KR102519354B1 - Microbial Agent comprising Psuedomonas gessardii and Manufacuring Method thereof - Google Patents

Abstract

The present invention relates to a microbial preparation for reducing odor of air conditioning equipment including Psuedomonas gessardii (Accession No. KCTC14253BP) and a method for producing the same, and more particularly, to a biomaterial screening process and Pseudomonas ger obtained through the process By using Sardi, the effect of reducing odors generated from air conditioning facilities can be expected, and it relates to a microbial preparation that provides safety from eye irritation, skin irritation and inhalation toxicity, and a manufacturing method thereof.

Description

Microbial agent comprising Psuedomonas gessardii and manufacturing method thereof for odor reduction in air conditioning equipment

The present invention relates to a microbial preparation for reducing odor of air conditioning equipment including Psuedomonas gessardii (Accession No. KCTC14253BP) and a method for producing the same, and more particularly, to a biomaterial screening process and Pseudomonas ger obtained through the process By using Sardi, the effect of reducing odors generated from air conditioning facilities can be expected, and it relates to a microbial preparation that provides safety from eye irritation, skin irritation and inhalation toxicity, and a manufacturing method thereof.

In the present invention, a biomaterial screening method for fundamentally solving odors generated from air conditioning facilities and a biomaterial that can be expected to reduce odor using Psuedomonas gessardii (Accession No. KCTC14253BP) obtained through the process It is intended to provide a microbial agent for reducing odor, a method for producing the same, and a method for treating the same.

Air conditioning refers to maintaining suitable conditions for people or goods in open or closed spaces that require control of temperature, humidity, airflow, cleanliness, and ventilation.

Among them, temperature is the most sensitive element depending on seasonal temperature or various environmental requirements, and it is classified as cooling or heating based on this, but it cannot be called perfect air conditioning except for humidity, air flow, cleanliness and ventilation.

That is, the air conditioning facility means a facility capable of controlling various factors including temperature with respect to a wide range of requirements for people or goods. Therefore, air conditioning equipment is referred to as HVAC (Heating, Ventilation and Air Conditioning).

Among the various elements constituting the air conditioner, the temperature that is felt the most is created by controlling the temperature through an air conditioner, which means a cooling facility, especially in summer when the temperature and humidity increase.

An air conditioner is a facility that cools and heats a room or purifies air by using a refrigeration cycle of refrigerant consisting of a compressor, condenser, expander, and evaporator, and is operated to lower the elevated indoor temperature and humidity.

Unlike the past, clean air is recognized as a basic requirement for health and well-being in the present society where air quality is getting worse due to fine dust and ultrafine dust. Interest in air conditioning is greatly increasing, and odors generated from air conditioning facilities including air conditioners are reported as a typical and chronic consumer complaint that occurs every year in the air conditioning field quality index.

In general, odors in air conditioners occur for two main reasons.

First, as the low-temperature refrigerant and higher-temperature air exchange heat with each other, condensation on the surface of the cooling fins located inside the evaporator causes water droplets to form, preventing dust and various bacteria or molds from entering from the outside from proliferating. It becomes a good environment.

Air containing volatile organic compounds (VOCs) generated in the process of growth of bacteria or fungi grown in such an environment is blown into the room, and the user senses the odor. The second reason for the generation of odor is due to the adhesion of substances that can generate odor to the surface of the evaporator, although there is a difference depending on the use environment.

In order to solve this problem, it is known that manufacturers are implementing pre-application by using chemical substances that are clearly identified to the extent that safety problems do not occur in order to prevent odor in the production process.

In addition, in the case of general consumers, mold, odor removers, disinfectants and fragrances are used through on/offline stores, but it is difficult to see them as a complete solution because they have a limitation in that they cannot accurately remove the cause of the odor.

In addition, products using chemical biocidal substances are likely to be potentially harmful to the human body, and excessive use can cause problems with antibiotic-resistant bacteria or super bacteria. a lot of situations.

Furthermore, legal regulations on chemical substances have been strengthened at home and abroad due to human casualties caused by the use of products containing chemical biocidal substances, and consumers have also increased interest in safety issues. The situation is.

To this end, deodorants such as EM (Effective Microorganism) fermented liquid using natural substances or microorganisms have been released, but rather show problems such as the occurrence of mold.

Microorganisms form a biofilm as a protective mechanism, which is composed of one or several types of surface-attached microorganisms and the multimeric matrix they produce, including polysaccharides, proteins, lipids, and extracellular polymeric substances (EPS). It is reported as a social group behavior by a microbial community as a mucous three-dimensional structure, and the most representative example is dental plaque formed on the surface of teeth.

Types of microorganisms that create biofilms include bacteria, fungi, and protists. The advantage of this is that they share nutrients with each other, and various harmful environmental factors such as low nutrition, high and low temperatures, and antibiotics. At the same time, when the conditions for microorganisms to grow are met, they can be activated again and grow.

In fact, since biofilms by microorganisms can be formed on almost all types of solid surfaces, conventionally, during the infection process, epithelial cells, teeth, inner walls of blood vessels, etc., as well as catheters and various implants, have caused problems in the medical field. It is also reported that biofilms are formed in all kinds of artificial facilities accessible to microorganisms, such as water pipes, sewage pipes, water purifiers, and air purification facilities.

Biofilms formed in this way cause chronic infection and corrosion and functional degradation of various facilities, so biofilms are highly interested in not only microbiology but also in a wide range of fields such as medicine, dentistry, pharmacy, civil engineering, architecture, city, and environmental engineering, and are essential. It is commonly used as an object to be removed by .

In addition, prevalent is mainly used in biology-related fields, and refers to the state of a community or species that achieves the largest number of individuals in a specific environment by preferentially or dominating.

As a representative example of a dominant state, in the case of fermented foods, beneficial microorganisms in the food are induced to dominate, and beneficial microorganisms such as lactic acid bacteria that are harmless to the human body or furthermore, beneficial microorganisms such as lactic acid bacteria dominate, and suppress the generation and proliferation of harmful bacteria species such as those that cause spoilage. The storage period is longer than the state, and when ingested, effects such as helping the composition of the intestinal microflora in a beneficial way can be obtained.

In this way, chemical biocidal substances are not used to reduce odors generated from air conditioning facilities, and the advantages of biofilm formed by microorganisms themselves and the fundamental problem of odor generation by artificially creating a prevalent environment A solution to this is required.

Republic of Korea Patent Registration No. 10-2015472 (2019.08.22) Republic of Korea Patent Registration No. 10-2155264 (2019.08.22)

The present invention has been made to solve the above problems, and an object of the present invention is to use a biomaterial selection process and Pseudomonas gersardi obtained through the process, so that microorganisms self-form without using chemical biocidal substances. A microbial preparation for odor reduction in air conditioning facilities containing Pseudomonas gersardi that can solve the fundamental problem of odor generation in air conditioning facilities by artificially creating a prevalent environment and the advantage of a biofilm that reduces odor and manufactures the same method can be provided.

The present invention provides safety from eye irritation, skin irritation and inhalation toxicity by solving the short durability and safety problems of products using conventional chemical biocidal substances, and various commercial air conditioning facilities that utilize air conditioning facilities such as for home, vehicle, and industrial use. It is possible to provide a microbial preparation for reducing odor of air conditioning equipment containing Pseudomonas germardi that can be applied as and a method for manufacturing the same.

The objects of the present invention are not limited to the above-mentioned objects, and other objects not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the above object, the present invention provides a culture seed preparation step of preparing a culture seed by culturing Pseudomonas gessardii (Accession No. KCTC14253BP) in a nutrient medium; A mass production step of inoculating the culture seed into a fermentor and producing a large amount of Pseudomonas gersardi culture medium through main culture; a cell recovery step of recovering Pseudomonas gersardi cells from the Pseudomonas gersardi culture medium obtained in the mass production step using a centrifugal separator; A freeze-drying step of freeze-drying the Pseudomonas gersardi cells recovered in the cell recovery step at a temperature of -65 to -70 ° C at intervals of 5 to 10 ° C under a pressure of 700 mtorr and freeze-drying for a certain period of time; And pulverizing the freeze-dried Pseudomonas germs cells through the freeze-drying step to have a particle size of 1 to 3 mm to form a microbial material, 95 to 99% by weight of the microbial material and 1 to 5% by weight of galactosamine It is possible to provide a method for producing a microbial preparation for reducing odor of air conditioning equipment including Pseudomonas gersardi, including a freezing raw material preparation step of preparing a microbial preparation by mixing.

At this time, the odor reduction strain selection step of selecting odor reduction strains through sensory evaluation, low nutrient evaluation, biofilm formation ability evaluation, and biofilm dispersion evaluation for the culture solution sample generated by pure culture of Pseudomonas gersardi in a nutrient medium. It is characterized in that it further includes;

Here, in the step of selecting the strain for reducing odor, the odor intensity of the culture solution sample generated during the pure culture of Pseudomonas germardi is sensory evaluated to select the culture solution sample having a relatively weak odor intensity among the culture solution samples. In the sensory evaluation step, a portion of the culture sample selected in the sensory evaluation step is smeared on a solid nutrient medium and primary cultured at 24 to 28 ° C for 2 to 5 days, and then the solid nutrient medium used in the primary culture process Streaking the culture sample first cultured on a solid nutrient medium reduced by 20 to 75% than the first culture medium and secondly cultured under the same conditions as the first culture process, and colonies in the second culture sample ) A low nutrient growth evaluation step of selecting a culture medium sample having the largest formation, and a portion of the culture medium sample corresponding to the culture medium sample selected in the low image growth evaluation step among the pure cultured culture medium samples is aminoglycoside-based Antibiotics and beta-lactam antibiotics were spread on a solid nutrient medium to which antibiotics were added, cultured at 24 to 28 ° C for 2 to 5 days, and then the degree of biofilm formation among the cultured culture samples was observed to determine the level of biofilm. The biofilm formation ability evaluation step of selecting the culture medium sample, which is determined to have a relatively dense tissue formation and high biofilm formation ability, and the culture medium sample corresponding to the culture medium sample selected in the biofilm formation ability evaluation step among the pure cultured culture medium samples A portion of was smeared on a solid nutrient medium containing succinate, glutamate and glucose, and primary cultured at 24 to 28 ° C for 2 to 5 days, and then the degree of dispersion of the biofilm among the cultured culture samples was observed to determine the degree of biofilm. It is characterized in that it comprises a biofilm dispersity evaluation step of selecting the culture medium sample having a relatively high dispersibility.

In addition, a pre-freezing step of pre-freezing the Pseudomonas gersardi cells recovered in the cell recovery step before the freeze-drying step at -65 to -75 ° C. for 24 hours; characterized in that it further comprises.

On the other hand, it is possible to provide a microbial preparation for reducing odor in air conditioning equipment containing Pseudomonas gersardi prepared by the above-described manufacturing method.

At this time, it is characterized in that 0.001 to 5% by weight of the microbial agent is mixed with 93.5 to 99.899% by weight of sterile distilled water to which 0.1 to 1.5% by weight of calcium is added.

The present invention uses the biomaterial selection process and Pseudomonas gersardi obtained through the process to avoid the use of chemical biocidal substances and to obtain the advantages of a biofilm formed by microorganisms themselves and artificially prevalent environments. It has the advantage of being able to solve the fundamental problem of odor generation in air conditioning equipment.

In addition, the present invention provides safety from eye irritation, skin irritation and inhalation toxicity by solving the short durability and safety problems of conventional products using chemical biocidal substances, thereby providing various commercial air that utilizes air conditioning facilities such as home, vehicle, and industrial use. It has the advantage of being able to apply harmonization equipment.

1 is a process flow chart showing a method for manufacturing a microbial preparation for reducing odor in air conditioning equipment containing Pseudomonas germardi according to a preferred embodiment of the present invention.
Figure 2 is a process flow chart showing in detail the step of selecting strains for reducing odor shown in Figure 1.

Advantages and features of the present invention, and methods of achieving them, will become clear with reference to the detailed description of the following embodiments taken in conjunction with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in various different forms, only these embodiments make the disclosure of the present invention complete, and common knowledge in the art to which the present invention belongs. It is provided to fully inform the holder of the scope of the invention, and the present invention is only defined by the scope of the claims.

With reference to the accompanying drawings below, specific details for the practice of the present invention will be described in detail. Like reference numbers refer to like elements, regardless of drawing, and "and/or" includes each and every combination of one or more of the recited items.

Terms used in this specification are for describing the embodiments and are not intended to limit the present invention. In this specification, singular forms also include plural forms unless specifically stated otherwise in a phrase. As used herein, "comprises" and/or "comprising" does not exclude the presence or addition of one or more other elements other than the recited elements.

Unless otherwise defined, all terms (including technical and scientific terms) used in this specification may be used in a meaning commonly understood by those of ordinary skill in the art to which the present invention belongs. In addition, terms defined in commonly used dictionaries are not interpreted ideally or excessively unless explicitly specifically defined.

The microbial preparation for reducing the odor of air conditioning equipment of the present invention has the advantages of a biofilm formed by Pseudomonas gessardii itself, including Psuedomonas gessardii (Accession No. KCTC14253BP), and an artificially dominant (prevalent) environment. It is possible to solve the fundamental problem of odor generation in air conditioning equipment by forming a composition, and hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.

1 is a process flow chart showing a method for manufacturing a microbial preparation for reducing odor in air conditioning equipment containing Pseudomonas germardi according to a preferred embodiment of the present invention.

As shown in FIG. 1, in order to prepare the microbial preparation, a strain selection step for odor reduction (S10), a culture seed preparation step (S20), a mass production step (S30), a cell recovery step (S40), and a freeze-drying step (S50) and frozen raw materialization step (S60) are sequentially performed.

First, the step of selecting strains for reducing odor (S10) is a step of selecting the Pseudomonas gersardi used in the preparation of the microbial preparation as a strain for reducing odor in order to further increase the odor reduction effect of the microbial preparation, and the Pseudomonas gersardi Strain for odor reduction is selected through sensory evaluation, low nutrient growth evaluation, biofilm formation ability evaluation, and biofilm dispersity evaluation for the culture medium sample produced by pure culture of D. in a nutrient medium.

Pseudomonas gersardi selected as a strain for odor reduction in the odor reduction strain selection step (S10) is cultured in a nutrient medium in the culture seed preparation step (S20) to produce a culture seed.

The culture seed manufacturing step (S20) is a step of preparing seed microorganisms, that is, culture seeds, which are the basis for microbial culture using a fermentor prior to proceeding with the mass production process (S30), in a final volume of 1,000 L In order to prepare the culture seed having a volume corresponding to 0.25% to 1% with respect to (working volume), Pseudomonas gersardi selected as the odor reduction strain was added to the nutrient medium in a 1,500L to 5,000L plant scale fermentor. want to inoculate.

Specifically, first, in consideration of the aerobic metabolism of the Pseudomonas germdi, the culture solution sample generated by pure culture of the Pseudomonas germdi selected as the odor-reducing strain in the malodor-reducing strain selection step (S10) Based on the final working volume of the culture seed (working volume) 1,000L, the inoculation is prepared by dividing the seed into 1 to 3 having a volume of 0.25 to 0.5% and 3 to 5 having a volume of 0.7 to 1%.

At this time, preferably, the inoculum is prepared by dividing into 2 0.5% culture seeds and 4 1% culture seeds.

In addition, a plurality of 5L Erlenmeyer flasks are prepared, the nutrient medium corresponding to the divided culture seeds is prepared, and then sterilized for 15 to 25 minutes at a temperature of 120 to 125 ° C using an autoclave. proceed with

At this time, the sterilization conditions are preferably maintained at a temperature of 121 ° C. for 20 minutes.

In addition, the nutrient medium is yeast extract (Yeast extract) 1 to 2 parts by weight, glucose (Glucose) 0.5 to 1 parts by weight, galactosamine (galactosamine) 0.1 to 0.5 parts by weight), magnesium sulfate (Magnesium sulfate) 0.01 to 0.1 parts by weight, ferrous sulfate heptahydrate 0.01 to 0.1 parts by weight, potassium hydrogen phosphate 0.01 to 0.1 parts by weight, potassium dihydrogen phosphate 0.01 to 0.1 parts by weight and phosphoric acid It contains 0.01 to 0.1 parts by weight of calcium phosphate, preferably 1.5 parts by weight of yeast extract, 0.5 parts by weight of glucose, 0.5 parts by weight of galactosamine, and 0.05 parts by weight of magnesium sulfate. parts by weight, ferrous sulfate heptahydrate 0.05 parts by weight, potassium hydrogen phosphate 0.05 parts by weight, potassium dihydrogen phosphate 0.1 parts by weight and calcium phosphate 0.03 parts by weight It may contain parts by weight.

In addition, the volume of the nutrient medium should be prepared based on 1.5 to 3.0 L of the culture seed, and preferably, the volume of the nutrient medium should be prepared based on 2.5 L of the culture seed.

In the aseptic machine, the culture seed corresponding to 1% based on the volume of the culture seed is inoculated into the nutrient medium, and in a shaking incubator (shaking incubator) at a temperature condition of 30 to 32 ° C. at 3 to 150 rpm. The primary seed culture is carried out for 5 days, preferably 4 days.

After culturing for a predetermined time, the absorbance was measured at a wavelength of 600 nm, and the measured value was Lab. When the culture termination absorbance standard value derived through the scale experiment is reached, the culture is terminated to obtain the primary seed culture medium.

Thereafter, a new nutrient medium is prepared through a 1,500L fermentor, and sterilization is performed with an air filter for 15 to 25 minutes at a temperature of 120 to 125 ° C. At this time, the sterilization condition is maintained at a temperature of 121 ° C for 20 minutes. desirable.

After the sterilization process, the culture conditions of 30 to 32 ° C, 50 mL / min, 50 to 80 rpm and 0.5 kg / cm ² of the culture seed are controlled, and the primary seed culture medium is prepared for inoculation, and a flame ring is installed at the inoculation port. After releasing the pressure in the fermentor, the air supply is adjusted to 25 to 40 L/min, preferably 25 mL/min, and the primary seed culture solution is inoculated into the fermentor for 3 to 5 days, preferably 4 days. Seed culture is carried out.

After culturing for a predetermined time, the absorbance was measured at a wavelength of 600 nm, and the measured value was Lab. When the culture termination absorbance standard value derived through the scale experiment is reached, the culture is terminated to obtain the secondary seed culture medium.

After the completion of the secondary seed culture, the secondary seed culture solution is sampled to confirm whether or not contamination occurs, and 10ul of the secondary seed culture solution is smeared on a solid nutrient medium in a sterilizer, and incubator (incubator) Incubation is performed at 30 to 32 ° C. to confirm that cells are observed in addition to the seed culture medium.

After confirming whether contamination occurs, centrifugation and freeze-drying to lower the water activity of the cells in the seed culture medium and pulverize them, evaluate the yield, and finally obtain the powdered Pseudomonas gersardi cells as culture seeds. The culture seed preparation step (S20) ends.

Next, after the culture seed preparation step (S20), the culture seed is inoculated into a fermentor through the mass production step (S30) to produce a large amount of Pseudomonas gersardi culture solution through main culture.

Specifically, first, the culture seed corresponding to 0.5% of the final working volume was prepared, and 1,000 L of distilled water and an optimized nutrient medium were added to the fermenter to cultivate Pseudomonas gersardi through a 1,500 L fermentor. and sterilize the air filter.

At this time, sterilization is performed for 15 to 25 minutes at a temperature of 120 to 125 ° C, and preferably, the sterilization conditions should be maintained at a temperature of 121 ° C for 20 minutes.

In addition, prior to air filter sterilization, the condensed water present in the steam pipe of the fermenter is drained for 10 to 15 minutes, and after the air filter sterilization is performed, the air filter is dried for 30 minutes.

Here, the drain of the condensed water is to prevent contamination during the air filter process. When steam passes to the air filter in the presence of condensed water, foreign substances including iron contained in the stem pipe contaminate the filter. phenomenon appears.

On the other hand, when the air filter is sterilized, if the steam pressure entering the steam pipe is too high, it causes deformation of the air filter, so it must be adjusted to an appropriate pressure.

After sterilizing the fermenter and medium through the sterilization of the air filter, cooling is performed to the culture temperature of 30 to 32 ° C, and the culture conditions are 24 to 28 ° C, 50 L / min, 50 to 80 rpm, 0.5 to 0.8 kg / cm ² , prepare the primary seed culture solution for inoculation, install a flame ring at the inoculation port, release the fermentor pressure, adjust the air supply to 25 to 40 L/min, and transfer the culture seed to the fermentor. It is inoculated and cultured for mass production of the Pseudomonas Gersardi culture medium.

When the mass production step (S30) is completed, the cell recovery step (S40) is performed. In the cell cell recovery step (S40), the Pseudomonas gersardi culture solution obtained in the mass production step (S30) is used by using a centrifuge. Recover Pseudomonas Gersardi cells from

More specifically, cells are recovered from the Pseudomonas gersardi culture medium using a tubular centrifuge, and at this time, cell recovery rate is increased by changing conditions such as centrifuge RPM, culture medium transfer pump speed, and number of centrifugal film replacements. can be raised

Preferably, the speed of the centrifuge is set to 11,000 to 12,500 rpm, the culture medium transfer pump speed is set to 18 to 28 L/min, and the cell recovery film is replaced 1 to 3 times to obtain the Pseudomonas gersardi in the Pseudomonas gersardi culture medium. Cells must be recovered.

After the cell recovery step (S40), the freeze-drying step (S50) is performed. At this time, prior to proceeding with the freeze-drying step (S50), the Pseudomonas gersar recovered in the cell cell recovery step (S40) A pre-freezing step (not shown) of pre-freezing the cells using a deep-freezer at a temperature of -65 to -75 ° C, preferably at -70 ° C for 24 hours is carried out to be followed. The freeze-drying efficiency in the freeze-drying step (S50) can be increased.

On the other hand, in the freeze-drying step (S50), the Pseudomonas gersardi cells recovered in the cell recovery step (S40) are heated at an interval of 5 to 10 ° C from -65 to -70 ° C under a pressure condition of 700 mtorr and frozen for a certain time Dry.

Specifically, the Pseudomonas gersardi cells recovered in the cell recovery step (S40) by removing the centrifugal film are placed on a tray for a freeze dryer.

At this time, when the temperature sensor is placed between the Pseudomonas germs and the tray, the temperature deviation for each tray is large, so it should be installed in the thickest part of the Pseudomonas germs, preferably the pre-freezing step ( Not shown), the temperature sensor should be installed in the thickest part of the Pseudomonas gersardi cell beforehand.

Thereafter, the shelf temperature of the freeze dryer is set to -40 ° C, and when the set temperature is reached, the tray on which the Pseudomonas gersardi cells are placed is placed in a chamber and a cold-trap is operated to When the temperature reaches -65 to 70 ° C, a vacuum is applied by operating a vacuum pump.

Thereafter, the shelf temperature is increased at 5°C intervals where the pressure on the freeze dryer panel does not change to 700 mtorr or more, and when the shelf temperature is increased to 0°C or more, the pressure of the freeze dryer is increased at 10°C intervals at the line where 700 mtorr does not change. , and additionally increase the shelf temperature at 2-hour intervals.

After approximately 144 hours have elapsed since the start of the freeze-drying step (S50), the freeze-drying step (S50) is terminated and the powder of the Pseudomonas gersardi cell body is harvested. At this time, the Pseudomonas gersardi cell powder Drying time can be flexibly adjusted according to the degree of drying.

Next, in the freeze-raw material step (S60), the Pseudomonas gersardi cells freeze-dried through the freeze-drying step (S50) are pulverized to have a particle size of 1 to 3 mm in a clean area blocked from the outside to be used as a microbial material 95 to 99% by weight of the microbial material and 1 to 5% by weight of galactosamine, an amino sugar based on EPS, are mixed to form a microbial preparation.

In order to reduce the odor of air conditioning equipment, the microbial preparation is a diluted solution prepared by mixing 0.001 to 5% by weight with 93.5 to 99.899% by weight of sterile distilled water to which 0.1 to 1.5% by weight of calcium is added to help strengthen the biofilm. Preferably, the dilution solution is sprayed onto the surface of the evaporator of the air conditioning facility under conditions of a pressure of 0.5 to 1.3 kg/cm ² , a flow rate of 0.8 to 1.9 L/min, and a spray angle of 30 to 75 degrees, thereby increasing the odor reduction effect of the air conditioning facility. .

Figure 2 is a process sequence showing in detail the step of selecting strains for reducing odor shown in Figure 1.

2, the odor reduction strain selection step (S10) includes sampling and pure culture step (S11), sensory evaluation step (S12), low nutrient growth evaluation step (S13), biofilm forming ability evaluation step (S14) and biofilm It consists of detailed steps including a dispersion degree evaluation step (S15).

In the sample collection and pure culture step (S11), a sample is collected by scratching the surface of the evaporator of the air conditioning facility several times using a sterile cotton swab, and then the upper part of the sterile cotton swab tube from which the sample is collected and 10 ml of PBS are collected in an aseptic machine. Each is put into a 50ml conical tube, and the desorption process is performed 2 to 3 times for 3 to 4 minutes by setting the agitation speed of the vortex to the maximum. Perform the process of leaving for 2 minutes.

Then, using the PBS solution after each desorption process as a stock solution, 10 ul of the stock solution was taken in the aseptic container and smeared on a solid nutrient medium, respectively, for solid-phase culture. 3 to 5 days in an incubator at 28 ° C. carry out incubation. In addition, 1 ml of the stock solution is inoculated into each test tube prepared by adding 9 ml of sterilized R2A and NA medium, and incubated for 2 to 5 days in a shaking incubator at 24 to 28 ° C and 100 rpm.

When the culture of each test tube inoculated with the desorption stock solution is completed, 10ul of each culture medium is streaked on a solid nutrient medium, and 100ul of the culture diluent (10 ^-5 to 10 ^-8 ) is plated (Spreading) and cultured for 2 to 5 days in an incubator at 24 to 28 ° C. Through this process, solid/liquid culture is performed, and colonies with different colors/shapes are selected from plates obtained. , In order to obtain a single strain, a colony is taken with platinum lice and smeared on a new solid nutrient medium to perform pure culture.

For the single strain of the culture solution sample obtained through the pure culture process, it can be used to prepare a liquid medium for the culture solution sample to select the strain for odor reduction, and also, for the single strain obtained through the pure culture process, long-term preservation solution can be prepared. It can be used for the preparation of liquid medium for

Specifically, sterilization was performed at 121 ° C for 15 minutes using a sterilizer, and colonies were taken from the streaking plate where pure culture was performed, and inoculated with R2A medium (broth), and inoculated at 28 ° C in a shaking incubator , Culture is carried out for 3 to 5 days under 100 rpm conditions. At this time, the long-term preservation solution is prepared by preparing 20% glycerol (glycerol) with distilled water in a volume ratio (v / v) and using it through a sterilization process, and the R2A medium for liquid culture is prepared in a 500ml Erlenmeyer flask, but the preparation of the long-term preservation solution For this purpose, 1 ml of the culture solution is collected from the Erlenmeyer flask where the culture has been completed in a sterile field into a sterilized 1.5 ml tube, and centrifuged at 10,000 rpm for 30 seconds. Add each, suspend with a pipet, and store in a cryogenic freezer (-70 ° C).

Next, the sensory evaluation step (S12) is a step of preferentially selecting a strain for reducing odor in the culture sample by sensory-evaluating the odor intensity of the culture sample generated during the pure culture of Pseudomonas germardi, Sensory evaluation is performed to determine the degree of odor intensity during the cultivation process of microorganisms isolated as a single strain.

At this time, in order to minimize the intensity of odor generated in the liquid medium, the amount of low nutrient medium used is reduced by 30 to 75% to prepare and sterilize the medium, and inoculate a part of the culture medium sample prepared with the long-term preservation liquid in a shaking incubator. After culturing for 3 to 5 days at 100 rpm at a temperature of 28 ° C, the selected microbial material is awarded 5 points based on the odor intensity generated during the cultivation process through a sensory evaluation group consisting of 10 or more people through the first or second rounds. Selection is carried out through a scoring method.

Here, in the case of the scoring criteria of the '5-point scoring method', 0 points for no smell, 1 point for very weak odor (smell that is difficult to detect), 2 points for weak odor (smell that is difficult to distinguish), and odor (type classification) This possible odor) is scored as 3 points, strong odor as 4 points, and very strong odor as 5 points. The culture solution sample is first selected as a strain for reducing odor.

Next, in the low-nutrient growth evaluation step (S13), a portion of the culture medium sample selected in the sensory evaluation step (S12) was plated on a solid nutrient medium and primary cultured for 2 to 5 days at 24 to 28 ° C. After that, the culture medium sample firstly cultured on the solid nutrient medium reduced by 20 to 75% than the solid nutrient medium used in the first culture process is smeared and the second culture is carried out under the same conditions as the first culture process, This is a process of selecting a culture medium sample having the largest colony in the culture medium sample cultured with the primary culture.

Specifically, prepare a plate in which the culture sample is cultured, select one with a large colony when incubated for the same time, and take one colony on a new solid nutrient medium as a platinum ear and perform a smear. After primary culture is performed in an incubator at 24 to 28 ° C for 2 to 5 days, a solid nutrient medium is prepared by reducing the amount of nutrient medium used by 20 to 75%, and the culture medium sample is cultured on the plate. ), stroke smear was performed, and secondary culture was carried out in an incubator at 24 to 28 ° C for 2 to 5 days.

Thereafter, the culture medium sample in which a large colony is formed when cultured for the same time on the secondly separated plate is selected.

At this time, by securing basic data necessary for mass production in the future through repetition of the 1st to 2nd culture process 2 to 3 times, it is possible to utilize this to optimize the culture conditions for productivity improvement.
On the other hand, the culture medium sample cultured and used for evaluation in the low nutrient growth evaluation step (S13) is used only for low nutrient growth evaluation, and when the evaluation step is finished, immediately kill all strains in the culture medium sample Discard all of the culture medium samples.

Next, the biofilm formation ability evaluation step (S14) is a portion of the culture solution sample corresponding to the culture solution sample selected in the low nutrient growth evaluation step (S13) of the pure cultured culture solution samples aminoglycoside antibiotics and After the primary culture for 2 to 5 days at 24 to 28 ° C. by spreading on a solid nutrient medium to which β-lactam antibiotics were added, the degree of biofilm formation of the cultured samples was observed to form a tissue of a biofilm. This step is to select the culture medium sample that is formed relatively densely and is determined to have high biofilm formation ability.

As is well known, the antibiotic is defined as an agent that inhibits or kills the growth of specific microorganisms, among which aminoglycoside antibiotics, such as streptomycin, tobramycin, and neo When cultured at one or a combination of two or more constant concentrations of Neomycin and Gentamicin, it can help form a biofilm. In addition, when cultured under conditions of a certain concentration of β-lactam antibiotics, a polysaccharide-based alginate biofilm is formed.

Therefore, in the biofilm formation ability evaluation step (S14), 0.01 to 400 ug/ml of aminoglycoside antibiotics and 0.01 to 400 ug/m of β-lactam antibiotics are added, respectively, in the process of preparing a solid nutrient medium. 100 ul of the selected culture medium is plated on each prepared antibiotic-based solid nutrient medium, and culture is performed in an incubator at 24 to 28 ° C. for 2 to 5 days.

Thereafter, the degree of biofilm formation of the cultured culture samples is primarily determined using CFU counting, dry mass, and strain, and fluorescent staining is performed on the selected microbial material, and confocal laser Nutrient analysis of biofilms was performed through a scanning microscope (CLSM, confocal laser scanning microscopoe), or the degree of biofilm formation was determined through visual observation of the biofilms in the gel through a microfluidic channel embedded biofilm model using a general microscope. Among the culture samples, a relatively dense tissue biofilm is formed, that is, a culture sample having a high ability to form a biofilm is selected.
On the other hand, the culture medium sample cultured and used for evaluation in the biofilm formation ability evaluation step (S14) is used only for evaluation of biofilm formation ability, and when the evaluation step is completed, immediately kill all strains in the culture medium sample, and then the culture medium sample Abolish power.

Next, the biofilm dispersity evaluation step (S15) is a part of the culture solution sample corresponding to the culture solution sample selected in the biofilm forming ability evaluation step (S14) of the pure cultured culture solution sample succinate, glutamate and glucose After the first incubation for 2 to 5 days at 24 to 28 ° C. by spreading on a solid nutrient medium to which was added, the degree of biofilm dispersion of the cultured culture samples was observed to select the culture solution sample having a relatively high degree of dispersion of the biofilm. It is a step to

It is well known that the sudden increase in availability of carbon substrates such as succinate, glutamate, and glucose induces biofilm dispersal, and glycolysis utilizes this to determine the dispersity of biofilm among culture medium samples. It is a step of selecting the culture solution sample having a high value as a strain for reducing odor.

Specifically, in the process of preparing a solid nutrient medium, 1 to 100 mM of succinate, glutamate, and glucose are added, respectively, and 100 ul of the culture sample selected as each of the prepared antibiotic-based solid nutrient medium is plated, and incubated at 24 to 28 ° C. , After culturing for 2 to 5 days, the degree of biofilm dispersion is primarily determined using CFU counting, dry mass, and staining, and fluorescence staining is performed, and confocal laser scanning A culture medium sample by determining the degree of biofilm dispersion by conducting nutrient analysis of biofilm through a microscope (CLSM, confocal laser scanning microscopoe) or observing biofilm in a gel under a general microscope through an embedded biofilm model using a microfluidic channel Among them, the culture solution sample having a relatively high dispersion of the biofilm is finally selected as a strain for reducing odor.
On the other hand, the culture medium sample cultured and used for evaluation in the biofilm dispersity evaluation step (S15) is used only for evaluation of biofilm dispersity, and when the evaluation step is finished, all strains in the culture medium sample are immediately killed and then the culture medium Discard all samples.

Hereinafter, the present invention will be described in more detail by manufacturing examples and experimental examples according to the method of manufacturing a microbial preparation for reducing odor in air conditioning equipment containing Pseudomonas germardi according to a preferred embodiment of the present invention.

manufacturing example

A diluted solution of a microbial preparation containing the Pseudomonas gersardi selected according to the present invention was prepared.

Here, the diluted microbial preparation was prepared in a mixing ratio of 98% (w/w) of the microbial preparation containing Pseudomonas germardi, 1% (w/w) of galactosamine and 1% (w/w) of calcium.

Experimental example

20 ml of a product whose main component is ethanol, which is sold as a deodorant for removing bad teeth in air conditioning equipment on the market, and the dilution of the microorganism preparation prepared in the manufacturing example according to the present invention and the commercially available deodorant for removing odor prepared as a comparative example, heat exchange in air conditioning equipment It was applied as an evaporator.

For the gas generated after setting the elapsed time and air conditioning equipment to cooling mode, use a pump (SIBATA, MP-30N) to collect gas samples with a 5L aluminum polyester bag and use a portable volatile organic compound detector (TESTAUCTION, SKT1050) The results of comparison after measurement are shown in Table 1.

elapsed time Microbial population (species) Odor reduction rate (%) manufacturing example comparative example manufacturing example comparative example immediately after processing

One

8 77 94 1 month 2

11 81 73 2 months 3

14 81 61 4 months 3

13 87 34 6 months 5

16 91 14

When comparing the preparation example and the comparative example as shown in Table 1, it was confirmed that the durability of the preparation example was far superior to that of the comparative example as one month passed after the treatment.

In particular, it was confirmed that the odor reduction performance of the comparative example was significantly lowered after 1 month. On the other hand, the preparation example was expected to have an odor reduction effect through the formation of a biofilm of microbial materials, so immediately after treatment, the comparative example Although it showed a lower odor reduction rate, it was confirmed that from the point of 1 month, the odor reduction effect was higher than that of the comparative example and was maintained steadily up to 6 months.

In addition, as a result of sampling air conditioning equipment evaporator specimens for each treatment period and examining the microbial community, the comparative example shows a decrease in the odor reduction rate and an increase in the number of microorganisms, while the manufacturing example shows a line that does not significantly exceed the number of microbial materials treated. , the microbial population was maintained and the odor reduction rate showed an increasing pattern.

Therefore, it can be seen from this that the microbial preparation for reducing odor in air conditioning equipment containing Pseudomonas germardi according to the present invention dominates and reduces the growth of mold or bacteria that cause odor, and consequently has an effect of reducing odor. can

Although the embodiments of the present invention have been described with reference to the above and accompanying drawings, those skilled in the art to which the present invention pertains can implement the present invention in other specific forms without changing the technical spirit or essential features. You will understand that there is Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

S10: Bacteria selection step for odor reduction
S20: culture seed manufacturing step
S30: mass production stage
S40: cell recovery step
S50: freeze drying step
S60: Frozen raw materialization step

Claims (7)

In the method for producing a microbial preparation for reducing odor of air conditioning equipment containing Pseudomonas germardi,
Pseudomonas Gersardi ( Psuedomonas gessardii , accession number KCTC14253BP) in a nutrient medium to prepare a culture seed culture seed preparation step;
A mass production step of inoculating the culture seed into a fermentor and producing a large amount of Pseudomonas gersardi culture medium through main culture;
a cell recovery step of recovering Pseudomonas gersardi cells from the Pseudomonas gersardi culture medium obtained in the mass production step using a centrifugal separator;
A freeze-drying step of freeze-drying the Pseudomonas gersardi cells recovered in the cell recovery step at a temperature of -65 to -70 ° C at intervals of 5 to 10 ° C under a pressure of 700 mtorr and freeze-drying for a certain period of time; and
The microbial material is formed by pulverizing the freeze-dried Pseudomonas gersardi cells to have a particle size of 1 to 3 mm through the freeze-drying step, and mixing 95 to 99% by weight of the microbial material and 1 to 5% by weight of galactosamine A method for producing a microbial preparation for reducing odor in air conditioning facilities, comprising a step of freezing raw materials to prepare a microbial preparation. According to claim 1,
Odor reduction strain selection step of selecting odor reduction strains through sensory evaluation, low nutrient growth evaluation, biofilm formation ability evaluation, and biofilm dispersion evaluation for the culture solution sample produced by pure culture of Pseudomonas gersardi in a nutrient medium. Method for producing a microbial agent for reducing odor in air conditioning equipment, further comprising: According to claim 2,
In the step of selecting strains for reducing odor,
A sensory evaluation step of selecting a culture solution sample having a relatively weak odor intensity among the culture solution samples by sensory-evaluating the odor intensity of the culture solution sample generated during the pure culture of Pseudomonas germardi;
A portion of the culture medium sample selected in the sensory evaluation step is smeared on a solid nutrient medium and primary cultured at 24 to 28 ° C for 2 to 5 days, and then 20 to 75 The culture medium sample first cultured in the solid nutrient medium with % reduction is streaked and the second culture is performed under the same conditions as the first culture process, and the colony is the most A low-nutrient growth evaluation step of screening a large culture sample;
Among the pure cultured culture samples, a portion of the culture sample corresponding to the culture sample selected in the low nutrient growth evaluation step is a solid nutrient medium to which aminoglycoside antibiotics and beta-lactam antibiotics are added After being smeared and cultured at 24 to 28 ° C. for 2 to 5 days, the degree of biofilm formation among the cultured culture samples was observed, and the tissue of the biofilm was formed relatively densely, so that the culture medium was judged to have high biofilm formation ability. A biofilm forming ability evaluation step of selecting a sample, a method for producing a microbial agent for reducing air conditioning odor, characterized in that it comprises. According to claim 3,
A portion of the culture sample corresponding to the culture sample selected in the biofilm forming ability evaluation step among the pure cultured culture samples was smeared on a solid nutrient medium to which succinate, glutamate and glucose were added, and at 24 to 28 ℃ conditions 2 to After the primary culture for 5 days, an air conditioner comprising a biofilm dispersity evaluation step of selecting the culture solution sample having a relatively high degree of dispersion of the biofilm by observing the degree of dispersion of the biofilm among the cultured culture fluid samples. A method for producing a microbial agent for reducing odor. According to claim 1,
A pre-freezing step of pre-freezing the Pseudomonas germs recovered in the cell recovery step before the freeze-drying step at -65 to -75 ° C for 24 hours; A method for preparing a microbial preparation. A microbial preparation for reducing odor in air conditioning equipment containing Pseudomonas gersardi produced by the manufacturing method according to any one of claims 1 to 5. It is characterized in that 0.001 to 5% by weight of the microbial agent prepared by the manufacturing method according to any one of claims 1 to 5 is mixed with 93.5 to 99.899% by weight of sterile distilled water to which 0.1 to 1.5% by weight of calcium is added. A microbial preparation for reducing odor in air conditioning equipment containing Pseudomonas germardi.

Images