KR102107989B1 - Mass production method by medium optimization of deodorized microorganisms for reducing odor in industrial or commercial air conditioners - Google Patents

Abstract

The present invention relates to a mass production method by medium optimization of deodorizing microorganisms for reducing odors in industrial or commercial air conditioners. The deodorizing microorganisms include Herbaspirillum huttiense Korea Ocean Bio Cluster 180411, Oleomonas sagaranensis Korea Ocean Bio Cluster 180716, Bacillus subtilis Korea Ocean Bio Cluster 180524, Methylobacterium aquaticum, Methylobacterium fujisawaense, Methylobacterium radiotolerans, Methylobacterium extorquens, Methylobacterium brachiatum, Arthrobacter atrocyaneus, Deinococcus apachensis and Deinococcus koreensis strains.

Description

Mass production method by medium optimization of deodorized microorganisms for reducing odor in industrial or commercial air conditioners}

The present invention relates to a mass production method by optimizing the medium of deodorized microorganisms for reducing odor of industrial or commercial air conditioners, and determining temperature and pH values corresponding to the optimization process and culture conditions of the medium components such as carbon source, nitrogen source, and inorganic salt. It is intended to mass-produce eco-friendly deodorizing microorganisms that can increase the production of deodorizing microorganisms, reduce production costs, and replace deodorants using conventional chemicals.

In modern times, the development of various diseases such as respiratory diseases caused by deterioration of air quality, such as air pollution, is a problem. In particular, the indoor living time of modern people occupies about 80% of the day time, and naturally the interest in comfort in the indoor environment has emerged.

 An air conditioner is a device that purifies, cools, reduces moisture, and controls the air condition, such as heating and humidification, for health, and is equipped with a function to install a blower and blow air to each room.

 The simplest air conditioner is a home air conditioner. The air conditioner is composed of a compressor, a condenser, an expander, and an evaporator (cooling fin) and is used to cool or purify a room using a refrigerant refrigeration cycle. In the summer, the indoor temperature rises easily due to the sun and the engine overheating of household appliances along with the rainy season. At this time, an air conditioner is used to lower the indoor temperature.

 When the air conditioner is used for a long time, the low-temperature refrigerant and air of a higher temperature exchange heat, causing condensation on the surface of the cooling fin and condensation. Accordingly, an environment in which bacteria or fungi from the outside are proliferated is created, and when air passing through the air filter enters the room, odor is generated by volatile organic compounds (VOCs) produced by microorganisms.

Hull baseupi rilrum allowed tienseu Korea Ocean Bio Cluster 180411 (Herbaspirillum huttiense Korea Ocean Bio Cluster 180411) used in this experiment (Microbiological Accession Number: KCTC18691P), Oleo Monastir Sagara linen sheath Korea Ocean Bio Cluster 180716 (Oleomonas sagaranensis Korea Ocean Bio Cluster 180716 ) (Microorganism Accession No .: KCTC18693P), Bacillus subtilis Korea Ocean Bio Cluster 180524 (Microorganism Accession No .: KCTC18692P), Methylobacterium aquaticum , Methylobacterium Methylobacterium fujisawaense , Methylobacterium radiotolerans, Methylobacterium extorquens, Methylobacterium brachiatum, Arttrobacter art Oh rosayi Neuss (Arthrobacter atrocyaneus), Day-no Cocu Apa Ken System (Deinococcus apachensis), Day Noko kusu Koren sheath (Deinococcus koreensis) strains are environmentally friendly natural-based materials to replace a chemical odor control agent used as the odor deodorant of the existing air conditioner as the microorganism adsorption.

 Therefore, the present invention is an optimization of the medium divided into a carbon source, a nitrogen source, and an inorganic salt to increase the production of the strains, and provides a method of increasing the production amount with these components.

Korean Registered Patent Publication No. 10-1935688 (Dec. 28, 2018) Korean Registered Patent Publication No. 10-1907994 (2018.10.08)

The object of the present invention is the Herbaspirillum huttiense Korea Ocean Bio Cluster 180411 (Microorganism Accession Number: KCTC18691P), Oleomonas sagaranensis Korea Ocean Biocluster 180716 ( Oleomonas sagaranensis Korea Ocean Bio Cluster) 180716 ) (Microorganism Accession No .: KCTC18693P), Bacillus subtilis Korea Ocean Bio Cluster 180524 (Microorganism Accession No .: KCTC18692P), Methylobacterium aquaticum , Methylobacterium Methylobacterium fujisawaense , Methylobacterium radiotolerans, Methylobacterium extorquens, Methylobacterium brachiatum, Arttrobacter art Arthrobacter atrocyaneus , Deinococcus It is to provide a method for determining the optimized culture medium of each strain of Deinococcus apachensis and Deinococcus koreensis to provide a method for large-scale analysis.

In order to solve the above problems, the present invention is to prepare a culture medium, a carbon source, a nitrogen source and inorganic salts, and then pass the components through the fine particle sorter 100 to make fine particles into fine particles (S1); A strain preparation step (S2) of preparing a deodorized microorganism strain preserved as a culture medium formed of microparticles in the fine particle selection step (S1); Experimental minimum medium preparation step (S3) for preparing a minimal broth; After adding any one of sugars or carbohydrates to a minimum medium prepared through the experiment minimum medium preparation step (S3) at a predetermined concentration, a predetermined in a shaking incubator with a predetermined temperature. After incubation for a period of time, the carbon source selection step (S4) for recovering the bacterial cells and lyophilizing them to confirm the production amount; From the carbon source identified through the carbon source selection step (S4), an organic nitrogen source of a predetermined concentration is added, incubated for a predetermined time with a shaking incubator at a predetermined temperature, and then centrifuged to recover the cells and lyophilized to confirm the production amount. A nitrogen source selection step (S5) of adding an inorganic nitrogen source of a predetermined concentration from the organic nitrogen source and incubating it with a shaking incubator at a predetermined temperature for a period of time to recover the cells by centrifugation and freeze-drying; After adding the inorganic salt at a predetermined concentration from the carbon source identified through the carbon source selection step (S4) and the nitrogen source identified through the nitrogen source selection step, incubate for a predetermined time with a shaking incubator at a predetermined temperature, centrifugation to recover the cells and freeze Inorganic salt selection step to check the production amount by drying (S6); Using the medium containing the carbon source determined through the carbon source selection step (S4) and the nitrogen source determined through the nitrogen source selection step (S5) and the inorganic salt selected through the inorganic salt selection step (S6), the culture temperature within a predetermined range is set to a predetermined section. The culture temperature determination step (S7) of determining the optimum culture temperature by dividing and checking the cell production amount for each section of the culture temperature; After the culture temperature determination step (S7), a medium containing a carbon source determined through the carbon source selection step (S4) and a nitrogen source determined through the nitrogen source selection step (S5) and inorganic salts determined through the inorganic salt selection step (S6) are used. And a pH value determination step (S8) for determining an optimal pH value by dividing the pH value within a predetermined range into predetermined sections and checking the cell production amount of each section of the pH value, and culturing the fine particle selection step (S1). The medium is formed in a powdered form as an LB medium, and the nitrogen source selection step (S5) is performed by adding an organic nitrogen source at a predetermined concentration from the carbon source identified through the carbon source selection step (S4) for a predetermined time in a shaking incubator at a predetermined temperature. Organic nitrogen source selection step of culturing and centrifugation (S5-1); In the state in which the organic nitrogen source identified through the organic nitrogen source selection step (S5-1) and the carbon source identified through the carbon source selection step (S4) are mixed, an inorganic nitrogen source is added at a predetermined concentration for a predetermined time in a shaking incubator at a predetermined temperature. It characterized in that it comprises a; inorganic nitrogen source selection step (S5-2) for centrifugation after cultivation, the fine particle sorter 100 of the fine particle selection step (S1) is to be supported on the ground to the empty interior It is formed to have a base portion 110, the inside of the base portion 110, the fine particle sorter 100 is a vibration unit 120 that vibrates, and the upper side of the base portion 110 A plurality of formed but the buffer member 130 supporting the bottom surface of the first particle selection unit 140, and located on the upper portion of the base portion 110, one side is opened so that the powder filtered by the filter net is discharged Is formed, but the other side is first The first particle selection unit 140 is formed with a sphere 141, and is located on the upper portion of the first particle selection unit 140, but one side is formed open and the other side is formed with a first filter network 151. Located on the upper side of the first filter network 151, the second particle selection unit 150 is formed on the second discharge port 152, and the second particle selection unit 150, but one side is open The third particle selection unit 160 is formed on the other side of the second filter network 161 and the third discharge port 162 is formed on the upper side of the second filter network 161. Located on the upper portion of the portion 160 is formed on one side is opened, the other side is a third filter network 171 is formed and the fourth filter outlet 172 is formed on the upper one side of the third filter network 171 Including the sorting unit 170, the first filter network 151, the second filter network 161 and the third filter network 171, the hole size of the first filter network 151, the second filter network ( 161), the third filter network 171 is formed to increase in order The third filter network 171 of the fourth particle selection unit 170, the second filter network 161 of the third particle selection unit 160, and the first filter network 151 of the second particle selection unit 150 It characterized in that through the sequential passage to be fine particles, the deodorant microorganism strain of the strain preparation step (S2) is Hulvaspirilum Hertience Korea Ocean Bio Cluster 180411 ( Herbaspirillum huttiense Korea Ocean Bio Cluster 180411 ) (microbial deposit number) : KCTC18691P), Oleomonas sagaranensis Korea Ocean Bio Cluster 180716 (microbial accession number: KCTC18693P), Bacillus subtilis Korea Ocean Bio Cluster 180524 ( Bacillus subtilis Korea Ocean Bio Cluster 180524 ) (Microbiology Accession No: KCTC18692P), a bacterium tumefaciens aqua tikum (Methylobacterium aquaticum) methyl, methyl Solarium Fu yen branch and the (Methylobacterium fujisawaense), Solarium bacterium methyl radical Toledo lance (Methylobacterium radiotolerans), tumefaciens X Toku Enschede (Methylobacterium extorquens), tumefaciens beuraki Atum (Methylobacterium brachiatum), are Trojan ah Neuss (Arthrobacter atrocyaneus) between a bakteo art as methyl, Day Noko kusu Apa Ken System ( Deinococcus apachensis ), Deinococcus koreensis ( Deinococcus koreensis ) provides a mass production method by optimizing the medium of a deodorizing microorganism strain, characterized in that any one.

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The present invention is a deodorant microorganism Hulbarspyrilum Hertience Korea Ocean Bio Cluster 180411 ( Herbaspirillum huttiense Korea Ocean Bio Cluster 180411 ) (microbial accession number: KCTC18691P), Oleomonas sagaranensis Korea Ocean Biocluster 180716 ( Oleomonas sagaranensis Korea Ocean Bio Cluster 180716 ) (Microorganism Accession No .: KCTC18693P), Bacillus subtilis Korea Ocean Bio Cluster 180524 (Microorganism Accession No .: KCTC18692P), Methylobacterium aquaticum , methylo Methylobacterium fujisawaense , Methylobacterium radiotolerans, Methylobacterium extorquens, Methylobacterium brachiatum, Arttrobacterium Arthrobacter atrocyaneus , Dino Deinococcus apachensis ( Deinococcus apachensis ), Deinococcus koensis ( Deinococcus koreensis ) provides an optimal medium for production of strains and is a very useful invention as an eco-friendly natural product-based material that replaces chemical deodorants.

1 is a flow chart of the present invention.
2 to 5 are views related to the fine particle selection step (S1) of the present invention.
6 to 8 are diagrams related to experimental examples of the present invention.

The terms or words used in the present specification and claims should not be interpreted as being limited to ordinary or lexical meanings, and the inventor can appropriately define the concept of terms in order to best describe his or her invention. Based on the principles, it should be interpreted as meaning and concept consistent with the technical idea of the present invention.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.

The deodorant microbial strains used in the present invention are Hulvaspirilum Hertiens Korea Ocean Bio Cluster 180411 ( Herbaspirillum huttiense Korea Ocean Bio Cluster 180411 ) (microorganism accession number: KCTC18691P), Oleomonas sagaranensis Korea Ocean Biocluster 180716 ( Oleomonas sagaranensis Korea Ocean Bio Cluster 180716 ) (Microorganism Accession No .: KCTC18693P), Bacillus subtilis Korea Ocean Bio Cluster 180524 (Microorganism Accession No .: KCTC18692P), Methylobacterium aquaticum , Methylobacterium fujisawaense , Methylobacterium radiotolerans, Methylobacterium extorquens, Methylobacterium brachiatum, Methylobacterium brachiatum, Arthrobacter atrocyaneus ), Deinococcus apachensis , and Deinococcus koreensis are eco-friendly natural materials based materials that replace chemical deodorants used as odor deodorants in existing air conditioners.

The present invention relates to a mass production method by optimizing the medium of a deodorizing microorganism strain, and a method of optimizing the medium divided into a medium, a carbon source, a nitrogen source, and an inorganic salt to increase the production of the above-mentioned strains to increase the production amount with these components Is to provide

Referring to Figure 1, the mass production method by maximizing the medium of the deodorizing microorganism strain of the present invention is a fine particle selection step (S1), a strain preparation step (S2), an experiment minimum medium preparation step (S3), and a carbon origination step (S4). ), Nitrogen source selection step (S5), inorganic salt selection step (S6), culture temperature determination step (S7), pH value determination step (S8).

S1) Fine particle selection step

The fine particle selection step (S1) is a step of preparing the culture medium, carbon source, nitrogen source, and inorganic salts used in the present invention and passing the components through the fine particle separator 100 to make fine particles.

 2 to 5, the fine particle sorter 100 is for filtering only fine particles by injecting a powdery substance, the base part 110, the vibration part 120, the buffer member 130, the agent It comprises a first selection member 140, a second selection member 150, a third selection member 160, a fourth selection member 170, and a cover portion 180.

The base 110 is intended to be supported on the ground and is formed to have an empty interior.

The vibrating unit 120 is located inside the base unit 110 so that the inputted components are sequentially filtered by the fine particle sorter 100 vibrating.

A plurality of buffer members 130 are formed on one side of the upper portion of the base portion 110 to support the bottom surface of the first particle selection unit 140.

The buffer member 130 is preferably formed of a spring, but may be formed in various forms in addition.

The first particle selection unit 140 is located on the upper portion of the base unit 110, but one side is opened so that the powder filtered by the filter net is discharged, and the other side is formed with a first outlet 141.

The second particle selection unit 150 is located on the upper portion of the first particle selection unit 140, but one side is formed open, and the other side is formed with a first filter network 151 and the first filter network 151. A second discharge port 152 is formed on one side of the upper portion.

The third particle selection unit 160 is located on the upper portion of the second particle selection unit 150, but one side is formed open, and the other side is formed with a second filter network 161 and the second filter network 161. A third discharge port 162 is formed on the upper side of the.

The fourth particle sorting unit 170 is located on the upper portion of the third particle sorting unit 160, and one side is opened and formed, while the other side is formed with a third filter network 171 and an upper portion of the third filter network 171. A fourth discharge port 172 is formed on one side.

3 is a view showing a state in which the inside of the fourth particle sorting unit 170 is visible as the cover portion 180 is removed. Referring to this, a support portion 173 is additionally formed on one lower side of the third filter network 171, and the support portion 173 can support the third filter network 171 to maintain robustness.

Although not shown in the drawing, a support part may be additionally formed to support each of the first filter network 151 and the second filter network 161.

The cover portion 180 is formed to cover the open portion of the fourth particle selection portion 180, but an inlet 181 is formed on one side.

Referring to FIG. 4, the hole sizes of the first filter network 151, the second filter network 161, and the third filter network 171 are the first filter network 151, the second filter network 161, and It is formed so as to increase in order of the three filter networks 171 (hole size of the first filter network 151 <hole size of the second filter network 161 <hole size of the third filter network 171).

In this way, as the specific component injected into the inlet 181 located at the top goes from the top to the bottom, the hole size of the filter network is narrowed to filter out the particles in each section so that the finest particles are discharged in the last sub-section. .

Referring to FIG. 5 in detail, specific components introduced in the a1 direction through the inlet 181 of the cover 180 are primarily particles through the third filter network 171 of the fourth sorting member 170. The filtered and filtered particles of the third filter network 171 move in the a2 direction, and the unfiltered particles are discharged in the a3 direction along the fourth discharge port 172.

The particles moved in the a2 direction are secondarily filtered through the second filter network 161 of the third selection member 160, and the particles filtered by the second filter network 161 are moved in the a4 direction and are not filtered. Particles are discharged in the a5 direction along the third discharge port 162.

Particles moved in the a4 direction are thirdly filtered through the third filter network 171, filtered particles are moved in the a6 direction, and unfiltered particles are discharged in the a7 direction along the second outlet 152.

Particles filtered in the a6 direction are finally discharged to the first outlet 141.

Through such a process, the carbon source, the nitrogen source, and the inorganic salts are each made into fine particles, thereby effectively creating a component balance in the culture medium, and thus having the advantage of maximizing the nutritional absorption of bacteria.

S2) Strain preparation stage

The strain preparation step (S2) is a step of preparing a deodorized microorganism strain preserved as a culture medium formed of fine particles in the fine particle selection step (S1).

The deodorant microbial strain prepared as a strain is Herbaspirillum huttiense Korea Ocean Bio Cluster 180411 (microbial accession number: KCTC18691P), Oleomonas sagaranensis Korea Ocean Biocluster 180716 ( Oleomonas sagaranensis Korea Ocean Bio Cluster 180716 ) (Microorganism Accession No .: KCTC18693P), Bacillus subtilis Korea Ocean Bio Cluster 180524 (Microbial Accession No .: KCTC18692P), Methylobacterium aquaticum , Methylobacterium fujisawaense , Methylobacterium radiotolerans, Methylobacterium extorquens, Methylobacterium brachiatum, Archiobacterium brachiatum, Arthrobacter atrocyane us ), Deinococcus apachensis , Deinococcus koreensis , and the like, and it is preferable to cultivate each of the above deodorant microorganism strains through the method of the present invention. It will be described collectively as a deodorizing microorganism strain.

The strains were strains preserved at -70 ° C using a glycerol stock method, and LB medium (Tryptone 1%, Yeast Extract 0.5%, NaCl 1%) was used as the culture medium.

S3) Minimum medium preparation stage

The minimum medium preparation step (S3) of the experiment is a step of preparing a minimal broth.

The minimum medium is a medium based on 0.5% peptone and 0.3% beef extract.

S4) Carbon source selection step

The carbon source selection step (S4) adds any one of sugars or carbohydrates to the minimum medium prepared through the experiment minimum medium preparation step (S3) to a predetermined concentration, and then shakes the incubator with a predetermined temperature. After incubation for a predetermined period of time in (shaking incubator), the cells are recovered and lyophilized to check the production.

The saccharides are dextrose, fructose, galactose, glucose, glycerol, lactose, maltose, mannitol , Soluble starch, sucrose, and xylose, which can be selected and used.

In the carbon source selection step (S4), a selected component among sugars or carbohydrate components is each 0.1 to 10% in concentration, incubated in a shaking incubator at 15 to 50 ° C (degree) for 1 to 10 days, and centrifuged. , Preferably incubated for 5 days in a shaking incubator at 30 ° C (degree) at a concentration of 1% each, and then centrifuged to recover the cells and freeze-dried to check the production amount and 1% with optimal conditions The carbon source can be determined.

That is, it is possible to find the optimal carbon source by repeating the carbon source selection step (S4) to confirm the production amount of each of the above-mentioned types of sugars.

S5) Nitrogen source selection step

In the nitrogen source selection step (S5), an organic nitrogen source of a predetermined concentration is added from the carbon source identified through the carbon source selection step (S4), incubated for a predetermined time with a shaking incubator at a predetermined temperature, and then centrifuged to recover the cells and lyophilized to produce It is a step of confirming the production amount by confirming, culturing for a predetermined time with a shaking incubator at a predetermined temperature by adding an inorganic nitrogen source at a predetermined concentration from the carbon source and the organic nitrogen source, and then centrifuging to recover the cells and freeze-drying.

The nitrogen source selection step (S5) may be divided into an organic nitrogen source selection step (S5-1) and an inorganic nitrogen source selection step (S5-2).

The organic nitrogen source selection step (S5-1) of the carbon source identified through the carbon source selection step (S4), that is, from 1% carbon source with optimum conditions, 0.1 to 3% of organic nitrogen source is added to 15 to 50 ℃ (degrees) After incubating for 1 to 10 days in a shake incubator, centrifugation is performed. Preferably, 0.5% of each organic nitrogen source is added in order to determine a nitrogen source from a 1% carbon source, followed by incubation for 5 days in a 30 ° C shake incubator, followed by centrifugation. By recovering the cells and lyophilizing them, it is possible to find the optimum organic nitrogen source through this.

The organic nitrogen source is beef extract, casein, malt extract, peptone, skim milk, soytone, tryptone, yeast There is a extract (yeast extract) and the like, and by repeating the organic nitrogen source selection step (S5-1), it is possible to find the optimum organic nitrogen source by checking the production amount of each of these organic nitrogen sources.

Inorganic nitrogen source selection step (S5-2) is the organic nitrogen source identified through the organic nitrogen source selection step (S5-1), that is, 0.5% organic nitrogen source with optimum conditions and 1% confirmed through the carbon source selection step (S4) In a state where the carbon source is mixed, the inorganic nitrogen source is added at a concentration of 0.1 to 3%, incubated for 1 to 10 days in a shaking incubator at 15 to 50 ° C (degree), and then centrifuged, preferably 1% carbon source and 0.5% organic matter Various inorganic nitrogen sources are added to each wish by adding 0.5% of each, followed by incubation for 5 days in a 30 ° C shake incubator, followed by centrifugation to recover the cells and freeze-drying to check the production amount. Through this, the optimal inorganic nitrogen source can be found.

The inorganic nitrogen source is monoammonium phosphate ((NH ₄ ) H ₂ PO ₄ ), ammonium chloride (NH ₄ Cl), ammonium nitrate (NH ₄ NO ₃ ), ammonium sulfate ((NH ₄ ) O ₂ SO ₄ ), potassium nitrate There are (KNO ₃ ), sodium nitrate (NaNO ₃ ), etc., and by repeating the inorganic nitrogen source selection step (S5-2), it is possible to find the optimal inorganic nitrogen source by confirming the production of each of these inorganic nitrogen sources.

S6) Inorganic salt selection step

In the inorganic salt selection step (S6), a predetermined concentration of inorganic salts are added from the carbon source identified through the carbon source selection step (S4) and the nitrogen source identified through the nitrogen source selection step, followed by incubation for a predetermined time with a shaking incubator at a predetermined temperature. This is a step to collect the cells by centrifugation and lyophilize to check the production.

In the inorganic salt selection step (S6), when the carbon source identified through the carbon source selection step (S4) and the nitrogen source identified through the nitrogen source selection step are mixed, inorganic salts are added at a concentration of 0.1 to 3% to 15 to 50%. After incubation for 1 to 10 days in a shaking incubator at ℃ (degrees), centrifugation is performed. Preferably, 0.5% each of various inorganic salts is added from a 1% carbon source and a 0.5% nitrogen source, and cultured for 5 days in a shaking incubator at 30 ° C. After that, the cells are recovered by centrifugation and lyophilized to check the production amount. Through this, the optimal inorganic nitrogen source can be found.

The inorganic salts include calcium chloride (CaCl ₂ ), dipotassium phosphate (K ₂ HPO ₄₎ , potassium chloride (KCl), monopotassium phosphate (KH ₂ PO ₄ ), magnesium sulfate (MgSO ₄ ), magnesium (2) sulfate (MnSO ₄₎ ), Sodium chloride (NaCl), potassium nitrate (KNO ₃ ), disodium phosphate (Na ₂ HPO ₄ ), ferrous sulfate (FeSO ₄ ), etc., and repeat the inorganic salt selection step (S6) to increase the production of each of these inorganic nitrogen sources. By checking, the optimum inorganic salt can be found.

S7) Culture temperature determination step

The culture temperature determination step (S7) is determined by using a medium containing a carbon source determined through the carbon source selection step (S4) and a nitrogen source determined through the nitrogen source selection step (S5) and inorganic salts determined through the inorganic salt selection step (S6). This step is to determine the optimum culture temperature by dividing the culture temperature within the range into predetermined sections and checking the cell production amount for each section of the culture temperature.

Preferably, in the culture temperature determination step (S7), each production amount is checked at 20 ° C, 25 ° C, 30 ° C, 35 ° C, and 40 ° C, and the production temperature is re-confirmed by subdividing the culture temperature in units of 1 ° C in a section with high production volume. By doing so, the optimum culture temperature is determined.

S8) pH value determination step

The pH value determination step (S8) is performed through the nitrogen source and inorganic salt selection step (S6) determined through the carbon source and nitrogen source selection step (S5) determined through the carbon source selection step (S4) after the culture temperature determination step (S7). This is the step of determining the optimum pH value by checking the cell production amount for each section of the pH value by dividing the pH value within a predetermined range using a medium containing a specified inorganic salt.

Preferably, the pH value determination step (S8) is adjusted to the pH of the medium to pH6, pH6.5, pH7, pH7.5, pH8 at the culture temperature determined through the culture temperature determination step (S7), and cultured for 5 days. After that, the cells can be recovered by centrifugation and freeze-dried to check the production amount, so that the optimum pH value can be confirmed.

Through this, it is possible to cultivate deodorizing microorganisms by checking optimized components, temperature, and pH values for mass production of the cells of the present invention.

[Experimental Example]

Below is an example of an experiment on the above method.

1. Strains and media

Hull baseupi rilrum allowed tienseu Korea Ocean Bio cluster 180411 (Herbaspirillum huttiense Korea Ocean Bio Cluster 180411) used in the present invention (microorganism accession number: KCTC18691P), Oleo Pseudomonas Sagara norbornene cis Korea Ocean Bio cluster 180716 (Oleomonas sagaranensis Korea Ocean Bio Cluster 180716 ) (Microorganism accession number: KCTC18693P), Bacillus subtilis Korea Ocean Bio Cluster 180524 (Microorganism accession number: KCTC18692P), Methylobacterium aquaticum , Methylobacterium Methylobacterium fujisawaense , Methylobacterium radiotolerans, Methylobacterium extorquens, Methylobacterium brachiatum, Arttrobacter atrobacterium Ah between Neuss (Arthrobacter atrocyaneus), Day-no Cocu Apa Ken System (Deinococcus apachensis), Day Noko kusu Koren sheath (Deinococcus koreensis) strains were stored at -70 as glycerol ℃ Stark (glycerol stock) method, a culture medium is an LB medium (Luria-Bertani broth medium, 1% Tryptone, Yeast Extract 0.5%, NaCl 1%) was used.

2. Medium component optimization

 In the present invention, NB medium (nutrient broth, 0.5% peptone, 0.3% beef extract) was used as a basic medium to determine the optimum conditions of medium composition for increasing the production of each strain, and each composition was added and used differently.

 To determine the carbon source of the production medium, dextrose, fructose, galactose, glucose, glycerol, which are sugars or carbohydrates as carbon source in NB medium (glycerol), lactose, maltose, mannitol, soluble starch, sucrose, xylose at a concentration of 1% for 30 ° C shaking incubator After incubation for 5 days, the cells were collected by centrifugation and lyophilized to confirm the production.

Table 1. Determine Carbon Sources Component A B C D E F G H I J K Control (NB) 8.4 8.8 7.3 10.8 9.7 5.7 9.5 7.5 8.3 9.3 7.9 Carbon source (1%, w / v) Dextrose 18.6 10.6 21.7 26.5 10.4 20.9 15.7 18.1 9.5 21.6 15.8 Fructose 10.8 15.7 18.4 15.1 11.2 18.5 10.8 8.5 11.2 14.2 9.0 Galactose 15.8 14.7 15.7 11.6 15.4 11.2 17.4 16.5 15.7 19.6 16.4 Glucose 22.9 16.2 10.9 11.1 13.7 7.5 12.8 10.9 18.1 11.2 22.8 Glycerol 10.7 15.1 18.5 10.0 13.5 18.4 15.3 14.2 15.3 13.7 10.7 Lactose 9.5 20.5 13.8 16.3 11.8 10.4 17.4 8.3 9.2 14.3 10.2 Maltose 15.5 18.9 10.1 13.9 16.6 15.8 18.4 15.5 15.5 17.6 11.7 Mannitol 11.6 15.9 14.8 17.5 15.7 8.4 12.1 16.9 11.2 15.1 17.5 Soluble starch 13.4 19.4 15.9 25.4 13.4 18.2 16.3 19.3 13.1 20.1 11.4 Sucrose 12.7 24.6 18.3 19.6 18.8 10.5 19.2 9.9 10.8 15.6 9.5 Xylose 13.8 10.8 10.8 13.6 11.5 15.4 15.5 10.3 11.4 16.7 15.5

 (Counting units: mg / 100 ml)

A: Herbaspirillum huttiense Korea Ocean Bio Cluster 180411

B: Oleomonas sagaranensis Korea Ocean Bio Cluster 180716

C: Bacillus subtilis Korea Ocean Bio Cluster 180524

D: Methylobacterium aquaticum

E: Methylobacterium fujisawaense

F: Methylobacterium radiotolerans

G: Methylobacterium extorquens

H: Methylobacterium brachiatum

I: Arthrobacter atorocyaneus

J: Deinococcus apachensis

K: Deinococcus koreensis

To determine the nitrogen source from the 1% carbon source specified above, various organic nitrogen sources beef extract, casein, malt extract, peptone, skim milk, and soyton ( Soytone), tryptone, and yeast extract were added in 0.5% increments, incubated for 5 days in a 30 ° C shaking incubator, and centrifuged to recover the cells and lyophilized to confirm the production.

From the specified 1% carbon source and 0.5% organic nitrogen source, the inorganic nitrogen source is monoammonium phosphate ((NH ₄ ) H ₂ PO ₄ ), ammonium chloride (NH ₄ Cl), ammonium nitrate (NH ₄ NO ₃ ), ammonium sulfate ((NH ₄ ) O ₂ SO ₄ ), potassium nitrate (KNO ₃ ), and sodium nitrate (NaNO ₃ ) were added in 0.5% increments, incubated for 5 days in a shaking incubator at 30 ℃, and centrifuged to recover the cells and lyophilized. To confirm production.

Table 2. Determine Nitrogen Sources Component A B C D E F G H I J K Control 22.9 24.6 21.7 26.5 18.8 20.9 19.2 19.3 18.1 21.6 22.8 Organic nitrogen source (0.5%, w / v) Beef extract 46.1 27.3 53.9 44.9 38.4 49.1 38.7 30.9 25.8 36.8 32.3 Casein 18.7 56.1 50.2 50.1 34.5 32.4 33.1 45.3 33.8 49.7 34.3 Malt extract 19.8 27.2 47.6 40.7 25.8 44.7 45.1 33.9 34.1 36.4 27.4 Peptone 37.9 33.2 50.7 59.6 45.5 45.2 20.5 44.9 26.7 50.7 25.8 Skim milk 47.9 45.3 56.6 35.4 22.0 30.8 36.2 23.7 36.4 26.7 33.7 Soytone 54.5 55.7 52.2 30.6 20.0 22.3 28.3 29.7 22.1 28.1 37.9 Tryptone 49.6 52.3 35.4 28.6 30.8 33.8 33.3 39.7 34.2 39.4 25.5 Yeast extract 59.7 41.6 39.7 61.6 19.7 45.3 44.6 50.8 31.0 57.9 34.7 Inorganic nitrogen source (0.5%, w / v) (NH ₄ ) H ₂ PO ₄ 74.7 69.6 58.6 62.8 52.6 58.6 51.3 51.4 40.5 60.3 46.2 NH ₄ Cl 62.6 62.5 60.8 75.6 56.8 56.6 53.2 51.9 53.1 61.8 38.7 NH ₄ NO ₃ 61.2 56.4 77.6 66.4 54.5 50.7 58.9 53.6 59.4 66.7 44.8 (NH ₄ ) O ₂ SO ₄ 65.7 71.8 57.6 69.2 51.8 53.9 56.3 56.8 46.8 63.9 43.2 KNO ₃ 61.8 56.9 64.1 63.7 61.6 62.3 51.5 54.3 38.2 59.1 42.3 NaNO ₃ 64.1 61.1 59.1 65.1 57.9 51.8 52.4 52.8 55.3 64.6 41.6

(Counting units: mg / 100 ml)

A: Herbaspirillum huttiense Korea Ocean Bio Cluster 180411

B: Oleomonas sagaranensis Korea Ocean Bio Cluster 180716

C: Bacillus subtilis Korea Ocean Bio Cluster 180524

D: Methylobacterium aquaticum

E: Methylobacterium fujisawaense

F: Methylobacterium radiotolerans

G: Methylobacterium extorquens

H: Methylobacterium brachiatum

I: Arthrobacter atorocyaneus

J: Deinococcus apachensis

K: Deinococcus koreensis

Various inorganic salts of calcium chloride (CaCl ₂ ), dipotassium phosphate (K ₂ HPO ₄₎ , potassium chloride (KCl), monopotassium phosphate (KH ₂ PO ₄ ), magnesium sulfate (MgSO ₄ ) at 1% carbon source and 0.5% nitrogen source specified above , Magnesium (2) sulfate (MnSO ₄ ), sodium chloride (NaCl), potassium nitrate (KNO ₃ ), disodium phosphate (Na ₂ HPO ₄ ), ferrous sulfate (FeSO ₄ ), 0.1% each, and shaking at 30 ℃ After incubation for 5 days, the cells were collected by centrifugation and lyophilized to confirm the production.

Table 3. Determine Mineral Sources Component A B C D E F G H I J K Control 74.7 71.8 77.6 75.6 61.6 62.3 58.9 56.8 59.4 66.7 46.2 Mineral Source (0.1%, w / v) CaCl ₂ 78.6 78.4 81.7 76.9 73.6 65.6 61.4 60.9 69.4 70.3 49.5 K ₂ HPO ₄ 94.7 75.7 89.2 91.5 67.1 68.4 66.8 63.1 64.8 88.9 54.5 KCl 76.7 81.7 90.6 80.3 75.5 75.6 72.3 58.4 63.1 75.5 63.3 KH ₂ PO ₄ 75.6 73.4 81.1 77.5 69.7 65.2 60.3 61.1 62.7 68.1 61.4 MgSO ₄ 78.1 71.8 94.8 98.3 71.1 78.3 75.1 66.7 65.3 83.3 48.2 MnSO ₄ 78.7 87.3 81.1 78.0 76.6 68.4 74.2 60.3 64.1 77.5 50.4 NaCl 83.2 93.6 85.6 75.9 65.8 71.2 75.1 57.3 59.8 71.1 53.9 KNO ₃ 89.5 89.1 88.5 79.1 67.3 68.2 64.1 57.2 66.4 69.8 47.9 Na ₂ HPO ₄ 83.6 74.3 85.6 76.5 64.2 71.6 60.2 58.9 63.1 71.9 57.9 FeSO ₄ 81.8 81.8 83.5 73.2 68.9 65.8 79.8 58.8 62.7 75.3 52.7

 (Counting units: mg / 100 ml)

A: Herbaspirillum huttiense Korea Ocean Bio Cluster 180411

B: Oleomonas sagaranensis Korea Ocean Bio Cluster 180716

C: Bacillus subtilis Korea Ocean Bio Cluster 180524

D: Methylobacterium aquaticum

E: Methylobacterium fujisawaense

F: Methylobacterium radiotolerans

G: Methylobacterium extorquens

H: Methylobacterium brachiatum

I: Arthrobacter atorocyaneus

J: Deinococcus apachensis

K: Deinococcus koreensis

3. Optimization of culture conditions

The culture temperature and pH were optimized using a medium containing a defined carbon source, nitrogen source, and inorganic salt.

In the case of culture temperature, after incubation for 5 days in shaking incubator at 20 ℃, 25 ℃, 30 ℃, 35 ℃, and 40 ℃, the cells were collected by centrifugation and lyophilized to confirm the production.

Table 4. Determination of incubation temperature of individual microorganisms Component A B C D E F G H I J K 20 ℃ 50.8 61.3 55.3 51.9 42.6 39.5 41.8 37.9 38.4 43.8 30.1 25 ℃ 90.5 89.8 91.3 89.1 70.8 74.6 80.8 65.4 68.2 83.4 57.4 30 ℃ 94.7 93.6 94.8 98.3 76.6 78.3 79.8 66.7 69.4 88.9 63.3 35 ℃ 85.7 86.6 87.1 84.6 67.3 69.2 75.1 60.1 64.8 80.8 55.3 40 ℃ 77.5 74.6 69.2 71.9 52.9 54.2 59.2 47.3 50.7 53.9 45.8

   (Counting units: mg / 100 ml)

A: Herbaspirillum huttiense Korea Ocean Bio Cluster 180411

B: Oleomonas sagaranensis Korea Ocean Bio Cluster 180716

C: Bacillus subtilis Korea Ocean Bio Cluster 180524

D: Methylobacterium aquaticum

E: Methylobacterium fujisawaense

F: Methylobacterium radiotolerans

G: Methylobacterium extorquens

H: Methylobacterium brachiatum

I: Arthrobacter atorocyaneus

J: Deinococcus apachensis

K: Deinococcus koreensis

After that, the culture temperature was checked in units of 1 ℃ in the section with the most production.

Table 5. Determination of incubation temperature of individual microorganisms Component A B C D E F G H I J K 25 ℃ 90.5 89.8 91.3 89.1 70.8 74.6 80.8 65.4 68.2 83.4 57.4 26 ℃ 91.6 89.9 91.5 93.8 72.7 75.1 81.5 66.0 68.3 84.9 58.9 27 ℃ 91.9 90.3 92.6 95.1 74.9 75.5 82.6 66.7 69.1 85.6 59.7 28 ℃ 92.5 91.8 93.7 101.8 77.5 76.9 78.5 69.1 69.1 86.1 60.5 29 ℃ 93.8 92.4 94.1 99.3 79.5 77.5 79.1 68.0 69.2 87.5 62.1 30 ℃ 94.7 93.6 94.8 98.3 76.6 78.3 79.8 66.7 69.4 88.9 63.3

 (Counting units: mg / 100 ml)

A: Herbaspirillum huttiense Korea Ocean Bio Cluster 180411

B: Oleomonas sagaranensis Korea Ocean Bio Cluster 180716

C: Bacillus subtilis Korea Ocean Bio Cluster 180524

D: Methylobacterium aquaticum

E: Methylobacterium fujisawaense

F: Methylobacterium radiotolerans

G: Methylobacterium extorquens

H: Methylobacterium brachiatum

I: Arthrobacter atorocyaneus

J: Deinococcus apachensis

K: Deinococcus koreensis

In the case of pH, after adjusting the pH of the medium to pH6, pH6.5, pH7, pH7.5, pH8 and incubating for 5 days at the specified culture temperature, the cells were collected by centrifugation and lyophilized to confirm the production.

Table 6. Determination of incubation temperature of individual microorganisms Component A B C D E F G H I J K pH6 83.8 81.6 88.7 94.6 62.8 67.5 61.9 43.8 41.7 68.6 45.2 pH6.5 92.4 93.6 94.8 100.9 78.7 77.4 81.3 68.2 67.8 87.6 61.5 pH7 94.7 92.7 93.4 101.8 76.1 76.8 79.7 69.1 69.4 88.9 63.3 pH7.5 90.1 90.5 92.6 100.3 79.5 78.3 82.6 65.9 66.1 87.4 60.8 pH8 87.5 84.6 82.9 97.5 71.9 72.7 73.5 61.9 64.2 85.3 57.6

(Counting units: mg / 100 ml)

A: Herbaspirillum huttiense Korea Ocean Bio Cluster 180411

B: Oleomonas sagaranensis Korea Ocean Bio Cluster 180716

C: Bacillus subtilis Korea Ocean Bio Cluster 180524

D: Methylobacterium aquaticum

E: Methylobacterium fujisawaense

F: Methylobacterium radiotolerans

G: Methylobacterium extorquens

H: Methylobacterium brachiatum

I: Arthrobacter atorocyaneus

J: Deinococcus apachensis

K: Deinococcus koreensis

3-1) Medium composition and culture conditions for each strain derived through the above method

A: Herbaspirillum huttiense  Korea Ocean Bio Cluster 180411

-Optimal medium composition: 1.4% Glucose, 0.8% Yeast extract, 1% (NH ₄ ) H ₂ PO ₄ , 0.1% K2HPO4

-Cultivation conditions: 30 ℃, pH7

B:  Oleomonas sagaranensis  Korea Ocean Bio Cluster 180716

-Optimal medium composition: 1.3% Sucrose, 0.5% Casein, 0.5% (NH ₄ ) O ₂ SO ₄ , 0.2% NaCl

-Cultivation conditions: 30 ℃, pH6.5

C: Bacillus subtilis Korea Ocean Bio Cluster 180524

-Optimal medium composition: 1.7% Dextrose, 0.6% Skim milk, 0.4% NH ₄ NO ₃ , 0.1% MgSO ₄

-Cultivation conditions: 30 ℃, pH6.5

D: Methylobacterium aquaticum

-Optimal medium composition: 1% Dextrose, 0.5% Yeast extract, 0.5% NH ₄ Cl, 0.1% MgSO4

-Cultivation conditions: 28 ℃, pH7

E: Methylobacterium fujisawaense

-Optimal medium composition: 1% Sucrose, 0.5% Peptone, 0.5% KNO ₃ , 0.1% MnSO ₄

-Cultivation conditions: 29 ℃, pH7.5

F: Methylobacterium radiotolerans

-Optimal medium composition: 1% Dextrose, 0.5% Beef extract, 0.5% KNO ₃ , 0.1% MgSO ₄

-Cultivation conditions: 30 ℃, pH7.5

G:  Methylobacterium extorquens

-Optimal medium composition: 1% Sucrose, 0.5% Malt extract, 0.5% NH ₄ NO ₃ , 0.1% FeSO ₄

-Cultivation conditions: 27 ℃, pH7.5

H: Methylobacterium brachiatum

-Optimal medium composition: 1% Soluble starch, 0.5% Yeast extract, 0.5% (NH4) O2SO4, 0.1% MgSO4

-Cultivation conditions: 28 ℃, pH7

I:  Arthrobacter atorocyaneus

-Optimal medium composition: 1% Glucose, 0.5% Skim milk, 0.5% NH ₄ NO ₃ , 0.1% CaCl2

-Cultivation conditions: 30 ℃, pH7

J: Deinococcus apachensis

-Optimal medium composition: 1% Dextrose, 0.5% Yeast extract, 0.5% NH ₄ NO ₃ , 0.1% K2HPO4

-Cultivation conditions: 30 ℃, pH7

K: Deinococcus koreensis

-Optimal medium composition: 1% Glucose, 0.5% Soytone, 0.5% (NH ₄ ) H ₂ PO ₄ , 0.1% KCl

-Cultivation conditions: 30 ℃, pH7

4. Comparison of commercially available media and production volume

Herbaspirillum huttiense Korea Ocean Bio Cluster 180411 , Oleomonas sagaranensis Korea Ocean Bio Cluster 180716 , Bacillus subtilis Korea Ocean Bio Cluster 180524 . Compared to mediums used for the determination of mediums for the production of mediums that are optimized for the production of mediums for comparison with the medium of the invention, the culture medium optimized for production You can.

The culture conditions were the same as those of the above experiment, and commercially available medium for bacterial culture was LB (Luria-Bertani broth) medium and R2A (Reasoner's 2A agar) medium.

 In the comparison method, the culture conditions were set in the same manner as in the above experiments, and the production was confirmed by lyophilization after culture.

Table 7. Production Comparison with Commercial Microbial Medium Component A B C D E F G H I J K Optimized medium 94.7 93.6 94.8 101.8 79.5 78.3 82.6 69.1 69.4 88.9 63.3 LB 71.6 75.6 69.6 94.6 71.8 68.4 71.7 54.8 48.6 62.5 58.4 R2A 84.5 79.4 78.1 102.6 79.8 74.5 85.9 73.4 71.5 89.6 65.8

 (Counting units: mg / 100 ml)

A: Herbaspirillum huttiense Korea Ocean Bio Cluster 180411

B: Oleomonas sagaranensis Korea Ocean Bio Cluster 180716

C: Bacillus subtilis Korea Ocean Bio Cluster 180524

D: Methylobacterium aquaticum

E: Methylobacterium fujisawaense

F: Methylobacterium radiotolerans

G: Methylobacterium extorquens

H: Methylobacterium brachiatum

I: Arthrobacter atorocyaneus

J: Deinococcus apachensis

K: Deinococcus koreensis

5. Response surface analysis according to the central synthesis plan

Herbaspirillum huttiense Korea Ocean Bio Cluster 180411 , Oleomonas sagaranensis Korea Ocean Bio Cluster 180716, Bacillus subtilis Korea Ocean Bio Cluster 180524 for better results compared to commercially available media Response surface analysis was performed with bacteria.

In order to determine the optimum concentration of the previously selected medium components, the effect of each concentration change of the medium components on the production of cells was analyzed using the Central Composite Design (CCD) method. The independent variables selected as a result of the above are carbon sources (g / l, X ₁ ), organic nitrogen sources (g / l, X ₂ ), inorganic nitrogen sources (g / l, X ₃ ), inorganic salts (g / l) , X ₄ ), and based on the PBD experiment plan, the center value and the experiment range were set to -2, -1, 0, 1, 2, respectively, and encoded in 5 steps. As the dependent variable (Y), which is influenced by the independent variable, the output was measured 3 times and the average value was used for the rare analysis, and 16 experiments including 4 center points were conducted. The response value of the dependent variable (Y, dry cell mass) for each independent variable is shown in [Table 8].

Table 8. Range of different variables for the central composite design and results for dried cell weight using factors Factor Symbol (unit) Coded values -2 -One 0 +1 +2 Glucose

(g/l)

(g / l) 5 12.5 20 27.5 35 Yeast extract

(g / l) 2.5 7.5 12.5 17.5 22.5 (NH ₄ ) H ₂ PO ₄

(g / l) 2.5 7.5 12.5 17.5 22.5 K ₂ HPO ₄

(g / l) 0.5 1.5 2.5 3.5 4.5 Runs

Response, Y (g / l) One -2  One  One  0 1.32333 2 -One -One  One -One 2.21667 3  One  0 -One -One 2.48001 4  One  0  0  One 1.07333 5  2 -One -One  0 1.40012 6 -One  0  One -One 2.06333 7  0 -One  0 -2 1.26679 8  One  0  0 -One 1.27088 9  0  0  One  0 1.55054 10  0  2  One  0 1.40333 11  0  One -One  0 1.07503 12 -One -One -One  One 1.41667 13 -One  0  0  One 1.23503 14  0 -2  2  2 1.33333 15  One -One  One  One 2.26333

Dried cell weight by Herbaspirillum huttiense Korea Ocean Bio Cluster 180411 cultivated at 30 ℃. The experiments were carried out in triplicate.

Response were dried cell weight from Herbaspirillum huttiense Korea Ocean Bio Cluster 180411

Factor Symbol (unit) Coded values -2 -One 0 +1 +2 Sucrose

(g/l)

(g / l) 0.5 1.0 1.5 2 2.5 Casein

(g / l) 0.6 1.0 1.4 1.8 2.2 (NH ₄₎ O ₂ SO ₄

(g / l) 0.6 1.0 1.4 1.8 2.2 NaCl

(g / l) 0.1 0.2 0.3 0.4 0.5 Runs

Response, Y (g / l) One -One  One -One -One 1.30488 2 -One  One -One  One 1.42643 3 -One -One  One -One 1.34800 4 -One  0  One  One 1.47236 5  0  0  0  0 1.36625 6  0  One  0  0 1.51472 7 -2  One  0  0 1.39229 8  2 -One  0  0 1.15123 9  0 -One  2 -One 2.23406 10  0  One -One -2 1.59536 11  One  0  0  2 2.22542 12  One  0  0 -One 1.44614 13  2  0 -One  One 1.05754 14 -One  One -One -One 1.32853 15  One  One  One  One 2.23705

Dried cell weight by Oleomonas sagaranensis Korea Ocean Bio Cluster 180716 cultivated at 30 ℃. The experiments were carried out in triplicate.

Response were dried cell weight from Oleomonas sagaranensis Korea Ocean Bio Cluster 180716.

Factor Symbol (unit) Coded values -2 -One 0 +1 +2 Dextrose

(g/l)

(g / l) 1.0 1.5 1.8 2 2.5 Skim milk

(g / l) 0.5 1.0 1.4 1.8 2.0 NH ₄ NO ₃

(g / l) 0.5 1.0 1.4 1.8 2.0 MgSO ₄

(g / l) 0 0.1 0.2 0.3 0.4 Runs

Response, Y (g / l) One -One  One -One -One 2.27696 2 -One  One -One  One 2.27420 3 -One  One  One -One 1.83190 4 -One -One  One  One 1.10646 5  0 -One  0  0 2.06130 6  0 -One  0  0 1.44492 7 -2  0  0  0 1.19315 8  2  0  0  0 2.26834 9  0  0  2  0 1.31944 10  0  0 -One -2 1.37368 11  0  2  0  2 1.66520 12  0  One  0 -One 1.78235 13  One  2 -One  One 1.61759 14  One -2 -One -One 1.92587 15  One  One  One  One 2.10979

Dried cell weight by Bacillus subtilis Korea Ocean Bio Cluster 180524 cultivated at 30 ℃. The experiments were carried out in triplicate.

Response were dried cell weight from Bacillus subtilis Korea Ocean Bio Cluster 180524.

The prediction of the model equation for the response value was analyzed using the Design expert program, and the regression equation for the dry cell mass is as follows.

T = β ₀ + β _One X _One + β ₂ X ₂ + β ₃ X ₃ + β ₄ X ₄ + β ₁₁ X _One X _One + β ₂₁ X ₂ X _One + β ₂₂ X ₂ X ₂ + β ₃₁ X ₃ X _One + β ₃₂ X ₃ X ₂ + β ₃₃ X ₃ X ₃ + β ₄₁ X ₄ X _One + β ₄₂ X ₄ X ₂ 6 + β ₄₃ X ₄ X ₃ + β ₄₄ X ₄ X ₄

As a result of variance analysis, the R-square (decision coefficient) was 0.9156, showing a value close to 1, showing a very high significance. Confirmed. In addition, the coefficient of variation (CV) was 13.45, indicating that the interaction between variables as well as the independent influence of variables on the production of cell mass was very high.

A plot showing the significance of the predicted and experimental values through the response surface analysis method is shown in FIG. 5, and the difference between the two values is very similar. You can.

5 (a) relates to Herbaspirillum huttiense Korea Ocean Bio Cluster 180411, FIG. 5 (b) relates to Oleomonas sagaranensis Korea Ocean Bio Cluster 180716, and FIG. 5 (c) relates to Bacillus subtilis Korea Ocean Bio Cluster 180524 .

6, each variable was confirmed by using a contour line to fix the production of each bacterium at one optimum. 7 is a result of confirming the interaction in a three-dimensional form using two remaining variables after fixing one independent variable among three variables to an optimal point to easily check the effect of each variable.

FIG. 6 (a) relates to Herbaspirillum huttiense Korea Ocean Bio Cluster 180411, FIG. 6 (b) relates to Oleomonas sagaranensis Korea Ocean Bio Cluster 180716, and FIG. 6 (c) relates to Bacillus subtilis Korea Ocean Bio Cluster 180524 .

7 (a) relates to Herbaspirillum huttiense Korea Ocean Bio Cluster 180411, FIG. 7 (b) relates to Oleomonas sagaranensis Korea Ocean Bio Cluster 180716, and FIG. 7 (c) relates to Bacillus subtilis Korea Ocean Bio Cluster 180524 .

Finally, the central synthesis planning method can be used to predict the concentration of independent variables that most affect production. The maximum dry cell mass predicted through the central synthesis planning method is Herbaspirillum huttiense Korea Ocean Bio Cluster 180411 : 2.03704 ± 0.210283 g / l, Oleomonas sagaranensis Korea Ocean Bio Cluster 180716 : 2.00564 ± 0.154973 g / l, Bacillus subtilis Korea Ocean Bio Cluster 180524 : It was predicted to be 2.0815 ± 0.145756 g / l.

The concentration of each variable at this time

A: Herbaspirillum huttiense Korea Ocean Bio Cluster 180411:

1.4% Glucose, 0.8% Yeast extract, 1% (NH ₄ ) H ₂ PO ₄ , 0.1% K ₂ HPO ₄

B: Oleomonas sagaranensis Korea Ocean Bio Cluster 180716:

1.3% Sucrose, 0.5% Casein, 0.5% (NH ₄ ) O ₂ SO ₄ , 0.2% NaCl

C: Bacillus subtilis Korea Ocean Bio Cluster 180524:

1.7% Dextrose, 0.6% Skim milk, 0.4% NH ₄ NO ₃ , 0.1% MgSO ₄

It was.

Three strains that have finally been optimized for concentration: Herbaspirillum huttiense Korea Ocean Bio Cluster 180411 , Oleomonas sagaranensis Korea Ocean Bio Cluster 180716 , Bacillus subtilis Korea Ocean Bio Cluster 180524 The comparison was made with the general bacterial media on the market.

Table 11. Final Production Verification Experiment Component A B C Optimized medium 106.7 100.8 103.7 LB 71.6 75.6 69.6 R2A 84.5 79.4 78.1

 (Counting units: mg / 100ml)

A: Herbaspirillum huttiense Korea Ocean Bio Cluster 180411

B: Oleomonas sagaranensis Korea Ocean Bio Cluster 180716

C: Bacillus subtilis Korea Ocean Bio Cluster 180524

As a result of the experiment, Herbaspirillum huttiense Korea Ocean Bio Cluster 180411 , Oleomonas sagaranensis Korea Ocean Bio Cluster 180716 , Bacillus subtilis Korea Ocean Bio Cluster 180524 The strains except the strains did not differ significantly in production. However, considering the cost, it is possible to produce the same production at a lower cost when using optimized ingredients.

And it can be seen that the three strains show more production compared to the production of the general bacterial medium.

 In view of these results, it was confirmed that the optimization experiment of the present invention was preferably performed.

Configurations shown in the embodiments and drawings described above are only the most preferred embodiments of the present invention and do not represent all of the technical spirit of the present invention, and various equivalents and modifications that can replace them It should be understood that there may be examples.

100 fine particle sorter
110 base
120 Vibration unit
130 Buffer member
140 First sorting member
141 First outlet
150 Second sorting member
151 First filter network
152 Second outlet
160 Third screening member
161 Second filter network
162 Outlet 3
170 Fourth sorting member
171 3rd filter network
172 4th outlet
180 cover
181 slot

Claims (3)

After preparing the culture medium, the carbon source, the nitrogen source and the inorganic salt, the microparticle sorting step (S1) to pass the components through the fine particle separator 100 into fine particles;
A strain preparation step (S2) of preparing a deodorized microorganism strain preserved as a culture medium formed of microparticles in the fine particle selection step (S1);
Experimental minimum medium preparation step (S3) for preparing a minimal broth;
After adding any one of sugars or carbohydrates to a minimum medium prepared through the experiment minimum medium preparation step (S3) at a predetermined concentration, a predetermined in a shaking incubator with a predetermined temperature. After incubation for a period of time, the carbon source selection step (S4) for recovering the bacterial cells and lyophilizing them to confirm the production amount;
From the carbon source identified through the carbon source selection step (S4), an organic nitrogen source of a predetermined concentration is added, incubated for a predetermined time with a shaking incubator at a predetermined temperature, and then centrifuged to recover the cells and lyophilized to confirm the production amount. A nitrogen source selection step (S5) of adding an inorganic nitrogen source of a predetermined concentration from the organic nitrogen source and incubating it with a shaking incubator at a predetermined temperature for a period of time to recover the cells by centrifugation and freeze-drying;
After adding the inorganic salt at a predetermined concentration from the carbon source identified through the carbon source selection step (S4) and the nitrogen source identified through the nitrogen source selection step, incubate for a predetermined time with a shaking incubator at a predetermined temperature, centrifugation to recover the cells and freeze Inorganic salt selection step to check the production amount by drying (S6);
Using the medium containing the carbon source determined through the carbon source selection step (S4) and the nitrogen source determined through the nitrogen source selection step (S5) and the inorganic salt selected through the inorganic salt selection step (S6), the culture temperature within a predetermined range is set to a predetermined section. The culture temperature determination step (S7) of determining the optimum culture temperature by dividing and checking the cell production amount for each section of the culture temperature;
After the culture temperature determination step (S7), a medium containing a carbon source determined through the carbon source selection step (S4) and a nitrogen source determined through the nitrogen source selection step (S5) and inorganic salts determined through the inorganic salt selection step (S6) are used. It includes a pH value determination step (S8) to determine the optimal pH value by checking the cell production amount of each section of the pH value by dividing the pH value within a predetermined range into a predetermined section,

The culture medium of the fine particle selection step (S1) is formed in a powdered form as LB medium,

The nitrogen source selection step (S5)
An organic nitrogen source selection step (S5-1) of adding a predetermined concentration of an organic nitrogen source from the carbon source identified through the carbon source selection step (S4) and incubating it for a predetermined time in a shaking incubator at a predetermined temperature;
In the state in which the organic nitrogen source identified through the organic nitrogen source selection step (S5-1) and the carbon source identified through the carbon source selection step (S4) are mixed, an inorganic nitrogen source is added at a predetermined concentration for a predetermined time in a shaking incubator at a predetermined temperature. Inorganic nitrogen source selection step of centrifugation after culturing (S5-2); characterized in that it comprises,

The fine particle sorter 100 of the fine particle selection step (S1) is
The base portion 110 is formed to have an empty interior to be supported on the ground,
It is located inside the base portion 110, the fine particle sorter 100, the vibration unit 120 and the vibration operation,
A plurality of buffer member 130 is formed on the upper side of the base portion 110 to support the bottom surface of the first particle selection unit 140,
Located on the upper portion of the base portion 110, the first particle selection unit 140 is formed on one side is opened so that the powder filtered by the filter net is discharged, the other side is a first outlet 141 is formed,
Located on the upper portion of the first particle selection unit 140, one side is formed open, but the other side is the first filter network 151 is formed and the second outlet (152) on the upper one side of the first filter network (151) ) Is formed second particle selection unit 150,
Located on the upper side of the second particle selection unit 150, one side is formed open, but the other side is formed with a second filter network 161 and a third outlet 162 on the upper one side of the second filter network 161 ) Is formed a third particle selection unit 160,
Located on the upper portion of the third particle selection unit 160, one side is open and formed, while the other side is formed with a third filter network 171 and a fourth outlet 172 is provided on the upper one side of the third filter network 171. Including the fourth particle selection unit 170 is formed,
The hole sizes of the first filter network 151, the second filter network 161, and the third filter network 171 are the first filter network 151, the second filter network 161, and the third filter network 171. ) Is formed so as to increase in order, but the third filter network 171 of the fourth particle selection unit 170, the second filter network 161 of the third particle selection unit 160, the second particle selection unit 150 Characterized in that to pass through the first filter network 151 in order to be fine particles,

The deodorant microbial strain in the step of preparing the strain (S2) is Herbaspirillum huttiense Korea Ocean Bio Cluster 180411 (microbial accession number: KCTC18691P), Oleomonas sagaranensis Korea Ocean Bio Cluster 180716 ( Oleomonas sagaranensis Korea Ocean Bio Cluster 180716 ) (Microorganism accession number: KCTC18693P), Bacillus subtilis Korea Ocean Bio Cluster 180524 ( Microbacteria accession number: KCTC18692P), Methylobacterium Aquaticum ( Methylobacterium aquaticum ), Methylobacterium fujisawaense , Methylobacterium radiotolerans , Methylobacterium extorquens , Methylobacterium extorquens , Methylobacterium extorquens brachiatum, Arthrobacter Atrocyaneus ), Deinococcus apachensis ( Deinococcus apachensis ), Denococcus korensis ( Deinococcus koreensis ), mass production method by media optimization of a deodorizing microorganism strain, characterized in that any one.
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