KR101821351B1 - Method for mass production of Poria cocos - Google Patents

Abstract

The present invention relates to a method for producing a microorganism which comprises culturing a bacterium strain in a liquid medium to obtain a bacterium mycelium, inoculating the obtained bacterium mycelium into a sawdust medium containing sawdust, rice bran, potato starch and corn starch, And a step of growing the seedlings.

Description

Method for Mass Production of Poria cocos

More particularly, the present invention relates to a method for mass production of bamboo shoots, which comprises culturing a bamboo shoot strain in a liquid medium to obtain a bamboo shoot mycelium, And a step of growing the inoculated benthic mycelium.

Poria cocos Wolf was mainly used in Oriental medicine, but recently it has been used as a functional food. It is a kind of fungi belonging to the taxonomic class. It belongs to the genus Bacillus subtilis.

After 3 to 5 years after the pine tree has been harvested, Bombyx mori occurs as a parasitic or by-produced brown rot fungus in the vicinity of its roots, forming an irregular sclerotia. Antitumor immunity, echinomycin ), Which is a medicinal resource containing a large amount of anticancer activity, is not well studied.

In the prior art regarding the cultivation method of bamboo shoots, the seeds were attached to the wood chips (10 to 30 cm in diameter and about 20 to 60 cm in length) using sawdust or grainy media and sealed with a black plastic bag, It is easy to harvest the bamboo shoots after 6 months by cultivating the bamboo shoots at a temperature of 15-25 ° C in a place where humidity control is easy (dark room) at a proper temperature of 70-80% A method of cultivating bamboo shoots has been disclosed (Korean Patent Publication No. 10-2005-0051801).

In addition, a method for cultivating non-woody non-reclaimed plastic bags in a soil for mass production of high-quality bamboo leaves has been disclosed (Korean Patent Publication No. 10-2011-0000089).

However, the above-mentioned prior arts have a problem in that there is a limitation in the mass production of bamboo shoots because no mycelia are used through liquid culture, no sawdust medium is used, or the specific composition of the sawdust medium is not disclosed.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made keeping in mind the above problems occurring in the prior art, and it is an object of the present invention to provide a method for rapidly growing bacillus strains and culturing the same in a liquid medium and a sawdust medium, .

In order to achieve the above-mentioned object, the present invention provides a method for producing a microorganism which comprises the steps of (a) culturing a bacterium strain in a liquid medium to obtain a bacteriological mycelium, (b) , And (c) growing the inoculated benthic mycelium. The mass production method comprises the steps of:

In addition, the present invention provides a method of producing the above-mentioned method.

The present invention also relates to a strain used as a bacterium for the production of bryon, wherein the strain is selected from the group consisting of Poria cocos GFRI2016PC01 (KFCC11592P), Poria cocos GFRI2016PC03 (KFCC11593P), Poria cocos GFRI2015PC07 (KFCC11619P), one kind or more strains selected from the group consisting of PO Ria Cocos (Poria cocos) GFRI2015PC09 (KFCC11620P) , PO Ria Cocos (Poria cocos) GFRI2015PC10 (KFCC11621P) and Po Liao Cocos (Poria cocos) GFRI2015PC17 (KFCC11622P) to provide.

According to the present invention, it is possible to rapidly propagate bacteriological strains and to produce large quantities of bacterium through culture in a liquid medium and a sawdust medium.

In addition, the artificial cultivation can be mass-produced all year round regardless of the season, by removing the conventional method of production that relies on natural extraction.

Figs. 1A to 1F show a sample mushroom fruiting body.
FIG. 2A shows a benthic strain (upper) and a proliferating body (lower) separated from the example of FIG. 1A, and FIG. 2B shows a benthic strain (upper) and a proliferating body (lower) separated from the example of FIG. 1B.
FIG. 3 shows the result of selection of the complex medium strain of Bokryeong.
Fig. 4 shows the results of investigating the pH characteristics of the goryeong solids medium.
FIG. 5 shows the results of investigation of the pH characteristics of the gyeongryong liquid medium.
Fig. 6 shows the amount of pH change in the gyeongryong liquid medium.
7 shows a process of culturing a large amount of mycelium in a gyeongryong liquid medium.
8A to 8F show a process of growing and culturing mycelia by inoculating a sawdust medium with a bamboo shoot, wherein FIG. 8A shows a cultivation step after liquid hyphae, FIG. 8B shows a state where mycelium of Byeong Ryong is activated, , FIG. 8D to FIG. 8F show a process of culturing bamboo shoots.

(B) culturing the bacterium strain in a liquid medium to obtain a bacterium mycelium; A step of inoculating the obtained benthic mycelium into a sawdust medium containing sawdust, rice bran, potato starch and corn starch, and (c) growing the inoculated benthic mycelium.

In the mass production method of the present invention, the bacterium strain is selected from the group consisting of Poria cocos GFRI2016PC01 (KFCC11592P), Poria cocos GFRI2016PC03 (KFCC11593P), Poria cocos GFRI2015PC07 (KFCC11619P) , Poria cocos GFRI2015PC09 (KFCC11620P), Poria cocos GFRI2015PC10 (KFCC11621P) and Poria cocos GFRI2015PC17 (KFCC11622P).

In the mass production method of the present invention, KFCC11592P and KFCC11593P were deposited on August 7, 2014, and KFCC11619P to KFCC11622P were deposited in Korean Microorganism Preservation Center on September 23, 2015, respectively.

In the mass production method of the present invention, the bacterium strain may preferably be Poria cocos GFRI2016PC01 (KFCC11592P) and / or Poria cocos GFRI2016PC03 (KFCC11593P).

In the mass production process of the Poria cocos of the present invention, the liquid medium of step (a) dextrose 25g / L, yeast extract _{5g / L, CaCl 2 2.2g /} L, MgSO 4 1.85g / L, FeCl 2 0.027g / L, 0.1 g / L of inositol and 0.001 g / L of thiamine.

In the mass production method of the present invention, the liquid medium of step (a) preferably contains 25 g / L of dextrose, 5 g / L of yeast extract, 2.2 g / L of CaCl ₂ , 1.85 g / L of MgSO ₄ , ₂ 0.027 g / L, inositol.

In the mass production method of the present invention, the cultivation in step (a) is carried out at a temperature of 18 to 22 캜, preferably 19 to 21 캜, more preferably 20 캜, 0.1 to 1.0 vvm, preferably 0.5 To 1.0 vvm, more preferably 0.7 vvm, and the cancer condition for 3 days to 7 days, preferably 4 days to 6 days, more preferably 5 days.

In the mass production method of the present invention, the term " dark condition " refers to a condition of a growth temperature of 10 to 12 DEG C or less and a humidity of 40% or less for the vitality of mycelial growth, , Mycelial growth can be promoted by temperature and humidity shocks.

In the mass production method of the present invention, the sawdust medium may further comprise buckwheat fermented product, magnesium sulfate, potassium nitrate, and calcium nitrate.

In the mass production method of the present invention, the sawdust medium comprises 30 to 35 parts by weight of rice bran, 20 to 30 parts by weight of potato starch, 5 to 10 parts by weight of corn starch, 0.1 to 1 part by weight of magnesium sulfate, 0.5 to 1.5 parts by weight of potassium nitrate, and 1.0 to 3.0 parts by weight of calcium nitrate.

In the mass production method of the present invention, the "buckwheat fermentation powder" refers to pulverized particles having a particle size of 3 to 0.3 μm in a state of being bound with an organic substance by enzymatic action of buckwheat.

In the mass production method of the present invention, the sawdust may be at least one kind selected from the group consisting of pine trees and various kinds of pine sawdust, fir sawdust, litha pine sawdust, pine wood sawdust and larch sawdust.

In the mass production method of the present invention, the sawdust may be pine sawdust.

In the mass production method of the present invention, the growth of the step (c) is carried out at a temperature of 12 to 18 캜, preferably 13 to 17 캜, most preferably 14 to 16 캜, To 30 days, preferably 23 to 27 days, more preferably 24 to 26 days.

Further, the present invention relates to a bamboo shoot produced by the above method.

The present invention also relates to a strain used as a bacterium for the production of bryon, wherein the strain is selected from the group consisting of Poria cocos GFRI2016PC01 (KFCC11592P), Poria cocos GFRI2016PC03 (KFCC11593P), Poria cocos GFRI2015PC07 (KFCC11619P), one kind or more strains selected from the group consisting of PO Ria Cocos (Poria cocos) GFRI2015PC09 (KFCC11620P) , PO Ria Cocos (Poria cocos) GFRI2015PC10 (KFCC11621P) and Po Liao Cocos (Poria cocos) GFRI2015PC17 (KFCC11622P) to provide.

Hereinafter, the present invention will be described in more detail by way of examples. However, the following examples are intended to illustrate the present invention, but the scope of the present invention is not limited to these examples.

≪ Example 1 > (Sclerotia collection and selection of strains)

The germ strains as shown in Figs. 1A to 1F were collected from Jangseon, Yangyang and Samcheok in Gangwon Province.

In order to examine mycelial elongation of the collected bacterium strains, 19.5 g of PDA medium (Difco ^® Potato Dextrose Agar) was sterilized in a 1 L Erlenmeyer flask, which had been dissolved in 500 mL distilled water, at 121 ° C for 15 minutes.

The inoculation of the bacterium strain was carried out by using a cork borer No. 3 (diameter: 7 mm), removing the six strains of the bacterium grown in the PDA to the same size, and inoculating the strains into the center of each PDA plate medium 2 was repeated.

In order to investigate the growth rate in the complex medium of Byeongryong, 8 complex media such as MCM, YM and YMPG as shown in the following Table 1 were used. In the production of the complex medium, 1 L of distilled water was added in g unit .

In the following Table 1, yeast extract, malt extract and Fenton were Difco ^® , and other reagents were Sigma ^® .

Each of the media prepared above was sterilized in a high-pressure sterilizer at 121 ° C for 15 minutes to prepare a plate culture medium based on the center point of the Petri dishes having lateral (a) and vertical (b) lines for growth.

division MCM YM YMPG MMM MALT YPSS GPM PDA Dextrose 20 10 Yeast extract 2 3 2 4 10 Malt extract 3 10 20 peptone 2 5 2 4 5 4 Shrubber 20 20 Starch 15 glucose 10 15 Asparagine One 2 KH ₂ PO ₄ 0.46 2 0.46 15 0.46 K ₂ HPO ₄ One MgSO ₄ 0.5 One One One One PDA 19.5 Baby 24 24 24 24 24 24 24 Buckwheat fermented powder 0.5 0.5 0.2

The culture conditions were 16 ° C to 22 ° C and 60% RH, and light conditions of 5,000 Lux were applied. After culturing for 30 days in an incubator, the cells were cultured on a line (a) and a vertical line (b) The growth of the mycelium grown was measured.

As a result of measurement of the mycelial growth, 82.6 mm, 88.4 mm, and 84.2 mm were found on the MMM, MALT, and YPSS media, respectively, of the eight kinds of complex media shown in Table 1, and the mycelial viability was excellent.

On the other hand, the average growth was 60.5 and 62.8mm in MMM and YPSS medium, respectively. In addition, the growth of the benthic strains in the complex medium showed a noticeably slower growth in the YM and GPM medium, and it was found that there was not much difference in growth in the other mushrooms.

The mycelial growth rate was the highest at 20 ℃ and the mycelial growth was generally between 16 ℃ and 22 ℃.

As can be seen from the above, the bacterium strains having excellent mycelial growth ability are referred to as Poria cocos GFRI2016PC01, Poria cocos GFRI2016PC03, Poria cocos GFRI2015PC07, and Poria cocos GFRI2015PC09 Poria cocos GFRI2015PC10 and Poria cocos GFRI2015PC17 were deposited with the Korean Society for Microbiological Research and deposited with KFCC11592P, KFCC11593P, KFCC11619P, KFCC11620P, KFCC11621P and KFCC11622P respectively.

<Example 2> Investigation of appropriate pH in solid medium of bamboo shoots

Solid media were prepared at pH 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0 and 8.5 in order to investigate the optimum pH of six strains according to Example 1.

PDA (Difco ^® Potato Dextrose Agar) was dissolved in 500 ml of distilled water. The pH was adjusted to 0.001 with NaOH and HCl, sterilized at 121 ° C for 15 minutes in a high pressure sterilizer, .

Inoculation of the strains was carried out using a cork borer No. 3 (diameter: 7 mm), removing the strains grown on the PDA to the same size, and repeating 5 times by inoculating five pieces of each piece into the center of the PDA plate medium. The cells were cultured in an incubator at a temperature of 22.0 ° C and a humidity of 60%, and the growth was observed at intervals of 7 days.

The culture conditions were 22.0 ° C and 60% RH, and light conditions of 5,000 lux were applied. After culturing for 30 days in an incubator, the culture medium was cultured at 7 days, 14 days, and 21 days after the inoculation, ) And length (b), respectively.

(A) and (b) were measured as the average growth rate of the mycelia grown from the center in 0.5 mm increments.

As shown in FIG. 4, when the pH of the medium was changed, the pH of the medium was maintained at about pH 4.0 to 7.0, and the pH was in the range of 7.0 to pH 8.0 , And it was confirmed that the growth of the hyphae mycelium was not greatly influenced by the change of pH.

&Lt; Example 3 > Measurement of optimum pH in a liquid medium of Bokryeong

For the pH study of liquid culture medium, 24 g of Difco ™ Potato Dextrose Broth (PDB) and 1 L of distilled water were dissolved and divided into 50 ml of 100 ml Erlenmeyer flask. Using NaOH and HCl, After the pH was adjusted, the inlet was closed and sterilized in a high-pressure sterilizer at 121 ° C for 15 minutes.

Inoculation was carried out by inoculating three pieces into each of three different Erlenmeyer flasks using pH 3 cork borer No. 3 (diameter 7 mm), followed by culturing in an incubator at a temperature of 23. ° C for 30 days, The optimum pH of six strains of Byeongryong was investigated.

As shown in Fig. 5, when the pH value of the third week of pH 7.5 was exceptionally taken into consideration, the optimum growth rate was observed at pH 4.0 to 0.0525 g, The growth at pH 6.0 was superior.

As a whole, it was confirmed that the pH of the medium was more preferably in the range of pH 5.0 to 6.5 than that of the alkaline medium of pH 8.0 to 8.5.

&Lt; Example 4 > Mass culture of mycelium of mycelium through liquid medium

In order to select the liquid culture medium of Byeongryong, similar hyphae growth rate and bacterium amount were shown in 5 different media (GPB, PDB, MCM, MEM, CoCos medium). However, CoCos medium (25 g dextrose, 5 g of the enzyme extract, 2.2 g of CaCl ₂ , 1.85 g of MgSO ₄ , 0.027 g of FeCl ₂ , 0.1 g of inositol, and 0.001 g of thiamine) were selected with optimal culture medium.

To investigate the effect of incubation time on mycelial culture in a 20 L liquid culture bottle, the amount of mycelium was measured from the 3rd day. The amount of mycelium decreased at an increased rate starting from a certain period, Was judged to be appropriate between 3 days and 7 days.

As a result of investigating the effect of aeration on the production of mycelium, the amount of dried cells increased with increasing the amount of aeration. However, since the increase of the aeration induces the evaporation of the water in the medium, the appropriate aeration amount is 0.7 vvm (vol.of air / vol.of medium / min).

&Lt; Example 5 > (Mass production of bamboo shoots through inoculation and growth of mycelium)

Sawdust medium was used to inoculate the bacterium mycelium cultured in the liquid medium of Example 4 above. The use of sawdust media is intended to compare the mycelial growth and developmental stability of the bamboo shoots.

The composition of the sawdust medium was 33 parts by weight of rice bran, 25 parts by weight of potato starch, 8 parts by weight of corn starch, 3 parts by weight of buckwheat fermented powder, 0.5 part by weight of magnesium sulfate anhydrous, 1 part by weight of potassium nitrate and 2 parts by weight of calcium nitrate were mixed and molded into spherical, hemispherical or hexahedral shapes and sterilized.

The sawdust medium was filled with sterilization pouches (30 × 32 × 11 cm) at a rate of 1500~1600 g and sterilized at 121 ° C. for 60 minutes.

The sterilized medium prepared from sawdust was cooled and inoculated with liquid seeds prepared in advance. Liquid cultured mycelia were inoculated into 200 ml of sterilized medium. The inoculated sawdust medium was cultured in a 15 ° C incubator for 25 days, and mass culture was performed.

Although the present invention has been described with reference to the preferred embodiments thereof, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit and scope of the invention as defined in the following claims. It can be understood that

According to the present invention, it is possible to rapidly propagate bacteriological strains and to produce large quantities of bacteriocins through cultivation in liquid medium and sawdust medium, as well as to overcome the conventional bacteriological production method which relies on natural harvesting, The present invention can be applied to a technical field to which the present invention belongs.

Korea Microorganism Conservation Center (Domestic) KFCC11592P 20140807 Korea Microorganism Conservation Center (Domestic) KFCC11593P 20140807 Korea Microorganism Conservation Center (Domestic) KFCC11619P 20150923 Korea Microorganism Conservation Center (Domestic) KFCC11620P 20150923 Korea Microorganism Conservation Center (Domestic) KFCC11621P 20150923 Korea Microorganism Conservation Center (Domestic) KFCC11622P 20150923

Claims (10)

(a). Culturing the bacterium strain in a liquid medium to obtain a bacterium mycelium, wherein the culture is carried out at a temperature of 18 to 22 DEG C, aeration amount of 0.1 to 1.0 vvm and cancer condition for 3 to 7 days;
(b). Inoculating the obtained benthic mycelium into a sawdust medium containing sawdust, rice bran, potato starch and corn starch; And
(c). And growing the inoculated benthic mycelium. The method according to claim 1, wherein the bacterium is selected from the group consisting of Poria cocos GFRI2016PC01 (KFCC11592P), Poria cocos GFRI2016PC03 (KFCC11593P), Poria cocos GFRI2015PC07 (KFCC11619P), poria cocos Poria cocos GFRI2015PC09 (KFCC11620P), Poria cocos GFRI2015PC10 (KFCC11621P) and Poria cocos GFRI2015PC17 (KFCC11622P). The method according to claim 1, wherein the liquid medium of step (a) is selected from the group consisting of dextrose 25 g / L, yeast extract 5 g / L, CaCl ₂ 2.2 g / L, MgSO ₄ 1.85 g / L, FeCl ₂ 0.027 g / g / L and thiamine 0.001 g / L. delete The mass production method according to claim 1, wherein the sawdust culture medium further comprises buckwheat fermentation powder, magnesium sulfate, potassium nitrate, and calcium nitrate. The method of claim 1, wherein the sawdust medium comprises 30 to 35 parts by weight of rice bran, 20 to 30 parts by weight of potato starch, 5 to 10 parts by weight of corn starch, 2 to 5 parts by weight of buckwheat fermented powder, 0.1 to 1 part by weight of magnesium, 0.5 to 1.5 parts by weight of potassium nitrate, and 1.0 to 3.0 parts by weight of calcium nitrate. The mass production method according to claim 1, wherein the sawdust is at least one selected from the group consisting of pine sawdust, fir sawdust, rigid pine sawdust, pine sawdust, and larch sawdust. The method according to claim 1, wherein the growth of step (c) is performed by culturing the inoculated benthic mycelium at a temperature of 12 to 18 ° C for 20 to 30 days. 9. The method according to any one of claims 1 to 7, delete

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