WO2007129866A1

WO2007129866A1 - Pediococcus pentosaceus st-01 for preventing microbial contamination and method for preparing the same

Info

Publication number: WO2007129866A1
Application number: PCT/KR2007/002293
Authority: WO
Inventors: Seok Tae Lee
Original assignee: Jung, Sung Su
Priority date: 2006-05-10
Filing date: 2007-05-10
Publication date: 2007-11-15
Also published as: KR100627428B1

Abstract

The present invention relates to microorganism strains for preventing the microorganism pollution and method of manufacturing the same, and more particularly, the present invention relates to novel Pediococcus sp. strain, method of isolating the same from soil, and a usage of the same for preventing microorganism pollution. The Pediococcus pentosaceus isolated with the method described above can effectively prevent the microorganism pollution which could be generated at the process of fermentation of microorganism, so as to remove secondary putrefaction and bad smell and restrain the development of pathogenic germ. Therefore, the Pediococcus pentosaceus can be widely developed into a livestock probiotics, a functional toilet paper, functional foods, nutrients, a functional feed additive, an environment improvement agent, various enzyme preparation, and the like.

Description

[DESCRIPTION] [Invention Title]

PEDIOCOCCUS PENTOSACEUS ST-Ol FOR PREVENTING MICROBIAL CONTAMINATION AND METHOD FOR PREPARING THE SAME [Technical Field]

<i> The present invention relates to microorganism strains for preventing the microorganism pollution and method of manufacturing the same, and more particularly, the present invention relates to novel Pediococcus sp. strain, method of isolating the same from soil, and a usage of the same for preventing microorganism pollution. [Background Art]

<2> There are many elements in the natural soil, and there are many unknown components among them. It is said that in the natural soil, in particular, there are 0.99 million elements which coincide with the elements consisting human. Therefore, as there is the order in the human, there is the order in the soil. 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, prevents 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 or above. However, any method using machines can not decompose the materials up to 300 mesh or above like this.

<5> We, the inventors, 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, we discovered that not only the bad smells are removed, but also the effect of decomposition is excellent, whereby we 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> In addition, recently, many people have deep interests in the health so that the recognition of harmful factor which may adversely affect the health is gradually increased, whereby the preference for foods having high safety is increased and the expenditure of the consumers places relatively greater importance to the safety of food. Therefore, a variety of methods which can minimize the use of the antibiotics are attempted and developed, and it is necessary to produce various novel products which introduced functionality to safety.

<7> In fact, in development of stock farm products, due to intensive raising methods, the possibility of infection of various pathogenic germs, 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. Therefore, the necessity of production of functional stock farming for improvement of health is increased so that the development of production technology of high quality safe stock farm products is required.

<8> In view of the situations described above, we, the inventors, continued to making an effort to separate beneficial microorganisms from the soil, and the result was that we separated the Pediococcus sp. strain, which prevents microorganism pollution, from the soil containing a lot of mineral, identified the Pediococcus sp. strain with the Pediococcus pentosaceus ST-Ol, and thereafter, confirmed that the strain can be effectively applied as a livestock probiotics, thereby successfully completing the present invention. [Disclosure] [Technical Problem] <9> The object of the invention is to provide a novel Pediococcus pentosaceus ST-Ol, a method of separating the same from the soil, and a usage of the same for preventing microorganism pollution.

[Technical Solution] <io> To achieve the object, the present invention provides a Pediococcus pentosaceus ST-Ol which prevents the microorganism pollution. <π> In addition, the present invention provides a usage of using the

Pediococcus sp. strain as a livestock probiotics, an environment improvement agent, functional foods, nutrients, various enzyme preparation, and the like.

[Description of Drawings] <12> Fig. 1 shows a growth curve of the Pediococcus pentosaceus ST-Ol of the present invention using an MRS broth.

<13> Fig.2 shows the activation of amylase, protease, cellulase, xylanase and phytase of the Pediococcus pentosaceus ST-Ol of the present invention detected on an MRS agar plate. <i4> Fig. 3 shows the acid resistance of the Pediococcus pentosaceus ST-Ol of the present invention detected in a range of pH of 2.0 to 5.0. <15> Fig. 4 shows the heat stabilities of the Pediococcus pentosaceus ST-Ol

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

<16> Fig. 5 shows the bile salt-tolerance of the Pediococcus pentosaceus ST-Ol of the present invention detected with a variety of concentration of bile salt .

<17> Fig. 6 shows a degree of growth inhibition of E-coil of the Pediococcus pentosaceus ST-Ol of the present invention detected by using the MRS broth. [Mode for Invention] <18> Now, the present invention will be described below in detail.

<19> The present invention provides a novel Pediococcus sp. strain.

<20> More specifically, the present invention provides the Pediococcus sp. strain separated from the soil, and preferably, the Pediococcus sp. strain separated from the mineral soil.

<2i> The Pediococcus sp. strain of the present invention is preferably the Pediococcus pentosaceus, and more preferably, the Pediococcus pentosaceus ST-Ol strain.

<22> The Pediococcus pentosaceus strain of the present invention was named the Pediococcus pentosaceus ST-Ol, and was deposited on April 7, 2006 to the Korean Culture Center of Microorganisms which is an internatonal deposit organization(deposit number : KFCC-11369P), and the Pediococcus pentosaceus ST-Ol was deposited to the same Center as^' a deposit number KCCM-10748P on May 4, 2006.

<23> In addition, the present invention provides a method of separating the Pediococcus pentosaceus ST-Ol from the soil.

<24> The Pediococcus pentosaceus ST-Ol of the present invention provides a usage of preventing the microorganism pollution. The Pediococcus pentosaceus ST-Ol of the present invention effectively prevents the secondary pollution of microorganisms including the Escherichia coli (E-coli). Here, the microorganisms may include all microorganisms having pathogenicity besides the E- coli .

<25> The present invention provides the livestock probiotics having the Pediococcus pentosaceus ST-Ol as an effective component. The livestock probiotics described above may be used as, a livestock feed additive.

<26> In addition, the present invention provides an environment improvement agent having the Pediococcus pentosaceus ST-Ol as an effective component. The Pediococcus pentosaceus ST-Ol may be used as a functional toilet paper.

<27> Now, the present invention will be described in further detail according to embodiments.

<28> It should be noted that the embodiments described below are only examples of the present invention, but the contents of the present invention are not limited to the embodiments.

<29>

<30> Embodiment 1. Isolation of the Pediococcus sp. strain from the soil and identification of the Pediococcus sp. strain

<3i> 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.

<32> 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 (Biomerieux. , 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.

<33> 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).

<34> 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 ( refer to sequence list). 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.

<35>

<36> Table 1

<37> A result of sugar fermentation experiment performed in the Pediococcus sp. strain by using the API 50 CHL Kit

(+ : positive, — "• negative)

<38> <39>

<40> Embodiment 2. Research of growth characteristics of the Pediococcus sp. strain

<41> In the present invention, to research of growth curve of the Pediococcus sp. strain, the preparations were sampled with an interval of 6 hours at 37 C by using the MRS broth without adjusting the pH in the jar fermenter, and cultured for 48 hours.

<42> 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 plate of the strain was cultured for 24 hours at 37 C, the colony forming unit was measured. <43> As a result of researching the optimum growth condition of the

Pediococcus 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 g 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.

<44>

<45> Embodiment 3. Research of enzyme creation of the Pediococcus sp. strain

<46> 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 Table^'s 1 and 2.

<47> 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.

<48> 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.

<49> <50> Table 2 <51> Screening media and dyes used for the detection of various enzyme act ivit ies

<52> <53>

<54> Table 3 <55> Composition of screening medium

<56>

<57>

<58> Embodiment 4. Acid resistance, heat stabilities and bile salt-tolerance of the Pediococcus sp. strain

<59> In the present invention, to detect the various characteristics of the Pediococcus sp. strain, the strains were seeded to the MRS broths by an

O 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. <60> In addition, the strain was cultured, and the culture solutions were

O O O O 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. <6i> 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

4 o 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.

<62>

<63> Embodiment 5. Research of effect to inhibit the growth of E-coil by Pediococcus sp. strain

<64> 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

O 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 .

<65> 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. [Industrial Applicability]

<66> As described above, the present invention relates to novel Pediococcus sp. strain, method of isolating the same from soil, and a usage of the same for preventing microorganism pollution. The Pediococcus pentosaceus strain and the like isolated with the method described above can effectively prevent the microorganism pollution which could be generated at the process of fermentation of microorganism, so as to remove secondary putrefaction and bad smell and restrain the development of pathogenic germ. Therefore, the Pediococcus pentosaceus strain and the like can be widely used as a livestock probiotics, a functional toilet paper, functional foods, nutrients, a functional feed additive, an environment improvement agent, various enzyme preparation, and the like. (Certificate of Deposit of Microorganism)

<68>

Claims

[CLAIMS] [Claim 1]

<69> A Pediococciis pentosaceus ST-Ol (Deposit No.: KCCM-10748P) for preventing microorganism pollution by being isolated from the soil.

[Claim 2]

<70> An environment improvement agent having, as an effective component, said Pediococcus pentosaceus ST-Ol as claimed in claim 1.

[Claim 3]

<7i> The environment improvement agent according to claim 2, wherein it is used as a functional toilet paper.