KR101595789B1 - Microorganisms having activities of decomposing residual pesticides or converting ginsenosides in the aerial part of ginseng and method for preparing a feed additive using same - Google Patents

Abstract

The present invention relates to a microorganism having a residual agrochemical decomposition activity or a ginsenoside conversion activity in a ginseng root part and a method for preparing a feed additive using the microorganism and the use of the same. Therefore, the ginseng root part can be safely used as a feed additive or the like, and the strain of Brevibacillus latexporus WR according to the present invention can be used to convert PT ginsenoside in the ginseng root part into PD ginsenoside Therefore, when used as a feed additive, it can improve the immunity of livestock to prevent diseases and improve productivity.

Description

TECHNICAL FIELD The present invention relates to a microorganism having a pesticidal decomposition activity or a ginsenoside conversion activity in a ginseng root, and a method for preparing the same, }

The present invention relates to a microorganism having a residual agrochemical decomposition activity or ginsenoside conversion activity in a ginseng root, and a method for preparing a feed additive using the same.

Ginseng is known to contain a large amount of saponins that are effective in enhancing the immune system, and research on pharmacological efficacy and nutrients has been progressing considerably. However, most of the studies are focused on the root portion of ginseng, and there is no research on the ginseng root portion, that is, leaf and stem.

The surface of ginseng is produced at a high rate of 25 ~ 35% of ginseng production, but it is discarded due to the problem of residual pesticides. However, recent studies have shown that the various nutrients in the surface of ginseng are not lacking, and that saponin is contained more in the ginseng root than in the ginseng roots. Therefore, there is a need for a method of utilizing the whole surface of the ginseng ground which is discarded (refer to Korean Patent Publication No. 2011-0105513).

Among the pesticides used in ginseng, diphenococonazole, which is the most commonly used, can be used up to 14 days before harvest, and the number of spraying is also five times higher. For this reason, concerns about residual pesticides on the surface of ginseng have been increased, and despite the fact that they contain active ingredients, most of the ginseng grounds are being disposed of without being used for medicinal or feed additions (approximately 6,300 tons ).

In addition, ginseng has protopanaxadiol (PD) ginsenosides and protopanaxatriol (PT) ginsenosides. PT ginsenosides have an excitatory effect, PD ginsenosides Side is known to sedate, and PD ginsenosides have been reported to be more effective in preventing disease through immunity enhancement. In the case of ginseng, it is reported that the ratio of PT ginsenoside is high in the root portion of ginseng, while the proportion of ginseng in the root portion is high in the root portion. In order to enhance the immunity of the ginseng root portion, PT ginsenoside It is advantageous to convert it into PD-based ginsenosides.

Studies have been conducted to find that ginsenosides are present in large amounts in the surface of ginseng to date. However, studies to solve the residual pesticides and studies to convert the PT ginsenoside into ginsenoside PD into ginsenoside PD I did.

[Patent Document 1] Korean Patent Publication No. 2011-0105513

Accordingly, an object of the present invention is to provide a novel microorganism having decomposition activity of the residual pesticide in the ginseng root part.

Another object of the present invention is to provide a novel microorganism having an activity of converting PT ginsenosides in the ginseng root part into PD ginsenosides.

It is still another object of the present invention to provide a method for removing residual pesticide residues in ginseng root by using the microorganism.

It is still another object of the present invention to provide a method for converting ginsenosides in the ginseng root using the microorganism.

Yet another object of the present invention is to provide a method for producing a feed additive from ginseng root using the microorganism.

To achieve the above object, the present invention provides a strain of Brevibacillus laterosporus WR (Accession No. KACC91946P).

In order to achieve the above other object, the present invention provides a Lactobacillus plantarum WL strain (Accession No. KACC91947P).

In order to achieve still another object of the present invention, the present invention provides a ginseng root part comprising a step of inoculating and cultivating a strain of Brevibacillus latratifolia WR (accession number: KACC91946P), a culture or a fermented product thereof, A method for removing residual pesticides is provided.

In order to accomplish the above another object, the present invention provides a ginseng root preparation (hereinafter referred to as " ginseng root preparation ") comprising a step of inoculating Lactobacillus plantarum WL strain (Accession No .: KACC91947P), a culture or fermented product thereof, Provides a method for converting protopanaxatriol (PT) ginsenosides to protopanaxadiol (PD) ginsenosides.

In order to accomplish the above-mentioned further object, the present invention provides a method for culturing a ginseng root, comprising the step of inoculating and cultivating a strain of Brevibacillus latratifolia WR (accession number: KACC91946P), a culture or a fermented product thereof, And drying the culture. The present invention also provides a method for producing a feed additive.

In order to accomplish the above-mentioned further object, the present invention provides a method for producing a lactic acid bacteria strain, comprising the step of inoculating and culturing Lactobacillus plantarum WL strain (Accession No .: KACC91947P), a culture or fermented product thereof, And drying the culture. The present invention also provides a method for producing a feed additive.

In order to accomplish the above-mentioned further object, the present invention provides a method for culturing a ginseng root, comprising the step of inoculating and cultivating a strain of Brevibacillus latratifolia WR (accession number: KACC91946P), a culture or a fermented product thereof, Culturing the above culture after inoculating Lactobacillus planta WL strain (accession number: KACC91947P), its culture or fermentation product; And drying the culture. The present invention also provides a method for producing a feed additive.

The strain Brevibacillus laetrosporus WR according to the present invention can remove residual pesticides in the ginseng ground part and thus can be used for safely feeding the ginseng root part as a feed additive and the like. The Brevibacillus lateosporus WR The strain can convert the PT ginsenoside in the ginseng root part to the PD ginsenoside, so that when used as a feed additive, it can improve the immunity of the livestock, thereby helping prevent disease and improve productivity.

FIG. 1 shows the results of analysis of strains selected in Example 1 using an API 50 CHB kit (Biomerieux ^® , France).
FIG. 2 shows the results of analysis of the resolving power of the pesticide difenokonazole and boscalid in the strain selected in Example 1. FIG.
Fig. 3 is a photograph showing the band intensities of PT-family ginsenosides and PD-family ginsenosides by TLC analysis from the culture broth of the strain for screening for ginsenoside-converting bacteria.
FIG. 4 shows the results of analysis of strains selected in Example 5 using an API 50 CHB kit (Biomerieux ^® , France).
FIG. 5 is a graph showing the ratios of the feed additives 1, the feed additives 2 and the red ginseng, which are known to have an excellent immunity, to the rats, and then measuring the growth rate at intervals of one week.
FIG. 6 is a graph showing the growth rates of feed additives 1, feed additives 2, red ginseng, a vaccine control, a positive control, and a negative control to birds at intervals of one week.
FIG. 7 is a graph showing the effect of the feed additive 1, the feed additive 2, red ginseng, the vaccine control, the positive control, and the negative control on the protective effect against the Salmonella attack.
FIG. 8 is a graph showing antibody values measured after administration of Feed Additive 1, Feed Additive 2, Red Ginseng, Vaccine Control, Positive Control, and Negative Control.

The present invention provides a strain of Brevibacillus lateosporus WR (Accession No .: KACC91946P). The strain is also referred to herein as the " WR strain ". The WR strain was deposited on May 16, 2014 under the name of "Brevibacillus laterusporus WR" (Accession No. KACC91946P).

The WR strain has the form and the biochemical characteristics shown in Table 1 of the present specification and shows the enzyme activity shown in Table 2. < tb >< TABLE > In addition, the WR strain has the 16s rRNA gene sequence shown in SEQ ID NO: 1, and the BruB Bacillus lactosporus And 99.2% homology with the standard strain.

The WR strain according to the present invention grows well in a culture medium for bacterial growth such as Lubria-Bertani agar (LB medium) and Bennett agar plate. In addition, the WR strain of the present invention grows well at a temperature of 27 ° C to 37 ° C (optimal growth temperature: about 30 ° C) and grows well in a medium of pH 6.5 to pH 7.5 (optimum growth pH: 6.8).

The WR strain according to the present invention has an excellent decomposing activity against residual pesticides in the ginseng root part. Examples of the pesticide decomposable by the WR strain include, but are not limited to, diphenococonazole, boscalid, cyazofamy, pyraclostrobin or triflocystrobin used in the cultivation of ginseng.

The WR strain according to the present invention can be usefully used for the ginseng root part. For example, when the ginseng ground part is to be used as a feed additive, the WR strain according to the present invention is treated on the surface of ginseng in advance of a conventional feed additive manufacturing process to remove the residual pesticide in the ginseng root part, Can be excluded. Although it has been exemplified in the present invention that the WR strain can be used when preparing the feed additive from the ginseng root portion, those skilled in the art can use the WR strain of the present invention to remove or reduce the residual pesticide residues in the ginseng root portion Will recognize.

Accordingly, the present invention provides a method for removing residual pesticide residues in the ginseng root part, comprising the step of inoculating a ginseng root part with a culture broth or fermented product of Brevibacillus latratosporous WR strain (accession number: KACC91946P) to provide. This method will be described in more detail in the method of preparing the feed additive described later.

On the other hand, the present invention provides Lactobacillus plantarum WL strain (Accession No .: KACC91947P). The strain is also referred to herein as a " WL strain ". The WL strain was deposited on May 16, 2014 under the name "Lactobacillus plantata WL" under the name of strain KACC91947P.

The WL strains have the form and biochemical characteristics shown in Table 10 of the present specification and show the enzyme activity shown in Table 11. In addition, the WL strain has the 16s rRNA gene sequence shown in SEQ ID NO: 2 and shows a homology of 98.2% with the lactobacillus plantarum standard strain.

The WL strains according to the present invention can be used in a pH range from 4.5 to 7.5, preferably from 6.0 to 7.0, and in a medium such as MRS, ATP, Elliker, Rogosa SL, Todd Hewitt, It grows well at a temperature of 20 ° C to 40 ° C, preferably at 37 ° C.

The WL strain according to the present invention can convert protopanaxatriol (PT) ginsenoside into ginsenoside protopanaxadiol (PD) ginsenoside.

Examples of the PD-based ginsenosides include Rg1, Re, Rf, Rh1, and Rg2. Examples of the PD-based ginsenosides include Rb1, Rc, Rb2, Rb3, Rd, Rg3, Rh2, .

The WL strain according to the present invention can be usefully used for the ginseng root part. For example, when the ginseng ground part is to be used as a feed additive, the WL strain according to the present invention is treated on the surface of the ginseng in advance of a conventional process for preparing a feed additive, so that PT ginsenoside in the ginseng root part is converted into PD ginsenoside The immunity enhancement effect can be improved. In other words, the ratio of PD-based ginsenoside having a high immunity-enhancing effect to the root portion of ginseng is higher than that of PT-based ginsenoside, whereas the ratio of PT-ginsenoside to PD- The WL strain of the present invention can change the ratio of the ginsenosides in the ginseng root portion to the ginsenoside in the root portion of the ginseng root. Although it has been exemplified in the present invention that WL strain can be used when preparing a feed additive from the ginseng root portion, those skilled in the art will appreciate that the WL strain of the present invention can be used to remove or reduce the residual pesticide residues in the ginseng root portion Will recognize.

Accordingly, the present invention relates to a method for producing protopanaxyl triol PT agar in the ginseng ground part, comprising the step of inoculating a lactobacillus planta WL strain (accession number: KACC91947P), a culture thereof or a fermented product thereof, And converting the senoside to the PD-based ginsenoside. This method will be described in more detail in the method of preparing the feed additive described later.

On the other hand, the present invention provides a method for preparing a feed additive using any one or both of the two microorganisms described above.

In a first embodiment of the present invention, there is provided a method for cultivating a strain of Brevibacillus lactisporus WR (accession number: KACC91946P), a culture thereof or a fermented product thereof, after inoculation into a ginseng root part; And drying the culture. ≪ Desc / Clms Page number 5 >

In addition, in a second embodiment of the present invention, there is provided a method for producing lactam, comprising the steps of: inoculating and culturing Lactobacillus plantarum WL strain (accession number: KACC91947P), a culture or fermented product thereof, And drying the culture. ≪ Desc / Clms Page number 5 >

Furthermore, in a third embodiment of the present invention, there is provided a method for culturing a ginseng root, comprising the step of inoculating and cultivating a strain of Brevibacillus latratifolia WR (accession number: KACC91946P), a culture or a fermented product thereof, Culturing the above culture after inoculating Lactobacillus planta WL strain (accession number: KACC91947P), its culture or fermentation product; And drying the culture. ≪ Desc / Clms Page number 5 >

The method for preparing a feed additive according to the first embodiment specifically includes the following process.

(a) Preparation of ginseng root

Prior to the feed additive manufacturing process, the ginseng root portion is dried at room temperature for 2 to 3 days to adjust the water content to 5 wt% to 20 wt%, preferably 10 wt% or less. It is preferable to use the ginseng ground part having a particle size of 5 mm or less after the grinding process before use. If the particle size is 5 mm or less, the mixing ratio of the ginseng ground part with other excipients can be increased.

(b) seed culture process

Prior to the feed additive manufacturing process, the WR strain or WL strain of the present invention can be pre-cultured and produced into seeds.

Specifically, the WR strain can be pre-cultured aerobically for 24 hours at 30 DEG C in TS medium (10 g / L casein pancreatic decomposition product, 5 g / L yeast extract and 10 g / L NaCl).

The strain WL was cultured in an MRS medium (10 g of Proteose Peptone No. 3, 10 g of beef extract, 5 g of yeast extract, 20 g of dextrose, 1 g of polysorbate 80, 2 g of ammonium citrate, 5 g of sodium acetate, 0.1 g of magnesium sulfate, 0.05 g of sulfate and 2 g / L of diphosphate phosphate) at 37 占 폚.

(c) Inoculation and culture of WR strain: Residual pesticide decomposition process

The ginseng ground part prepared in step (a) is added to the TS medium in an amount of 5 wt% to 20 wt%, preferably about 7 wt%, and sterilized at 121 캜 for 15 min. Thereafter, the culture medium of the WR strain pre-cultured in step (b) is inoculated in an amount of 0.5 wt% to 3 wt%, preferably 1 wt%, and aerobically cultured at 30 DEG C for 2 days.

(d) Addition of excipients and drying

The culture obtained in step (c) is heat-treated at 100 ° C. for 30 minutes to inactivate the WR strain, and then the ginseng root part concentration is adjusted to 10% by adding excipients (eg, flour or embryo). Thereafter, it is vacuum-dried at 60 DEG C and then pulverized to prepare a feed additive.

The method for producing a feed additive according to the second embodiment includes the following process.

(a) Preparation of ginseng root

Is the same as the feed additive method according to the first embodiment.

(b) seed culture process

Prior to the feed additive manufacturing process, the WL strain of the present invention can be pre-cultured and produced as seeds.

Specifically, the strain WL was cultured in an MRS medium (10 g of proteospeptone No. 3, 10 g of beef extract, 5 g of yeast extract, 20 g of dextrose, 1 g of polysorbate, 2 g of ammonium citrate, 5 g of sodium acetate, 0.05 g of manganese sulfate, 2 g / L of diphosphate phosphate) at 37 占 폚.

(c) Inoculation and culture of WL strain: Ginsenoside conversion step

The ginseng ground part prepared in step (a) is added to the MRS medium in an amount of 5 wt% to 20 wt%, preferably about 7 wt%, and sterilized at 121 캜 for 15 min. Thereafter, the WL strain pre-cultured in step (b) is added at a concentration of 0.5 wt% to 5 wt%, preferably about 2 wt%. The excipient is added and mixed so that the content of the ginseng ground part in the culture medium is 5 wt% to 20 wt%, preferably 10 wt%, and water is further added so that the total moisture content is 40 wt% to 60 wt% . Thereafter, air was shut off at 32 ° C to 37 ° C, followed by solid fermentation for about 2 to 5 days, followed by vacuum drying at 50 ° C to 70 ° C to prepare a sample.

The method for preparing a feed additive according to the third embodiment includes the following process.

(a) Preparation of ginseng root

Is the same as the feed additive method according to the first and second embodiments.

(b) seed culture process

Is the same as the feed additive method according to the first and second embodiments.

(c) Inoculation and culture of WR strain: Residual pesticide decomposition process

Is the same as the feed additive method according to the first embodiment.

(d) Inoculation and culture of WL strain: Ginsenoside conversion step

In order to grow the WL strain in the culture medium prepared in step (c), the MRS medium is added in an amount corresponding to the liquid amount and sterilized, and then the WL strain pre-cultured in step (b) is added in an amount of 0.5 wt% to 5 wt% By weight to about 2% by weight. WL is added, and the water content is further adjusted to 40% by weight to 60% by weight of the total water content by adding the excipient so that the content of the ginseng root part of the culture solution containing WL is 5% to 20% by weight, preferably 10% %. Thereafter, air was shut off at 32 ° C to 37 ° C, followed by solid fermentation for 2 to 5 days, followed by vacuum drying at 50 ° C to 70 ° C to prepare a sample.

The feed additives prepared in the above first to third embodiments can be commercialized by adjusting the concentration of the ginseng root part to 5 to 20 wt% and to 10 wt%. The feed additive prepared in the present invention may be added to the feed in an amount of 0.1 to 1.0% by weight and used as an animal feed. The feed containing the feed additive according to the present invention can contribute to prevention of diseases or improvement of productivity by enhancing immunity.

[Example]

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1: Isolation of pesticide-degrading strains

<1-1> Isolation of strain

Soil samples were collected from Geumsan - gun, Chungcheongnam - do, and 10 ginseng fields near Ganghwa Island in Incheon. (10%, Bogard, Syngenta Korea) and boskalide (10%) were added to TS (Tryptic Soy, Bacto) medium to select strains resistant to diphenococonazole and boscalid 47%, Kantus, Dongbu Hannong) was added to 100 ppm to prepare a culture medium. 1% (g / v) of the soil collected in the medium was added, cultured at 30 ° C and 200 rpm for 48 hours, plated on a TS agar plate medium and cultured at 30 ° C for 24 to 48 hours to form colonies 15 strains were selected first.

In order to confirm the resolving ability of the primary selected strains to difenoconazole and boscalid, the strain was inoculated into TS medium to which 100 ppm of diphenococonazole and boscalid were respectively added at a concentration of 100 ppm. After the culture medium was cultured at 30 DEG C and 200 rpm for 24 hours, the degraded pesticides were analyzed by HPLC under the following conditions.

(1) Diphenoconazole

- Column: Thermo hypersil-keystone aquasil c18 250x4.6mm

- Detector: UV detector wavelength 245 nm

- Injection amount: 10ul

- mobile phase: acetonitrile / D.W = 65/35

- Congestion time: 15 minutes

(2) Boss Khalid

- Column: Kromasil 100-5 c18 150 x 4.6 mm

- Detector: UV detector Wavelength 225nm

- Injection volume: 20ul

- mobile phase: MeOH / D.W = 70/30

- Retention time: 5.5 minutes

As a result of the above analysis, among the 15 strains selected firstly, three strains which simultaneously decompose di-phenoconazole and boscalid were identified, and one strains having the highest pesticide resolution were selected (see Fig. 2).

<1-2> Identification of the strain

The morphology and biochemical characteristics of the selected strains were analyzed and the results are shown in Table 1 below.

Morphology and biochemical characteristics reaction Colony Shape circle Colony color orange color Shape Sweet bacillus Gram staining voice Expiratory condition Flexible aerobic Endospore voice Catalase test voice

The enzyme activity of the strain was analyzed by an API ZYM kit (Biomerieux ^® , France). The results are shown in Table 2 below.

enzyme Degree of reaction Alkaline phosphatase (Alkaline phosphatase) 5 Esterase C4 4 Esterase Lipase &lt; RTI ID = 0.0 &gt; 4 Lipase 0 Leucine arylamidase &lt; RTI ID = 0.0 &gt; 5 Valine arylamidase &lt; RTI ID = 0.0 &gt; 4 Cystine arylamidase 0 Trypsine 4 alpha-chymotrypsin &lt; / RTI &gt; 4 Acid phosphatase 5 Naphthol-AS-BI-phosphohydrolase
(Naphtol-AS-BI-phosphohydrolase) 5 galactosidase &lt; RTI ID = 0.0 &gt; 0 ? -galactosidase 0 Glucuronidase &lt; RTI ID = 0.0 &gt; 0 Glucosidase &lt; RTI ID = 0.0 &gt; 0 ? -glucosidase 0 N-methyl-β-glucosamidase (Glucosamidase) 5 Mannosidase &lt; RTI ID = 0.0 &gt; 0 Fucosidase &lt; RTI ID = 0.0 &gt; 0

Further, the results of analysis of the sugar availability of the strain using an API 50 CHB kit (Biomerieux ^® , France) are shown in FIG. As a result of the kit analysis, it was confirmed that the strain was a strain belonging to Brevibacillus laetosporus as shown in Table 3.

Strip 0-19
Tube / substrate +/- Strips 20-39
Tube / substrate +/- Strip 40-49
Tube / substrate +/- 0 control group - 20 [alpha] -methyl-D-mannoside (Mannoside) - 40 D-Turanose - 1 glycerol - 21 [alpha] -methyl-D-glucoside - 41 D-Lysose (LYXose) - 2 Erythritol - 22 N-acetyl-glucosamine - 42 D-TAGatose - 3 D-arabinose - 23 Amygdalin + 43 D-Fucose - 4 L-arabinose - 24 Arbutin (ARButin) + 44 L-Fuchos - 5 Ribose - 25 Esculin + 45 D-albitol (ArabitoL) - 6 D-Xylose (XYLose) - 26 Salicin (Salicin) + 46 L-Alvitol - 7 L-xylose - 27 CELlobiose + 47 GlucoNaTe - 8 Adonitol (ADOnitol) - 28 MALtose + 48 2-keto-gluconate - 9? -Methyl-D-xylose - 29 Lactose (LACtose) - 49 5-keto-gluconate - 10 Galactose (GALactose) - 30 MELIBIOSE - 11 Glucose (GLUcose) + 31 Sucrose - 12 FRUCTOSE + 32 TREHALOSE + 13 MANOS + 33 Inulin (INUlin) - 14 SroBose - 34 MeLeZitose - 15 RHAmnose - 35 RAFfinose - 16 Dulcitol - 36 starches - 17 Inositol (INOsitol) - 37 Glycogen (GLYcoGen) - 18 MANITOL - 38 Xylitol (XyLiTol) - 19 Sorbitol - 39 Gentiobiose +

As a result of analysis of the 16s rRNA gene sequence of the strain, it was confirmed to have the nucleotide sequence of SEQ ID NO: 1. As a result of confirming the homology based on the 16s rRNA nucleotide sequence, the strain was identified as a novel strain belonging to Brevibacillus latusosporus having 99.2% homology with the Brevibacillus latexporus standard strain.

The strain was designated as "Brevibacillus latexporus WR" (hereinafter referred to as "WR" strain) and deposited on May 16, 2014 under accession number KACC91946P.

Example 2: Confirmation of the agrochemical decomposition activity of WR strain

The decomposing activity of the WR strain identified in Example 1 was tested for pesticides (diphenococonazole, boscalid).

 Specifically, diphenococonazole and boscalid pesticide were added to the TS medium at concentrations of 100, 200, 500, 1000, 2000, 3000 and 5000 ppm, respectively, and then the WR strain of Example 1 was inoculated and cultured at 30 ° C for 24 hours Lt; / RTI &gt; After completion of the cultivation, the residual pesticide concentration was measured from the culture solution under the conditions described in Example <1-1>, and the content of the pesticide decomposed with respect to the initial addition amount was calculated. The results are shown in Table 4 below.

density
(ppm) Decomposition concentration ratio (%) Difenokonazole Boss Khalid 100 81.1 35.5 200 76.3 39.8 500 63.7 40.2 1000 62.3 39.0 2000 67.7 - 3000 34.6 -

As shown in Table 4, it was confirmed that the WR strain was able to degrade various concentrations of difenoconazole and boscalid pesticide.

Example 3: Confirmation of the safety of decomposition of pesticide in WR strain

In order to confirm whether or not the pesticide sample decomposed by the WR strain in the Example 2 was safe in the body, a mouse oral administration test for diphenoconazole was conducted.

Diphenoconazole is known to have an LD _{50 of} 2,000 mg / kg based on OECD standards. Since the maximum oral dose of the mouse is 1 ml on an average weight of 20 g, 40 mg / ml (40,000 ppm) is the LD ₅₀ value of diphenococonazole. Prior to the above experiment, uninoculated difenoconazole was administered to the mice at various concentrations. As a result, LD ₅₀ The value was confirmed to be 8,000 ppm, which is 1/5 of the OECD standard.

Diphenoconazole concentration (ppm) 2,500 5,000 8,000 10,000 20,000 40,000 death rate(%) 0 0 50 80 100 100

Based on the above results, the toxicity of the degraded samples was confirmed by using the WR strain obtained in Example 2 above.

As shown in Table 4, since the maximum growth potential of the WR strain is 2,000 ppm of difenoconazole, a medium having an initial concentration of diphenococonazole of 2,000 ppm was prepared and the strain was cultured and then concentrated to 4 times and 5 times. After culturing the medium at an initial concentration of 2,000 ppm, the result of the analysis showed that the concentration of diphenococonazole was 842.2 ppm. The identified samples were pulverized by lyophilization and LD ₅₀ of the initial diphenococonazole concentration (8000ppm) and 10,000ppm (4 times and 5 times less than the initial amount), respectively. The results are shown in Table 6.

sample Diphenoconazole concentration (ppm) Treatment group 1
(Corresponding to 8,000 ppm) Treatment group 2
(Equivalent to 10,000 ppm) Early 2,094.4 After 24 hours 842.2 Final concentration 3,368.8
(4 times) 4,211.0
(5 times) Mortality (%) 0 0

The degraded samples showed no mortality in samples incubated at 8000 ppm and mortality did not occur in decomposition samples at 10,000 ppm, which showed the previous mortality rate of 80%. Therefore, it was presumed that diPhenoconazole sample decomposed by WR strain during ingestion was safe in the body.

Example 4: Confirmation of resolving ability of WR strain of pesticide residue at the top of ginseng

In order to confirm whether the WR strain can decompose the pesticide residues in the ginseng root part, 7 wt% of the ginseng ground part (leaf) was mixed in the TS medium and then the WR strain was inoculated and cultured at 30 ° C for 48 hours. The culture broth was completely dried in vacuo at 60 ° C and then pesticide-treated ground ginseng crushed and ground pesticide-treated ginseng ground crushed products were submitted to SGS Korea for measurement of residual pesticide content.

The results of the analysis are shown in Table 7.

Detection pesticide Residual content (mg / kg) Pesticide treated ginseng ground Ginseng root cultured with WR strain Boss Khalid 14.8 2.15 Cyazofamie 1.27 Not detected Pyraclostrobin 7.88 1.13 Trifloxystrobin 0.64 Not detected

As shown in Table 7, only two kinds of residual pesticides were detected in the ginseng ground part cultured with the WR strain compared to the pesticide treated ginseng ground part. Also, in the ginseng root cultured with WR strain, 85.5% of boschalide and 85.6% of pyraclastrobin were reduced compared to that of ginseng root which was not incubated with WR strain.

The above results show that the WR strain of the present invention has excellent pesticide resolution against pesticides treated at the ginseng root portion.

Example 5: Isolation of PD family ginsenoside converting bacteria

<5-1> Isolation of strain

In order to isolate strains that convert PT ginsenoside into PD ginsenosides from soil samples collected from ginseng fields such as Ganghwa and Geumsan, soil samples were added to each MRS or TS medium containing 20 wt% of ginseng ground part (G / v), and then cultured at 30 ~ 37 ℃ for 24 ~ 48 hours.

The selected strains were inoculated in each MRS or TS medium supplemented with 20% of the ginseng root part to a concentration of 1% by weight and cultured at 30 to 37 DEG C for 48 hours. The culture was completely dried at 60 ° C. 1 g of the dried sample was added to 50 ml of methanol, followed by 2 times of hot water extraction at 80 ° C, followed by concentration, and then methanol was added to a final volume of 10 ml.

From the sample, ginsenosides were analyzed by TLC analysis. The distribution of PT-derived ginsenosides (Rg1, Re, Rf, Rh1 and Rg2) and the PD-based ginsenosides (Rb1, Rc, Rb2, Rb3, Rd, Rg3, Rh2 and Compound- One strain that clarifies the band of the PD-based ginsenoside was selected instead of the weakened band of the senoside (see Fig. 3).

HPLC analysis was carried out according to the conditions shown in Table 8 below in order to quantitatively determine whether the PD-based ginsenosides were increased through the fermentation of the strain according to the results of TLC analysis.

Item Condition Analytical instrument HPLC (Agilent 1260) column Nova-Pak C18 (3.9 X 150 mm, 4 um) Eluent (A) -acetonitrile, (B) -water Flow rate 1 minute / mL gradient ~ 10 minutes (20% (A)) 10 to 40 minutes (20 to 32% (A)) 40 to 48 minutes (32 to 42% (A)) 48 to 50 minutes (42 to 100% (A)) 50 to 60 minutes (100% (A)) * Analysis by sample Pre 10 min (20% (A)) Column temperature 35 ℃

The results of HPLC experiments are shown in Table 9 below.

Control e1 e2
(WL) e3 e4 e5 e6 Rg 1 11.4 10.0 10.1 9.6 4.2 11.8 5.8 Re 20.6 17.2 15.0 19.4 18.8 18.6 16.4 Rf 1.5 0.5 0.2 0.2 0.9 0.5 1.3 Rh 1 1.9 1.4 1.5 1.5 0.1 1.1 0.2 Rg 2 5.2 3.0 3.1 3.1 0.2 0.7 0.4 Rb 1 5.0 0.1 3.0 0.1 3.7 0.7 6.9 Rc 2.3 0.2 0.3 0.3 2.7 2.7 1.3 Rb 2 4.1 0.4 0.5 0.4 1.0 1.4 0.1 Rb 3 1.4 0.3 3.3 0.6 0.2 0.7 0.3 Rd 2.3 3.9 7.8 3.9 0.3 8.0 0.7 Rg 3 2.3 1.2 1.2 1.3 0.2 1.5 0.5 Rh 2 0.9 2.6 3.7 1.7 0.2 0.00 0.1 compound-K 0.8 1.5 2.5 1.6 0.4 0.4 0.1 Total (mg / g)
(PT / PD) 59.8
(68/32) 42.1
(76/24) 52.1
(57/43) 43.3
(78/22) 32.7
(74/26) 4.80
(68/32) 34.1
(71/29)

As shown in Table 9, the strain (e2) having a higher proportion of the PD lineage than the PT line ginsenoside was selected as in the TLC result. At this time, control represents the ginseng ground part which has not undergone the fermentation of the strain.

<5-2> Identification of the strain

The morphology and biochemical characteristics of the selected strains were analyzed and the results are shown in Table 10 below

Morphology and biochemical characteristics reaction Colony Shape circle Colony color cream color Shape Sweet bacillus Gram staining voice Expiratory condition Flowable anaerobic Endospore voice Catalase test voice

The enzyme activity of the strain was analyzed by API ZYM kit (Biomerieux ^® , France). The results are shown in Table 11 below.

enzyme Degree of reaction Alkaline phosphatase (Alkaline phosphatase) One Esterase C4 2 Esterase Lipase &lt; RTI ID = 0.0 &gt; One Lipase 0 Leucine arylamidase &lt; RTI ID = 0.0 &gt; 5 Valine arylamidase &lt; RTI ID = 0.0 &gt; 5 Cystine arylamidase 0 Trypsine 0 alpha-chymotrypsin &lt; / RTI &gt; 0 Acid phosphatase 4 Naphthol-AS-BI-phosphohydrolase
(Naphtol-AS-BI-phosphohydrolase) 4 galactosidase &lt; RTI ID = 0.0 &gt; 0 ? -galactosidase 5 Glucuronidase &lt; RTI ID = 0.0 &gt; 0 Glucosidase &lt; RTI ID = 0.0 &gt; 5 ? -glucosidase 4 N-methyl-β-glucosamidase (Glucosamidase) 3 Mannosidase &lt; RTI ID = 0.0 &gt; 0 Fucosidase &lt; RTI ID = 0.0 &gt; 0

Further, the sugar availability of the strain was analyzed using API 50 CHB kit (Biomerieux ^® , France), and the results are shown in Table 12 and FIG. As a result of the kit analysis, it was confirmed that the strain was a strain belonging to Lactobacillus plantarum.

Strip 0-19
Tube / substrate +/- Strips 20-39
Tube / substrate +/- Strip 40-49
Tube / substrate +/- 0 control group - 20 [alpha] -methyl-D-mannoside (Mannoside) - 40 D-Turanose - 1 glycerol + 21 [alpha] -methyl-D-glucoside - 41 D-Lysose (LYXose) - 2 Erythritol - 22 N-acetyl-glucosamine + 42 D-TAGatose - 3 D-arabinose - 23 Amygdalin + 43 D-Fucose - 4 L-arabinose + 24 Arbutin (ARButin) + 44 L-Fuchos - 5 Ribose + 25 Esculin + 45 D-albitol (ArabitoL) - 6 D-Xylose (XYLose) - 26 Salicin (Salicin) + 46 L-Alvitol - 7 L-xylose - 27 CELlobiose + 47 GlucoNaTe + 8 Adonitol (ADOnitol) - 28 MALtose + 48 2-keto-gluconate - 9? -Methyl-D-xylose - 29 Lactose (LACtose) + 49 5-keto-gluconate - 10 Galactose (GALactose) + 30 MELIBIOSE + 11 Glucose (GLUcose) + 31 Sucrose + 12 FRUCTOSE + 32 TREHALOSE + 13 MANOS + 33 Inulin (INUlin) - 14 SroBose - 34 MeLeZitose - 15 RHAmnose - 35 RAFfinose - 16 Dulcitol - 36 starches - 17 Inositol (INOsitol) - 37 Glycogen (GLYcoGen) - 18 MANITOL + 38 Xylitol (XyLiTol) - 19 Sorbitol + 39 Gentiobiose +

As a result of analysis of the 16s rRNA gene sequence of the strain, it was confirmed to have the nucleotide sequence of SEQ ID NO: 2. As a result of confirming homology based on the 16s rRNA base sequence, the strain showed 98.2% homology with the standard strain of Lactobacillus plantarum and was confirmed as a novel strain belonging to Lactobacillus plantarum.

This strain was named "Lactobacillus plantata WL" (hereinafter referred to as "WL" strain) and deposited on May 16, 2014 with the deposit number KACC91947P.

Example 6: Growth characteristics of WL strain according to the content of the ginseng root part

 Growth characteristics of WL strain were investigated by the concentration of ginseng root.

Specifically, the ginseng ground part for fermentation was added to the medium with increasing 5 wt% to 5 wt% from 5 wt% to 25 wt%, respectively, and WL strain was inoculated to the medium at 2 wt%. The embryo was used as the excipient. For water activity at the time of fermentation, the final weight of the MRS medium was mixed so that the moisture content was 40 to 60%. The medium was incubated at 37 DEG C for 48 hours to proceed with fermentation, and the viable cell count was then measured. The results are shown in Table 13.

Additive ratio of ginseng top part (% by weight) Number of living cells (CFU / g) 5 wt% 2.5 x 10 ⁹ 10 wt% 2.7 × 10 ⁹ 15 wt% 9.0 × 10 ⁸ 20 wt% 5.6 x 10 ⁸ 25 wt% 1.2 × 10 ⁷

As shown in Table 13 above, when the ginseng ground part was included in the medium in an amount of 5 to 10% by weight, the growth of the WL strain was found to be almost similar. When the content of the ginseng root part exceeded 10% by weight, Was decreased. Based on the above results, the most preferable content of the ginseng root part was determined to be 10% by weight at the time of solid fermentation of the WL strain.

Example  7: Ginseng root over fermentation time Gin Senocide PT / PD  Series change

Based on the results of Example 6, the final concentration of the ginseng ground part was added so as to be 10 wt%, followed by solid culture.

Specifically, in order to produce 1 kg of the product, 100 g of ginseng ground part was put into 500 ml of water and heat treatment was performed at 80 ° C. for 1 hour to dissolve ginsenoside, and dissolved in 300 ml of MRS medium (about 600 g of water: about 33 g) The embryo bark, a post-excipient, was mixed well with 900 g. The mixed sample was incubated at 100 ° C for 1 hour, aseptically in 20 ml of the WS seed culture, and then anaerobically cultured at 30-37 ° C. The ginsenoside PT / PD system content change was measured in the same manner as in Example 5 -1 &gt; were used. The changes in the content of ginsenoside PT / PD series with fermentation time are shown in Table 14 below.

Elapsed time Total content of ginsenoside (mg / g) Sequence change (PT / PD) 0 5.95 67/33 12 5.89 66/34 24 5.67 64/36 36 5.43 61/39 48 5.26 60/40 60 5.14 59/41 72 5.10 58/42 84 5.13 58/42 96 5.14 58/42

As shown in Table 14, the content of PT / PD fraction of ginsenoside was the highest at 72 hours after solid fermentation, and there was no significant change after 72 hours. Considering the economical efficiency at the time of mass production, it is considered to be advantageous to ferment the WL strain for about 72 hours.

Example 8: Preparation of feed additive

<8-1> Preparation of Feed Additive 1

The ginseng ground part was dried at room temperature for 2 to 3 days to adjust the water content to 10 wt% or less and then pulverized to a particle size of 5 mm or less. On the other hand, the WR strain was pre-aerobically cultured in TS medium (10 g / L casein pancreatic decomposition product, yeast extract 5 g / L and NaCl 10 g / L) at 30 ° C for 24 hours.

The above-ground ginseng ground part was added to the TS medium in an amount of about 7% by weight and sterilized at 121 캜 for 15 minutes. Then, the pre-cultured WR strain culture was inoculated in an amount of 1% by weight, And cultured on a miracle. The culture broth was heat treated at 100 ° C. for 30 minutes to inactivate the WR strain, and then the embryonic leaf was added to adjust the concentration of the ginseng root part to 10%. After that, the culture was vacuum-dried at 60 ° C and then pulverized to prepare a feed additive. The feed additive was named &quot; feed additive 1 &quot;.

&Lt; 8-2 > Preparation of Feed Additive 2

WL strain was cultured in MRS medium (10 g of proteospeptone No. 3, 10 g of beef extract, 5 g of yeast extract, 20 g of dextrose, 1 g of polysorbate 80, 2 g of ammonium citrate, 5 g of sodium acetate, 0.1 g of magnesium sulfate, g and di-potassium phosphate 2 g / L) at 37 &lt; 0 &gt; C anaerobically.

Then, to grow the WL strain in the culture solution prepared in Example <8-1>, the MRS medium was added in an amount corresponding to the liquid amount and sterilized. Then, the pre-cultured WL strain was added at a concentration of about 2% by weight. The embryogenic foil was added to the culture broth such that the content of the ginseng root part was 10% by weight, and water was further added to adjust the total moisture content to 50% by weight. Thereafter, air was blocked at 32 ° C-37 ° C, and the mixture was subjected to solid fermentation for about 4 days, followed by vacuum drying at 60 ° C to prepare a feed additive. The feed additive was designated as &quot; feed additive 2 &quot;.

Example 9 Effect of Administration of Feed Additives

<8-1> Determination of the growth rate and immunity of the feed additive in rats

In order to compare the weight gain and immunity of the feed additive, five females were fed at 5 weeks of age. Feed additives 1, 2, and red ginseng, which are known to have an excellent effect on immunity, were fed 0.2% of feed to the experimental group, which was prepared by mixing excipients with 10% of body weight, and the body weight and feed intake were measured at intervals of one week. The initial growth rates are shown in Table 15 and Figure 5 (in the case of the control group, only the feed was fed). In addition, the cytokine IL-2, which is known to be effective for the inhibition of tumor metastasis by inducing differentiation, proliferation and activity of T cells as a T cell growth factor and inducing the formation of NK cells and antibody production of B cells, After 4 weeks of feeding, blood was collected and analyzed using an assay kit (IL-2, Bioscience). The results are shown in Table 16.

Growth rate (%) 1 week 2 weeks 3 weeks 4 weeks Control group 17.96 28.83 42.33 45.39 Feed additives 1 16.56 26.71 41.33 43.15 Feed Additives 2 21.62 33.33 47.57 51.78 Red ginseng 17.56 29.17 43.24 48.63

IL-2 (pg / ml) Control group 141.479 Feed additives 1 328.419 Feed Additives 2 311.535 Red ginseng 285.005

As shown in Table 15 and FIG. 5, the feed ratio of the feed additive 2 to the rats was the highest, but as shown in Table 16, it was confirmed that the feed additive 1 had the best immunity.

<8-2> Pancreatic pre-clinical experiment

Poultry experiments were conducted at 2 weeks of age and fed diets as shown in Table 17 below to compare body weight trends per week. In order to evaluate the vaccine and defense effects, vaccination was given at 3 weeks (nobilis salenvac-T), salmonella ( salmonella enterica Typhimurium ) at a dose of 4.5 x ^{10 9} CFU / dose.

division Feed mix Mari / County Vaccination Attack inoculation G1 (feed additive 1) 0.4% 10 O O G2 (feed additive 1) 0.4% 10 O O G3 (red ginseng) 0.4% 10 O O G4 (vaccine control) - 10 O O G5 (positive contrast) - 10 - O G6 (voice contrast) - 10 - -

As shown in FIG. 6, G1 and G3 were increased by 13.9% as shown in FIG. 6, after 1 week of vaccination based on 100% of G4 supplemented with only normal diet, G2 showed a growth rate of 8%. After 1 week of the inoculation, the weight gain of each group also increased by 16.2% and 11.9% in G1 and G3, respectively, and by 7.8% in G2. Therefore, it was confirmed that each experimental group had an effect on body weight gain compared with the control group.

The protective effect against attack of Salmonella was shown by the re - separation of attacked inocula in the spleen. As shown in FIG. 7, the values of CFU (log10) were G1 3.57 and G2 4.19, which were 1.61 times and 0.99 times lower than those of the control inoculation group G5, respectively. G1 and G2 were 1.33 times and 0.71 times And it was confirmed that the increase of immunity was more affected than that of red ginseng.

In addition, the evaluation of antibody titers after feeding of the feed additive and red ginseng was confirmed by administering the vaccine, and the results were as shown in FIG. G1, G2, and G3 groups were significantly higher than the control group (0.31, 0.45, and 0.32 times higher than the control group, respectively) after the feeding of the feed additive and red ginseng before vaccination. Serum levels of G1, G2 and G3 were 3.07, 3.2 and 3.15 times higher than negative control (G5 and G6), respectively, compared with the vaccine control group (G4) 2.81 times higher than that of the control group. Among them, G1 and G2 were measured higher than G4 till 2 weeks after vaccination, especially G1 was significantly increased.

As a result of the above results, it was confirmed that the feed additive 2 in the growth rate and the feed additive 1 in the immunity were the most effective in the test results of the rats. In the poultry experimental results, the feed additive 1 has the greatest effect on the immunity and the growth rate It is judged that feed additives that can be applied according to the livestock fed are different.

Center for Agricultural Genetic Resources KACC91946P 20140516 Center for Agricultural Genetic Resources KACC91947P 20140516

<110> CHEIL BIO, CO., LTD. <120> MICROORGANISMS HAVING ACTIVITIES OF DECOMPOSING RESIDUAL          PESTICIDES OR CONVERTING GINSENOSIDES IN THE AERIAL PART OF          GINSENG AND METHOD FOR PREPARING A FEED ADDITIVE USING SAME <130> FPD / 201405-0054 / C <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 980 <212> DNA &Lt; 213 > Brevibacillus laterosporus WR <400> 1 ggggggaacg gttatcggat ttattgggtt taagggtccg taggcggatc tgtaagtcag 60 tggtgaaatc tcacagctta actgtgaaac tgccattgat actgcaggtc ttgagtgttg 120 ttgaagtagc tggaataagt agtgtagcgg tgaaatgcat agatattact tagaacacca 180 attgcgaagg caggttacta agcaacaact gacgctgatg gacgaaagcg tggggagcga 240 acaggattag ataccctggt agtccacgcc gtaaacgatg ctaactcgtt tttggaatgt 300 aagtttcaga gactaagcga aagtgataag ttagccacct ggggagtacg aacgcaagtt 360 tgaaactcaa aggaattgac gggggcccgc acaagcggtg gattatgtgg tttaattcga 420 tgatacgcga ggaaccttac caaggcttaa atgggaaatg acaggtttag aaatagactt 480 ttcttcggac atttttcaag gtgctgcatg gttgtcgtca gctcgtgccg tgaggtgtta 540 ggttaagtcc tgcaacgagc gcaacccctg tcactagttg ccatcattaa gttggggact 600 ctagtgagac tgcctacgca agtagagagg aaggtgggga tgacgtcaaa tcatcacggc 660 ccttacgcct tgggccacac acgtaataca atggccggta cagagggcag ctacactgcg 720 aagtgatgca aatctcgaaa gccggtctca gttcggattg gagtctgcaa ctcgactcta 780 tgaagctgga atcgctagta atcgcgcatc agccatggcg cggtgaatac gttcccgggc 840 cttgtacaca ccgcccgtca agccatggaa gtctggggta cctgaagtcg gtgaccgtaa 900 caggagctgc ctagggtaaa acaggtaact agggctaatc tagaggaggg ggccccctct 960 ataaaataac ctttttgccc 980 <210> 2 <211> 1032 <212> DNA <213> Lactobacillus plantarum WL <400> 2 ggttgacgtt gcccggatta ttcgggcgta agcgagcgca ggcggttttt taagtctgat 60 gtgaaagcct tcggctcaac cgaagaagtg catcggaaac tgggaaactt gagtgcagaa 120 taggacagag gaactccatg tgtagcggtg aaatgcgtag atatatggaa gaacaccagt 180 ggcgaaggcg gctgtctggt ctgtaactga cgctgaggct cgaaagtatg ggtagcaaac 240 aggattagat accctggtag tccataccgt aaacgatgaa tgctaagtgt tggagggttt 300 ccgcccttca gtgctgcagc taacgcatta agcattccgc ctggggagta cggccgcaag 360 gctgaaactc aaaggaattg acgggggccc gcacaagcgg tggagcatgt ggtttaattc 420 gaagctacgc gaagaacctt accaggtctt gacatactat gcaaatctaa gagattagac 480 gttcccttcg gggacatgga tacaggtggt gcatggttgt cgtcagctcg tgtcgtgaga 540 tgttgggtta agtcccgcaa cgagcgcaac ccttattatc agttgccagc attaagttgg 600 gcactctggt gagactgccg gtgacaaacc ggaggaaggt ggggatgacg tcaaatcatc 660 atgcccctta tgacctgggc tacacacgtg ctacaatgga tggtacaacg agttgcgaac 720 tcgcgagagc aagctaatct cttaaagcca ttctcagttc ggattgtagg ctgcaactcg 780 cctacatgaa gtcggaatcg ctagtaatcg cggatcacat gccgggggaa aaaacctttc 840 ccggggcctt tgtacacccc gcccgtcaca ccatgaagag tttgtaacac ccaaaagtcc 900 ggtgggggta acctttttag gaaccagccc gcctaaaggt gggacagatg attaggtgat 960 tagaacagaa gggcccccaa aaacaaacac aaaaaaaaga gaagagggag cgggggggtt 1020 tttttttaaa aa 1032

Claims (9)

Brevibacillus laterosporus WR strain (Accession No .: KACC91946P).
delete A method for removing residual pesticide residues in the ginseng root part, comprising the step of inoculating a culture broth or a fermented product of Brevibacillus latexporus WR strain (Accession No .: KACC91946P) into a ginseng root part and culturing.
4. The method of claim 3, wherein the residual pesticide is difenoconazole, boscalid, cyazopamy, pyraclostrobin or triplocystrobin.
delete delete Culturing the Brevibacillus Laterosporus WR strain (Accession No .: KACC91946P), its culture or fermentation product after inoculation on the surface of ginseng root; And
And drying the culture. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt;
delete Culturing the Brevibacillus Laterosporus WR strain (Accession No .: KACC91946P), its culture or fermentation product after inoculation on the surface of ginseng root;
Culturing the above culture after inoculating Lactobacillus planta WL strain (accession number: KACC91947P), its culture or fermentation product; And
And drying the culture. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt;

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