to eliminate the difference between the efficacy and the absorption rate in the body by the conversion of the metabolite, the ginsenoside absorption rate of the body is increased through the conversion of the ginsenoside in the ginseng by the microorganism, There is a need for development of fermented ginseng which is strengthened en masse.

Korean Patent No. 10-1331171

Therefore, the present inventors have isolated and identified a novel strain for the production of fermented ginseng having enhanced function, which can enhance the body absorption rate of ginsenoside and improve the useful physiological activity of ginsenoside in the body, The present invention has been accomplished by establishing a process for producing fermented ginseng using a strain.

Accordingly, an object of the present invention is to provide a novel strain of Bacillus subtilis 4-1-1 (Accession No. KCCM11953P).

Another object of the present invention is to provide a method for producing fermented ginseng using the novel strain Bacillus subtilis 4-1-1.

Another object of the present invention is to provide fermented ginseng produced by the method of the present invention.

It is still another object of the present invention to provide a health functional food composition comprising the fermented ginseng of the present invention as an active ingredient.

In order to achieve the above object, the present invention provides a novel strain of Bacillus subtilis 4-1-1 (Accession No. KCCM11953P).

In one embodiment of the present invention, the strain may be isolated from the chungkukjang.

The present invention also relates to a process for fermenting ginseng using a novel strain of Bacillus subtilis 4-1-1 (Accession No. KCCM 11953P), wherein the content of panaxatriol (PPT) Wherein the content of Panaxadiol (PPD) is increased in a ratio of 3 to 5.

In one embodiment of the present invention, the Bacillus subtilis strain 4-1-1 (Accession No. KCCM11953P) may be inoculated in an amount of 1 to 5 parts by weight based on 100 parts by weight of ginseng.

In one embodiment of the present invention, the fermentation may be performed at a temperature of 35 to 37 DEG C for 5 to 10 days.

In one embodiment of the present invention, the content of ginsenosides Rb1, Rc, Re and Rg1 may be decreased and the content of ginsenosides Rd and Rg2 + Rh1 may be increased through the fermentation process.

In one embodiment of the present invention, the ginsenoside Rd may be the one converted at the ginsenoside Rc or Rb1.

The present invention also provides fermented ginseng produced by the method of the present invention.

In one embodiment of the present invention, the fermented ginseng may have a content of panaxadiol (PPD) of 3 to 5 relative to the content of panaxatriol (PPT).

In one embodiment of the present invention, the content of ginsenosides Rb1, Rc, Re and Rg1 in the fermented ginseng is decreased, and the content of ginsenosides Rd and Rg2 + Rh1 is increased.

In one embodiment of the present invention, the ginsenoside Rd may be the one converted at the ginsenoside Rc or Rb1.

In one embodiment of the present invention, the fermented ginseng may have thrombolytic activity.

The present invention also provides a health functional food composition containing the fermented ginseng as an active ingredient of the present invention.

In one embodiment of the present invention, the composition may have the effect of preventing or ameliorating atherosclerosis, ischemic heart disease, or stroke due to thrombosis.

The fermented ginseng fermented using Bacillus subtilis strain 4-1-1 (KCCM: 11953P) according to the present invention had a content of panaxadiol (PPD) relative to the content of panaxatriol (PPT) And the content of ginsenoside Rd having various physiological activities is particularly improved. Accordingly, the fermented ginseng of the present invention has improved functionality compared to the conventional fermented ginseng to improve the health of consumers, and can be used as a new raw material for health functional foods.

FIG. 1 is a photograph showing the surface of a culture medium after inoculation of a fresh strain Bacillus subtilis KCCM 11953P according to the present invention and a control strain in a ginseng solid medium.
FIG. 2 is a graph showing the results of confirming the degree of growth of bacteria according to the fermentation time after inoculation of the fresh strain Bacillus subtilis KCCM 11953P according to the present invention and the control strain into ginseng solid medium.
FIG. 3 shows the results of analysis of changes in ginsenosides in the process of fermenting ginseng using the new strain Bacillus subtilis KCCM 11953P and the control strain according to the present invention.
FIG. 4 shows the results of analysis of the content of impurities formed in the fermented medium during fermentation of ginseng using the novel strain Bacillus subtilis KCCM 11953P and the control strain according to the present invention.
FIG. 5 shows the results of analysis of viscosity change with fermentation time in the process of fermenting ginseng using the new strain Bacillus subtilis KCCM 11953P and the control strain according to the present invention.
FIG. 6 is a graph showing the results of analysis of thrombolytic activity of fermented ginseng prepared using the novel strain Bacillus subtilis KCCM 11953P according to the present invention and the control strain, according to fermentation time.

The present invention relates to a novel strain Bacillus subtilis 4-1-1 (KCCM: 11953P) which is suitable for the production of fermented ginseng having enhanced function so that the absorption efficiency of ginsenoside can be enhanced and the beneficial effects of ginsenoside can be improved. ) Is firstly isolated and identified, and a health functional food composition comprising the fermented ginseng, the fermented ginseng prepared by the method, and the fermented ginseng is provided.

The term "fermented ginseng" as used herein refers to a product obtained by inoculating microorganisms, particularly Bacillus subtilis 4-1-1 (KCCM: 11953P), into ginseng such as fresh ginseng, white ginseng and red ginseng and culturing them. The product may also be referred to as 'fermented ginseng'. The Bacillus subtilis 4-1-1 (KCCM: 11953P) strain of the present invention was isolated and identified from Chungkookjang. As a result of sequencing analysis, it was confirmed that the strain was a novel strain that was not disclosed until now. On December 01, he was deposited with the Korean Center for Microbial Resources and received the deposit number "KCCM 11953P".

The strain Bacillus subtilis 4-1-1 (KCCM: 11953P) according to the present invention is characterized by having biosynthesizing ability of saponin, that is, ginsenoside. Particularly, when ginseng is fermented using the same, fermented ginseng It was confirmed that the content of panaxadiol (PPD) was increased by 3 to 5 on the content of panaxatriol (PPT).

The ginseng to be produced in the fermented ginseng according to the present invention is not particularly limited and any of the ginseng itself and the processed ginseng can be used. For example, the ginseng itself and the processed ginseng product can be used. Fruit, ginseng leaf, ginseng roots, etc., or ginseng extract and ginseng powder can be used. The origin of the ginseng is not limited. For example, Korean ginseng, Korean ginseng, American ginseng, Panax ginseng, Himalayan ginseng, Vietnamese ginseng and the like can be used without limitation. The ginseng may be sterilized by a conventional method if necessary.

The form of ginseng used in the preparation of fermented ginseng is not particularly limited and may be, for example, in the form of an extract, a powder and an extract. The extract can be prepared by hot water extraction, alcohol extraction, and mixed extraction, and the powder can be prepared by pulverizing dry ginseng with a pulverizer or using an extract powder. The degree of powdering of ginseng is not particularly limited, and it is preferable that the strain of Bacillus genus derived from Cheonggukjang can penetrate into the tissue or fibrin of ginseng with high efficiency. For example, the degree of powdering of ginseng may be about 100-150 mesh. The extract can be produced by a vacuum distillation method or a thin film distillation method. More preferably, the ginseng may be pulverized and then an aqueous solution may be prepared and fermented.

In the present invention, the fermented ginseng using Bacillus subtilis 4-1-1 (KCCM: 11953P) strain is prepared by inoculating the above-mentioned ginseng into the above-mentioned ginseng and incubating the strain at a suitable fermentation temperature of 35 to 40 DEG C for 1 to 10 days Followed by fermentation at a temperature of 35 to 37 DEG C for 5 to 10 days.

The inoculation of the strain for fermentation can be carried out by inoculating 1 to 5% by weight of 100% by weight of ginseng, and the fermented ginseng prepared through the fermentation process can be used for the production of Panaxatriol (PPT) The content of Panaxadiol (PPD) is increased by 3-5.

As described above, there is a large amount of ginsenosides such as Rb1, Rb2, Rc and Rg1 in ginseng, but these ginsenosides do not absorb well in the body. Therefore, it is necessary to convert ginsenosides so that the ginsenosides can be easily absorbed in the body. In particular, in the present invention, the newly isolated novel strains of the present invention include ginsenosides Rd, Rg2, Rg3, Rh2, Rh1 and the like, it is possible to induce efficient ginsenoside conversion.

Therefore, the fermented ginseng fermented using the new strain Bacillus subtilis 4-1-1 (KCCM: 11953P) of the present invention has excellent absorption of ginsenoside in the body and thus has useful physiological activity of ginsenoside Can be further improved.

In one embodiment of the present invention, the change of ginsenoside before and after fermentation of fermented ginseng prepared by the method of the present invention was analyzed. As a result, the ginsenosides Rb1, Rc, Re and The content of Rg1 was decreased and the content of ginsenoside Rd and Rg2 + Rh1 was increased.

In particular, the ginseng fermented ginseng of the present invention showed an increase in the content of ginsenoside Rd. The ginsenoside Rd is useful for improving arthritis, cartilage regeneration, collagen formation, anticancer activity, adrenocorticotropic action and anti-obesity Respectively. However, since ginsenoside Rd is generally contained in less than 1% of ginseng, many studies have been conducted to improve the content thereof. In this respect, the novel strain of the present invention can effectively enhance the content of ginsenoside Rd.

The ginsenoside Rd contained in the fermented ginseng of the present invention is a ginsenoside Rb1 or Rc converted to Rd through fermentation. This can be converted to Rd by the ginsenoside-α-arabinofuranase enzyme during fermentation, and Rb1 can be converted to Rd by β-glucosidase.

The fermented ginseng produced according to the production method of the present invention can be used directly as food without passing through another tablet. That is, the fermented ginseng obtained according to the production method of the present invention can be concentrated or lyophilized and pulverized according to a conventional method, and can be directly applied to foods. The fermented ginseng obtained by concentrating or freeze- , Or the concentrate or lyophilized supernatant obtained by centrifuging the aging solution may be applied to food as it is.

In addition, the fermented ginseng of the present invention can be generally made into a final product by adding and blending various components in order to increase the flavor or to produce a product having excellent taste and flavor (for example, food, medicine, etc.). The term " product " means a natural product or a processed product containing one or more nutrients. The product is not limited in its kind. The product includes Bacillus subtilis 4-1-1 (KCCM: 11953P) There are no particular restrictions on the addition of other ingredients other than those containing the concentrate of the culture medium, the dried material of the culture medium, and the like. Examples of the ingredients to be added and compounded include various natural extracts, saccharides, emulsifiers, sweeteners, acidulants, and juices.

Accordingly, the present invention can provide a health functional food composition containing the fermented ginseng as an active ingredient of the present invention.

Further, the fermented ginseng prepared by fermenting with Bacillus subtilis 4-1-1 (KCCM: 11953P) of the present invention has an excellent thrombolytic activity.

Because thrombus plays a central role in the development and progression of circulatory disease, preventing thrombus formation and effectively eliminating thrombosis is a key element in the treatment of vascular disease. The platelets are disk-shaped hemocytes having a diameter of about 2 to 4 탆. The blood platelet is a hemocyte having a diameter of about 2 to 4 탆. The blood platelet is a hemocyte, a thrombus plug and a coagulation process by blood coagulation factors. And a biologic defense device for preventing the biologic attack. However, adhesion, aggregation and release of platelets by platelet activation in the occluded blood vessels lead to thrombus formation and are also involved in diseases such as arteriosclerosis, ischemic heart disease, or stroke.

In this regard, it was confirmed that the fermented ginseng prepared using the Bacillus subtilis 4-1-1 (KCCM: 11953P) strain of the present invention had better thrombolytic activity than the fermented ginseng fermented using other microorganisms Accordingly, it has been found that the food composition containing the fermented ginseng or the fermented ginseng according to the present invention may have an effect of preventing and improving atherosclerosis, ischemic heart disease, or stroke due to thrombosis.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited to these examples.

< Example  1>

For ginseng fermentation Bacillus genus  Strain isolation and identification

<1-1> Conventional Chungkukjang analysis suitable for ginseng fermentation

Before separating microorganisms suitable for fermentation of ginseng from freshly fermented chongkukjang, the present inventors firstly tried to select traditional chongkukjang suitable for ginseng fermentation by using the conventional Chongkugajang (Chunbuk Soonchang) ) Were purchased, and the ginseng was fermented by using Bacillus subtilis strain contained in each Cheonggukjang as a fermenting bacteria. To this end, 12.5 g of 4-year-old ginseng ginseng powder was placed in a 250 mL bottle, and 3 times of distilled water was added to the powder to absorb all of the distilled water. Then, the mixture was sterilized at 121 ° C for 15 minutes, Cheonggukjang was added in an amount of 10% and 20% by weight (w / w) based on the weight of the ginseng powder absorbed in distilled water, mixed and fermented at 37 캜 for 72 hours. After the fermentation was completed, ginsenosides were analyzed according to the fermentation process. At this time, the following control group was used in which the chungkukjang was not treated.

As a result, as shown in Table 1, Rb1 decreased significantly ( p <0.05) compared to the control group after fermentation in the other treatment groups except for the addition of 10% by weight of chungkukjang prepared in Soonchang and Nonsan. Rc and Rg3 decreased in all treatments with the difference from the control group. Especially, Rc decreased with increasing amount of added Chungkukjang ( p <0.05). On the other hand, Rd was higher in the four treatment groups except the treatment group added with Chungkookjang prepared in Soonchang, Jeonbuk, and the higher the content of added chungkukjang was, the higher the Rd content was ( p <0.05) .

Based on these results, it was found that the content of Rb1, Rc, and Rg3 was decreased while the content of Rd was increased. It was found that Chungkukjang made in Nonsan and Cheongyang in Chungnam was suitable for increasing the content of Rd. When the present Bacillus sp. Strains were used, it was found that the content of Rd having anticancer action, adrenocortical hormone stimulating action and anti-obesity effect could be effectively promoted by switching to ginsenoside.

<1-2> Suitable for fermentation of ginseng Bacillus  Isolation of genus strains

In order to separate Bacillus spp. From Chongkukjang made in Nonsan in Chungnam and Chungkukjang in Chungnam province, which was suitable for ginseng fermentation, Chungkukjang was first suspended in sterilized water and boiled in water at 100 ° C for 20 minutes, LB broth was cultured on a nutrient broth medium until a single colony was formed. Gram stain and spore staining were performed to obtain 10 strains which were spore-forming bacteria as gram-positive bacilli. . In order to select the strains which can be used for ginseng fermentation, the saponin conversion ability was tested in the liquid LB medium supplemented with crude saponin. For this purpose, 5 ml of LB liquid medium was prepared in a test tube, and the sterilized crude saponin solution was dissolved in 3000 ppm &Lt; / RTI > The strain was inoculated into LB liquid medium containing crude saponin and cultured at 37 ° C. After 48 hours, a 200 μl sample of the culture supernatant containing saponin was centrifuged and the saponin extraction method of ethyl ether and water-saturated butanol was carried out using a shibata method in a 1.5 ml tube. The resulting extract was dissolved in 40% acetonitrile HPLC analysis was carried out.

As a result, strains having the highest ability to convert ginsenoside Rd were selected and named "Bacillus subtilis 4-1-1".

<1-3> Suitable for fermentation of ginseng Bacillus  Identification of the genus Escherichia coli

In order to identify the strain "Bacillus subtilis 4-1-1" isolated from <1-2>, 1ul of a single colony of the candidate Bacillus subtilis 4-1-1 strain obtained by the above cultivation was collected in a loop The slides were inoculated on the slides and the CCHA matrix solution was dropped to 1 μl, and the microorganisms were identified by placing them in a VITEK MS MALDI-TOF instrument. In addition, 16S rRNA gene sequencing analysis was commissioned by Biophot.

As a result of the analysis, the identified strain was identified as a spore-forming strain of Gram-positive bacilli, and the gene sequence of 16S rRNA was compared with the NCBI database and the identified "Bacillus subtilis 4-1-1" The inventors of the present invention deposited the above strain on the Korean microorganism resource center on Dec. 1, 2016, and found that the microorganism deposited with the deposit number &quot; KCCM 11953P &quot; . The nucleotide sequence of 16S rRNA of the identified strain &quot; Bacillus subtilis 4-1-1 &quot; is shown in SEQ ID NO: 1.

< Example  2>

Bacillus Subtilis  Of strain 4-1-1 Growth ability  And ginsenoside Switchability  analysis

<2-1> Ginseng  Appearance analysis of strain growth

The strain Bacillus subtilis 4-1-1 (KCCM 11953P) isolated and identified in Example 1 was inoculated into a ginseng solid medium and the degree of growth was analyzed. To this end, ginseng solid medium was prepared by adding 3 times as much weight of distilled water (75 g) to 25 g of 4-year-old ginseng powder grown in Geumsan and sterilized at 121 ° C for 15 minutes. Bacillus subtilis 4-1-1 of the present invention was inoculated at 1 wt% based on the weight of the medium and incubated at 37 ° C for 0-10 days to confirm the surface and properties of the medium. At this time, Bacillus subtilis KCCM 41459, another strain belonging to the genus Bacillus, was used as a control in the same conditions as the strain of the present invention.

As a result of analysis, viscous material was produced in all media after 3 days of fermentation. More strains were grown on the surface of the medium of the test group inoculated with the strain of the present invention than Bacillus subtilis KCCM 41459, and after 5 days of fermentation, , The bacterial growth was continued on the surface of the medium and the viscous substance was decreased. From 7th day of fermentation, the surface was glossy and no viscous substance was observed. After 10 days from the start of fermentation, the color of the medium became thicker, and when the fermentation vessel was tilted with a lot of luster, the medium had enough properties to flow the medium. When the two strains belonging to the genus Bacillus were compared, it was found that the strain of the present invention was significantly proliferated in the ginseng solid medium (see FIG. 1).

<2-2> Bacterium count

After the inoculation, the number of bacteria was 2.92-3.74 log CFU / g. However, after 3 days of fermentation, the number of bacteria was increased to 9.22 log CFU / g regardless of the type of bacteria. Thereafter, the number of bacteria was gradually increased to 10.05-10.47 log CFU / g until 7 days of fermentation, and slightly decreased to 9.83-10.43 log CFU / g after 10 days of fermentation, but no significant difference was observed (see FIG.

Therefore, it can be seen from the results that the strain of the present invention is progressing smoothly during the ginseng fermentation process.

<2-3> Ginsenoside analysis

In order to analyze the degree of conversion of ginsenosides in ginseng fermented using the isolates of the present invention, the components and contents of ginsenosides were measured by HPLC analysis. To this end, the ginseng medium was lyophilized and pulverized according to the fermentation period, and 10 g of methanol was added to 0.5 g of the fermented ginseng powder, followed by ultrasonic extraction for 30 minutes. The supernatant was centrifuged at 3000 rpm for 10 minutes, and the supernatant was filtered through a 0.45 μm PTFE filter and used for HPLC analysis.

As a result of the analysis, as shown in Tables 2, 3 and 3, Rb1, Rc, Re and Rg1 decreased in the group using the strain of the present invention, while Rd and Rg2 + Rh1 increased Respectively. However, Rb, Rb3, Rd, Rg3 and Rg2 + Rh1 tended to increase slightly in the control group, while Rc, Re and Rg1 decreased ( p <0.05).

As the ginseng fermentation progressed, the conversion of ginsenoside was found to occur. Especially, Rc and Rd of the PPD series decreased while the PPT series Re and Rg1 decreased and Rg2 + Rh1 increased.

Accordingly, the present inventors calculated the PPD / PPT ratio and compared the degree of increase and decrease in order to quantitatively confirm the degree of conversion of ginsenoside. As a result, as shown in Table 2, the PPD / PPT ratio immediately after inoculation was 2.01 to 2.05 in both the control and the experimental groups, but the PPD / PPT ratio after the 7th day of fermentation was 4.35 Whereas the control group was 3.71, which was significantly lower than that of the strain of the present invention. In addition, the group treated with the strain of the present invention showed that PPD / PPT ratio remained much higher than that of the control group even after 10 days of fermentation, so that the strain of the present invention showed a high PPD / PPT ratio even during long fermentation time in ginseng As well as the ability to keep it constantly.

< Example  3>

Evaluation of physical properties and functionality

<3-1> Slime  Content analysis

During the ginseng fermentation process using Bacillus subtilis 4-1-1 (KCCM 11953P) of the present invention, the content of the viscous substance in the fermented ginseng culture was examined. To this end, the same amount of distilled water was added to the ginseng medium and homogenized at 25 ° C. for 30 minutes. Then, the mixture was centrifuged at 3000 rpm for 15 minutes to obtain 5 mL of supernatant, which was evaporated to dryness at 105 ° C., Respectively. As a control group, the group treated with Bacillus subtilis KCCM 41459 used in Example 2 was used.

As a result, as shown in FIG. 4, the content of the viscous substance in the group using the present invention and the control group was 7.120% and 6.253% after 3 days of fermentation, respectively, and 3.553% and 2.773% Respectively. On the other hand, from the 7th day of fermentation, it was confirmed that there was no viscous substance in the medium. When the content of the viscous substance was compared, it was found that the viscous substance of the medium fermented with the strain of the present invention was about 1.14 to 1.27 times as many as that of the culture medium fermented by the strain of the control group, so that when the strain of the present invention was used, .

For reference, the above-mentioned viscous substance is composed of a fructan-type levan which is a polymer of fructose and γ-polyglutamic acid (PGA) in which about 5,000 glutamic acid is polymerized as a fermentation metabolite of Bacillus sp. PGA is a kind of microbial polymer substance, and it has physiological activity functions such as immunity enhancement effect and anticancer effect. Therefore, PGA is useful as functional material of cosmetics because it is excellent in moisture retention as well as food and medicine. Therefore, when the strain of the present invention is used, the formation of a viscous substance having various physiologically active functions can be promoted, and thus it can be provided as a raw material for various products having superior functionality.

<3-2> Comparison of Viscosity

The ginseng solid medium was sampled at the fermentation time and used for viscosity measurement. The viscosity of the medium was measured at 25 ° C using a rheometer (Ar2000, TA instruments, New Castle, DE, USA). Shear rate was measured at 100 points in the range of 0 ~ 100 1 / s.

As a result, as shown in FIG. 5, the viscosity gradually decreased and stabilized to a constant viscosity as the shear rate was increased, regardless of the type of bacteria used for fermentation. In the case of the control group, the fermented medium had an initial viscosity of 399.85 Pa.s, but as the fermentation progressed, it gradually decreased to 224.9 Pa.s, 115.3 Pa.s, 83.4 Pa.s and 66.9 Pa.s, In the case of one case, it was 362.6 Pa.s immediately after inoculation, and it was 258.5 Pa.s after 3 days of fermentation, 96.8 Pa.s after 5 days of fermentation, 75.8 Pa.s after 7 days of fermentation, and 78.01 Pa.s after 10 days of fermentation The medium inoculated with the inventive bacterium showed that the initial viscosity was lowered as the fermentation progressed.

<3-3> Thrombolysis ability  analysis

The fibrinogen activity of the ginseng solid medium was measured by modifying the method of Astrup et al. (1952). Specifically, fibrinogen was completely dissolved in 50 mM phosphate buffer (pH 7.4, containing 0.15 M NaCl) to a final concentration of 0.3% 5 mL of the same solution was added with 5 mL of the same amount of 2% agarose (sterile agar solution) and mixed. 500 μl of thrombin (100 NIH units / mL) was added to the mixed solution, and the mixture was thoroughly mixed, immediately poured into a petri dish, and solidified at room temperature for 60 minutes to prepare a fibrin plate. For activity measurement, 7 holes of 5 mm in diameter were made with a pasteur pipette on a fibrin plate, and 4 mL of distilled water was added to 1 g of the medium. After mixing for 30 minutes, 20 μL of the supernatant was injected and centrifuged. And the area of the generated transparent ring was calculated. Plasmmin (plasmin; 1.0 unit / mL), a purified thrombolytic enzyme, was used as a control. The thrombolytic activity of the extract was calculated in terms of the relative ratio of the dissolution area of the sample to the dissolution area of the control.

As a result of the analysis, as shown in FIG. 6, the thrombolytic ability was not confirmed immediately after inoculation irrespective of the kind of bacteria. The fermentation medium of Bacillus subtilis KCCM 41459 as a control group showed 85.0% after 3 days of fermentation. As the fermentation continued, the thrombolytic activity was increased or decreased slightly but not significantly. However, the fibrinolytic activity of the ginseng fermented with the strain of the present invention for 3 days was 100.0%, which was the same as that of the positive control group, plasmin 1.0 unit / mL. After 5 days of fermentation, 97.5% It was 2.5% lower than that of the first day, but it was confirmed that it remained until the 10th day of fermentation. Therefore, it can be seen from the above results that when the strain of the present invention is used, fermented ginseng having a high fibrinolytic effect can be produced.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Korea Microorganism Conservation Center (overseas) KCCM11953P 20161201

<110> Korea food research institute <120> Novel Bacillus subtilis 4-1-1 and method for fermented ginseng          using the same <130> NPDC59887 <160> 1 <170> KoPatentin 3.0 <210> 1 <211> 1401 <212> DNA <213> Artificial Sequence <220> <223> KCCM11953P 16s RNA sequence <400> 1 atgcagtcga gcggacagat gggagcttgc tccctgatgt tagcggcgga cgggtgagta 60 acacgtgggt aacctgcctg taagactggg ataactccgg gaaaccgggg ctaataccgg 120 atggttgttt gaaccgcatg gttcaaacat aaaaggtggc ttcggctacc acttacagat 180 ggacccgcgg cgcattagct agttggtgag gtaacggctc accaaggcga cgatgcgtag 240 ccgacctgag agggtgatcg gccacactgg gactgagaca cggcccagac tcctacggga 300 ggcagcagta gggaatcttc cgcaatggac gaaagtctga cggagcaacg ccgcgtgagt 360 gatgaaggtt ttcggatcgt aaagctctgt tgttagggaa gaacaagtac cgttcgaata 420 gggcggtacc ttgacggtac ctaaccagaa agccacggct aactacgtgc cagcagccgc 480 ggtaatacgt aggtggcaag cgttgtccgg aattattggg cgtaaagggc tcgcaggcgg 540 tttcttaagt ctgatgtgaa agcccccggc tcaaccgggg agggtcattg gaaactgggg 600 aacttgagtg cagaagagga gagtggaatt ccacgtgtag cggtgaaatg cgtagagatg 660 tggaggaaca ccagtggcga aggcgactct ctggtctgta actgacgctg aggagcgaaa 720 gcgtggggag cgaacaggat tagataccct ggtagtccac gccgtaaacg atgagtgcta 780 agtgttaggg ggtttccgcc ccttagtgct gcagctaacg cattaagcac tccgcctggg 840 gagtacggtc gcaagactga aactcaaagg aattgacggg ggcccgcaca agcggtggag 900 catgtggttt aattcgaagc aacgcgaaga accttaccag gtcttgacat cctctgacaa 960 tcctagagat aggacgtccc cttcgggggc agagtgacag gtggtgcatg gttgtcgtca 1020 gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaacccttg atcttagttg 1080 ccagcattca gttgggcact ctaaggtgac tgccggtgac aaaccggagg aaggtgggga 1140 tgacgtcaaa tcatcatgcc ccttatgacc tgggctacac acgtgctaca atggacagaa 1200 caaagggcag cgaaaccgcg aggttaagcc aatcccacaa atctgttctc agttcggatc 1260 gcagtctgca actcgactgc gtgaagctgg aatcgctagt aatcgcggat cagcatgccg 1320 cggtgaatac gttcccgggc cttgtacaca ccgcccgtca caccacgaga gtttgtaaca 1380 cccgaagtcg gtgaggtaac c 1401

Claims (14)

A novel strain of Bacillus subtilis 4-1-1 (Accession No. KCCM11953P). The method according to claim 1,
The strain of Bacillus subtilis 4-1-1 (Accession No. KCCM11953P), which has been isolated from Chungkukjang. And a step of fermenting ginseng using a novel strain of Bacillus subtilis 4-1-1 (accession no. KCCM11953P), wherein the fermentation process comprises the step of dissolving panaxadiol (PPT) Wherein the content of Panaxadiol (PPD) is increased by a ratio of 3 to 5. The method of claim 3,
Wherein the Bacillus subtilis strain 4-1-1 (accession number KCCM11953P) is inoculated in an amount of 1 to 5 parts by weight based on 100 parts by weight of ginseng to effect fermentation. The method of claim 3,
Wherein the fermentation is carried out at a temperature of 35 to 37 DEG C for 5 to 10 days. The method of claim 3,
Wherein the content of ginsenosides Rb1, Rc, Re and Rg1 is decreased and the content of ginsenosides Rd and Rg2 + Rh1 is increased through the fermentation process. The method according to claim 6,
Wherein the ginsenoside Rd is converted at the ginsenoside Rc or Rb1. Fermented ginseng produced by the method of claim 3. 9. The method of claim 8,
The fermented ginseng is characterized in that the content of panaxadiol (PPD) relative to the content of panaxatriol (PPT) is increased by 3 to 5. 9. The method of claim 8,
Wherein the content of ginsenosides Rb1, Rc, Re and Rg1 is decreased, and the content of ginsenoside Rd and Rg2 + Rh1 is increased in the fermented ginseng. 11. The method of claim 10,
Wherein the ginsenoside Rd is converted at the ginsenoside Rc or Rb1. 9. The method of claim 8,
Wherein the fermented ginseng has a thrombolytic activity. A health functional food composition comprising the fermented ginseng of claim 8 as an active ingredient. 14. The method of claim 13,
Wherein the composition has an effect of preventing or ameliorating atherosclerosis, ischemic heart disease, or stroke due to thrombosis.

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