WO2008111803A1

WO2008111803A1 - Method in which microorganisms is cultivated in the blocks of wood and the wood chips cultivated with the method

Info

Publication number: WO2008111803A1
Application number: PCT/KR2008/001413
Authority: WO
Inventors: Seok Tae Lee
Original assignee: Seok Tae Lee
Priority date: 2007-03-14
Filing date: 2008-03-13
Publication date: 2008-09-18
Also published as: KR20080084052A; KR100881720B1

Abstract

The present invention relates to a method of culturing microorganisms in a piece of wood comprising the steps of : collecting miscellaneous woods such as waste wood, fell wood, and wood root; cutting the collected woods into a predetermined length by putting the collected woods into a crusher; sorting the pieces of cut woods according to the size of the pieces of woods by putting the woods; mixing a predetermined quantity of rice bran into the pieces of sorted wood; poring a strain culture solution into a mixture, to which said rice bran is mixed, so that a predetermined water content ratio is obtained; poring a mixture, to which said strain culture solution is pored, into a closed space, supplying an oxygen, and fermenting said strain culture solution for a predetermined period at a predetermined temperature! and producing a strain culture wood chip after fermenting the microorganisms for a predetermined period, and also relates to a microorganism culture wood chip which is cultured according to the steps for culturing described above.

Description

[DESCRIPTION] [Invention Title]

METHOD IN WHICH MICROORGANISMS IS CULTIVATED IN THE BLOCKS OF WOOD AND THE WOOD CHIPS CULTIVATED WITH THE METHOD [Technical Field]

<i> The present invention relates to a method of culturing microorganisms on a piece of wood, and a microorganism culturing wooden chip cultured using the method, and more particularly, to a method of culturing a beneficial indigenous microorganism by seeding the indigenous microorganisms to collected miscellaneous wood, waste wood, fell wood, wood root and the like, and a microorganism culturing wooden chip cultured using the method. [Background Art]

<2> There are many elements in the natural soil, and there are many unknown components among them. In particular, indigenous microorganisms in the soil live by continuously transforming the condition and the environment and adapting themselves to the condition and the environment by feeding the water and the oxygen, and without the water and the oxygen, the microorganisms are not in the activity and come into anesthetic state.

<3> A mineral enzyme obtained by completely fermenting the soil by microorganism decomposition can be said to be a soil enzyme obtained by decomposing the soil by changing the environment while feeding the water and the oxygen until the soil is decomposed by the microorganism up to 300 mesh or above. The mineral enzyme is a type of enzyme which both the human and the crops absorb. As described above, the soil is stabilized, proceeds the decomposition by means of mineral enzyme, presents the decay and removes the bad smell, thereby making clean environment.

<4> In fact, two hundred million or more microorganisms live in a spoonful soil and these microorganisms proceed the procedure of fermenting the soil itself by changing the environment. Satisfactorily activating the microorganisms living in the soil enables all the materials to be decomposed into fine grains of 300 mesh of above. However, any method using machines can not decompose the materials up to 300 mesh or above like this.

<5> I, the inventor, performed a method of treating sources of pollution, which is to train and to culture the microorganisms each acting its role, as they are, in the natural environment, with the result that, I discovered that not only the bad smells are removed, but also the effect of decomposition is excellent, whereby I confirmed that the simple method of conforming to the nature adapts to the environment without adverse effect so as to effectively restrain secondary pollution and the development of pathogenic germ due to the microorganisms.

<6> Recently, in development of stock farm products, due to intensive raising methods, the possibility of infection of various pathogenic germs, a decrease of disease-resisting force, a danger in an overdose of antibiotics for prevention of diseases, an increase of accumulation of environmental hormone and residual antibiotics, and the pollution of feed resources due to the environmental pollution have become great problems.

<7> Therefore, the necessity of production of functional stock farming is increased so that the mass proliferation and production of microorganism is required. [Disclosure] [Technical Problem]

<8> The object of the present invention is to provide a method of culturing microorganisms on a piece of wood, which is capable of mass proliferation and production of microorganisms by seeding the microorganism culture solution on a piece of wood and fermenting it.

<9> In addition, the object of the present invention is to provide a microorganism culturing wooden chip, which can activate the microorganisms by supplying the water to the narcotized microorganisms, in case it is desired that anyone can easily mass deposit the microorganisms and use the deposited microorganisms as an environment improvement agent or various enzyme products, through the mass proliferation and production of the microorganisms. [Technical Solution] <io> To achieve the above object, a method of culturing microorganisms in a piece of wood of the present invention is characterized in that it comprises the steps of: <π> collecting miscellaneous woods such as waste wood, fell wood, and wood root; <12> cutting the collected woods into a predetermined length by putting the collected woods into a crusher! <13> sorting the pieces of cut woods according to the size of the pieces of woods by putting the woods; <14> mixing a predetermined quantity of rice bran into the pieces of sorted wood! <15> poring a strain culture solution into a mixture, to which said rice bran is mixed, so that a predetermined water content ratio is obtained; <16> poring a mixture, to which said strain culture solution is pored, into a closed space, supplying an oxygen, and fermenting said strain culture solution for a predetermined period at a predetermined temperature; and <17> producing a strain culture wood chip after fermenting the microorganisms for a predetermined period. <18> In addition, the method of the present invention is characterized in that it further comprises the step of removing the water from the microorganism culture wood chip, and bringing the microorganisms into anesthetic state, after the production step. <i9> In addition, the method of the present invention is characterized in that it further comprises the step of activating the microorganisms by supplying the water and a broth to the narcotized microorganisms. <20> In addition, the method of the present invention is characterized in that strain of said strain culture solution is one of Bacillus sp. strain,

Pediococcus acidilactici strain, and Pediococcus pentosaceus strain. <2i> To achieve the above object, a microorganism culture wood chip of the present invention is characterized in that it is cultured according to the method of culturing microorganisms in a piece of wood of the present invent ion. [Advantageous Effects]

<22> According to the present invention, the microorganisms can be mass proliferated and produced by seeding the microorganism culture solution on a piece of wood and fermenting it.

<23> In addition, the microorganisms can not only be more easily mass stored by using the microorganism culturing wooden chip cultured by seeding the microorganism culture solution on a piece of wood and fermenting it, but also if the microorganisms are to be used as an environment improvement agent or the like, the microorganisms can be widely used in the industry by being developed into functional foods, nutrients, functional feed additives, environment improvement agent, various enzyme preparation or the like by using the fermented microorganisms through activating the narcotized microorganisms. Furthermore, the microorganisms can be applied to solving the problems of avian influenza and environmental pollution which presently seriously occur. [Description of Drawings]

<24> Fig. 1 shows, as an embodiment of the present invention, a procedure of culturing the microorganisms on a piece of wood.

<25> Fig. 2 shows the activation of amylase, protease, cellulase, xylanase and phytase of Pediococcus sp. strain used in the present invention detected on an MRS agar plate.

<26> Fig. 3 shows the acid resistance of the Pediococcus pentosaceus strain used in the present invention detected in a range of pH of 2.0 to 5.0.

<27> Fig. 4 shows the heat stabilities of the Pediococcus pentosaceus strain

O O used in the present invention detected in a range of 30 C to 6OC.

<28> Fig. 5 shows the bile salt-tolerance of the Pediococcus pentosaceus strain used in the present invention detected with a variety of concentration of bi Ie salt .

<29> Fig. 6 shows a degree of growth inhibition of E-coil of the Pediococcus pentosaceus strain used in the present invention detected by using the MRS broth. [Mode for Invention]

<30> Now, the present invention will be described below in detail with reference to embodiments. However, it should be noted that the embodiments are only to exemplify the present invention, and the contents of the present invention is not limited to the embodiments.

<3i> Fig. 1 shows, as an embodiment of the present invention, a procedure of culturing the microorganisms on a piece of wood, Fig. 2 shows the activation of amylase, protease, cellulase, xylanase and phytase of Pediococcus sp. Strain used in the present invention detected on an MRS agar plate, Fig. 3 shows the acid resistance of the Pediococcus pentosaceus strain used in the present invention detected in a range of pH of 2.0 to 5.0, Fig. 4 shows the heat stabilities of the Pediococcus pentosaceus strain used in the present

O O invention detected in a range of 30 C to 6OC, Fig. 5 shows the bile salt- tolerance of the Pediococcus pentosaceus strain used in the present invention detected with a variety of concentration of bile salt, and Fig. 6 shows a degree of growth inhibition of E-coil of the Pediococcus pentosaceus strain used in the present invention detected by using the MRS broth.

<32> As shown in Fig. 1, miscellaneous woods such as waste wood, fell wood, thinned wood, orchard pruned wood, and wood root are collected and put into a crusher (SlO, S20). The put in miscellaneous woods are cut into diameter of 2 to 10 and length of 5 to 60, the pieces of cut wood are put into a sorter, and the pieces of woods having diameter of 2 to 10 cm and length of 5 to 60 cm are sorted (S30). The pieces of wood outside of the above range may not sufficiently achieve the effect of the present invention, and furthermore, if chips made of the pieces of wood are too large, the chips may be caught by a conveyor belt or the like at the time of conveying, and if the chips are too small, the treatment of them may be complicated.

<33> The sorted pieces of woods are mixed with rice bran to the ratio of 7:3, and the strain solution is poured into the mixture to have a predetermined water content ratio (S50). At this time, the water content ratio should be 50 to 70%, and preferably 60% in the present embodiment.

<34> The microorganisms used in the strain solution of the present invention is preferably the microorganisms naturally existing in the soil or cultured in the soil .

<35> There are many elements in the soil, and there are many unknown components among them. In particular, indigenous microorganisms in the soil live by continuously transforming the condition and the environment and adapting themselves to the condition and the environment by feeding the water and the oxygen, and without the water and the oxygen, the microorganisms are not in the activity and come into anesthetic state.

<36> Therefore, the soil is taken from the depth of 5 to 10 cm from the surface of soil of various uncontaminated regions, and actinomycetes are separated by using three kinds of broths for separation of actinomycetes.

<37> The separated microorganisms are trained to live for themselves in any environmental condition. Probiotics in the soil are intensively cultured by using a mineral broth of a microorganism cultured body which generates a lot of anions since a lot of germanium is contained in the indigenous microorganisms.

<38> The microorganisms are induced to come to a condition of inactivity by,

O first, agitating a drum closed and having the culture temperature of 37 C for 7 hours while supplying an oxygen, and by repeatedly culturing the microorganisms while supplying an oxygen by using an air_blower in a closed space until the water content is lowered to 20% or lower.

<39> Next, a process of inducing an activation of the microorganisms by supplying again the water to the microorganisms, agitating and culturing using the above described process, and supplying an oxygen in a closed space is repeated for about 3 times.

<40> As a result, it can be confirmed that in the microorganisms cultured by removing the water, bring into anesthetic state, and next culturing with a mineral broth, the secondary contamination is substantially further reduced than and the degree of the offensive smell and the decay is lower than in the microorganisms cultured with the mineral broth directly after the separation.

Also it could be seen that since the development of the pathogenic germ is less, when adding the microorganisms to the feed, the infection of the pathogenic germ to the bred animals is further reduced than the feed to which the microorganisms are not added. <4i> To deposit the cultured microorganisms, the microorganisms are kept in a closed space by putting the microorganisms into a piece of wood or wood root having a plenty of fibroid material. <42> One of Bacillus sp. strain, Pediococcus acidilactici strain, and

Pediococcus pentosaceus strain which are the microorganisms cultured as such with the mineral broth is used as the microorganism of an embodiment of the present invention. <43> The characteristics of the nature of Pediococcus pentosaceus strain among Bacillus sp. strain, Pediococcus acidilactici strain, and Pediococcus pentosaceus strain is described below.

<44>

<45> Isolation of the Pediococcus sp. strain from the soil and identification of the Pediococcus sp. strain

<46> In the present invention, to isolate the Pediococcus sp. microorganisms from the soil, a soil preparation was spread on a MRS agar broth which

O contains 0.02% NaN₃, and cultured for 24 hours at 37 C, and five strains were

firstly selected.

<47> By using the firstly selected strains, five strains were secondly isolated based on the colony type and gram positive, etc.. One strain showing excellent growth was selected from them, a sugar fermentation experiment was performed on it by using the API 50 CHL Kit (Biomer ieux. , France), and it was identified with the Pediococcus sp. strain. The selected Pediococcus sp. strain was activated through 3 to 4 times by subculture. The strain was cultured by using the MRS broth, cultured for 24 hours at 37 C, then was preserved by means of glycerol stock for the next experiment.

<48> Specifically, the Pediococcus sp. strain isolated from the present invention was confirmed to be a typical anaerobic strain and gram positive coccus. In addition, it was confirmed to be the Pediococcus pentosaceus (99.9%), in particular, among the Pediococcus sp. strain (refer to table 1).

<49> Furthermore, as the result of the analysis of the 16S rRNA sequence of the Pediococcus sp. strain, it was again confirmed to be the Pediococcus pentosaceus strain in view of the sequence of 1,501 bases of the Pediococcus sp. strain. Therefore, it was seen that the Pediococcus sp. strain of the present invention is a lactic acid which is excellent in safety and can be used as a microorganism preparation.

<50>

<5i> Table 1

<52> A result of sugar fermentation experiment performed in the Pediococcus sp. strain by using the API 50 CHL Kit

(+ - positive, — : negative)

<53> <54>

<55> Research of growth characteristics of the Pediococcus sp. strain <56> In the present invention, to research of growth curve of the Pediococcus sp. strain, the preparations were sampled with an interval of 6

O hours at 37 C by using the MRS broth without adjusting the pH in the jar fermenter, and cultured for 48 hours.

<57> In addition, to measure the total viable cell count of the Pediococcus sp. strain, the culture solution was continuously diluted 10 times by using the 0.85% NaCl solution for each time, then MRS agar plate was smeared by dividing the culture solution by an amount of 0.1 mL. Then, the culture

O plate of the strain was cultured for 24 hours at 37 C, the colony forming unit was measured.

<58> As a result of researching the optimum growth condition of the Pediococciis sp. strain, it was found that, as shown in Fig.l, if the strain is cultured for 24 hours, it will reach the maximum point, that is, the

9 concentration of 4.3 x 10 CFU/ml . However, it was determined that it is preferable, in view of various activation, to use the strain after 6 to 12 hours which is the maximum growth period.

<59>

<60> Research of enzyme creation of the Pediococcus sp. strain

<6i> In the present invention, to research the enzyme creation of the Pediococcus sp. strain, the strain was researched in view of the secretion of particular enzyme by means of presence of clear zone which appears by using the screening media as suggested in Tables 1 and 2.

<62> Specifically, in case of the protease, xylanase and phytase, the presence of clear zone could be confirmed directly after the culture of the strain, and in case of the cellulase, it was dyed with 0.2%(w/v) congo red solution and washed with 1 M NaCl, thereby confirming the clear zone. In addition, in case of α -amylase, it was dyed with a solution containing 02.%(w/v) 12 and 4%(w/v) KI, thereby confirming the creation of clear zone.

<63> As a result, as shown in Fig. 2, in view of the size of clear zone on the screen media added with the substrate of each enzyme, it was confirmed that the Pediococcus sp. strain contains the activation of the amylase, cellulase, protease, xylanase and phytase. Since the activation of protease which can decompose protease, amylase and phosphorus is especially excellent, it is determined that their commercial use is possible.

<64>

<65> Table 2

<66> Screening media and dyes used for the detection of various enzyme activities

<67> <68>

<69> Table 3 <70> Composition of screening medium

<71>

<72>

<73> Acid resistance, heat stabilities and bile salt-tolerance of the Fediococcus sp. strain <74> In the present invention, to detect the various characteristics of the Pediococcus sp. strain, the strains were seeded to the MRS broths by an amount of 1%, cultured at 37 C for 24 hours, and then settled at pH of 5.0, 4.0, 3.0 and 2.0 by using 0.1 N HCl for 30 minutes, and the number of surviving microorganisms was measured, thereby detecting the acid resistance.

<75> In addition, the strain was cultured, and the culture solutions were settled at 3OC, 4OC, 50 C and 60 C for 10 minutes, respectively, and then the count of surviving microorganisms were measured, thereby detecting the heat stabilities. Furthermore, the strains were seeded to the MRS broths added with the bile salt by a concentration of 0, 0.1, 0.3, 0.5 and l%(w/v) , and

O settled at 37 C for 6 hours, then the MRS agar plates were smeared with 0.1 mL of each broth, and then the number of surviving microorganisms were measured, thereby detecting the bile salt-tolerance. <76> As a result, as shown in Fig. 3, the Pediococcus sp. strain perished at pH 2, however, the count of the strain of 3.38 x 10 CFU/ml was detected at pH 3, so that it is determined that the Pediococcus sp. strain can be used in industry. In addition, as shown in Fig. 4, the count of the strain of 2.67 x 10 CFU/ml was detected even at 6OC, so that it is determined that the

Pediococcus sp. strain has the heat stabilities. Furthermore, as shown in Fig. 5, the count of the strain was high for the bial salt of 1%, so that it is determined that the Pediococcus sp. strain has an excellent bial salt- tolerance.

<77>

<78> Research of effect to inhibit the growth of E-coil by Pediococcus sp. strain

<79> In the present invention, to detect the effect to inhibit the growth of E-coil by Pediococcus sp. strain, the Pediococcus sp. and E- coli which are the strains to be used for the detection of the effect to inhibit the growth of E-coil were cultured for 24 hours by using respectively corresponding broths. At this time, only the E-coli which is a control group was seeded to the nutrient broth, and the Pediococcus sp. strain and the E-coli were seeded to the MRS broth by 1% respectively. The culture solutions were collected with the interval of 3 hours while shake-culturing the seeded liquid broth at

O

150 rpm at 37 C, the collected solutions were diluted with the pasteurized physiological saline and smeared to the nutrient agar broth. The broth was cultured for 24 hours at 37 C, and the count of E-coli was measured from the generated colony, thereby detecting the degree of growth inhibition of E- coli .

<80> As a result, as in Fig. 6, in case of culturing the Pediococcus sp. strain of the present invention for 12 hours, E-coli were completely perished, and therefore, it is determined that the Pediococcus sp. strain of the present invention has excellent effect to inhibit the growth of E-coli.

<8i> In continuation, the steps following the step of pouring the strain culture solution into the mixture is described below.

<82> The Pediococcus sp. Strain culture solution is poured into the mixture to which the rice bran was mixed so that the water content ration is 60%, then the mixture into which the strain culture solution was poured is poured into the closed space by using the Baroque method, and the oxygen is supplied, then, after 3 days, the temperature rises to 6OC, and after further

3 days, rises to 70 to 8OC. At this time, the lignin and the cellulose of the wood chip are automatically decomposed and fermented due to thermo_nature, and in this decomposition and fermentation process, one of

Bacillus sp. strain, Pediococcus acidilactici strain, and Pediococcus pentosaceus strain is seeded (S60). <83> As described above, the microorganism culture wood chip having the microorganisms decomposed and fermented through the culture process is produced (S70). <84> In addition, if the water of the produced microorganism culture wood chip is removed, the seeded microorganisms come into anesthetic state

(narcotized state)) and no longer perform activity (S80). <85> When the water and the broth are supplied to the narcotized microorganisms, the microorganism come to the activation state from the anesthetic state and decompose and ferment the culture wood chip (S90).

Claims

[CLAIMS] [Claim 1]

A method of culturing microorganisms in a piece of wood comprising the steps of: collecting miscellaneous woods such as waste wood, fell wood, and wood root ; cutting the collected woods into a predetermined length by putting the collected woods into a crusher; sorting the pieces of cut woods according to the size of the pieces of woods by putting the woods! mixing a predetermined quantity of rice bran into the pieces of sorted wood; poring a strain culture solution into a mixture, to which said rice bran is mixed, so that a predetermined water content ratio is obtained; poring a mixture, to which said strain culture solution is pored, into a closed space, supplying an oxygen, and fermenting said strain culture solution for a predetermined period at a predetermined temperature; and producing a strain culture wood chip after fermenting the microorganisms for a predetermined period.

[Claim 2]

The method according to claim 1, further comprising the step of removing the water from the microorganism culture wood chip, and bringing the microorganisms into anesthetic state, after the production step.

[Claim 3]

The method according to claim 1, further comprising the step of activating the microorganisms by supplying the water and a broth to the narcotized microorganisms.

[Claim 4]

The method according to claim 1, wherein strain of said strain culture solution is one of Bacillus sp. strain, Pediococcus acidilactici strain, and Pediococcus pentosaceus strain.

[Claim 5]

The method according to claims 1 to 4, wherein the size of the pieces of wood is 2 to 10 cm in diameter, and 5 to 60 cm in length.

[Claim 6]

The method according to claims 1 to 4, wherein the mixture ratio of the pieces of wood and the rice bran is 7:3.

[Claim 7]

The method according to claims 1 to 4, wherein the predetermined

O temperature is 50 to 8OC, and the predetermined period is 3 to 9 days.

[Claim 8]

The method according to claims 1 to 4, wherein the water content ratio is 60%. [Claim 9]

A microorganism culture wood chip, which is cultured according to one of the methods of claims 1 to 4.