KR101339907B1 - Pseudomonas aeruginosa Ab7 to produce surfactant used in cultivation of jujube and method for production of surfactant therefrom - Google Patents

Abstract

The present invention relates to a novel microorganism having a surfactant producing ability and a method for producing a surfactant using the same, Pseudomanas Pseudomanas having a producing ability of the surfactant aeruginosa ) Ab7 is provided, the culture medium of this strain was concentrated and treated with fungi, such as gray mold and staphylococcus, when the antimicrobial effect was confirmed, it was confirmed that the inhibition of jujube.

Description

Pseudomonas aeruginosa Ab7 to produce surfactant used in cultivation of jujube and method for production of surfactant therefrom}

The present invention relates to a novel microorganism having a surfactant producing ability and a method for producing a surfactant using the same.

Jujube is suitable for the climatic environment of Korea and resistant to pests. It is not only easy to cultivate but also has a variety of uses, so it is important as a fruit tree.

Since the 1950s, however, various diseases such as broom disease have been widespread, and the cultivated area has sharply decreased. Broom disease is a fatal disease caused by bacteria of the genus Phytoplasma belonging to mycoplasma. In addition to jujube known diseases such as stem rot, anthrax, rust, leaf blight, various fungi.

Organic chemical pesticides commonly used to control plant diseases cause serious environmental problems such as ecosystem destruction and soil pollution. In addition to chemical pesticides, the various pesticide supplements used are chemically manufactured and thus are difficult to decompose or toxic in the environment, thereby causing an economic burden.

Among the pesticide supplements, surfactants that are frequently used have a surface activity in the organic material layer and the water layer, which are not mixed with each other, so that the two layers are relatively easily mixed. Applications as pesticide auxiliaries vary in emulsifiers, dispersants, electrodeposition agents, solubilizers, etc. and play an important role in determining the physical properties of pesticide products.

Chemically synthesized surfactants have many kinds and a wide range of applications, but are hardly decomposable and harmful to humans. Recently, biological surfactants derived from microorganisms have attracted attention.

Biosurfactants can be used as materials for removing contaminated oils, food additives, and medical materials in cosmetic moisturizers, petroleum recovery and paper industries due to their useful properties such as biodegradability, non-toxicity and biocompatibility. Due to the high commercial use is sluggish.

Biosurfactants are surfactants produced by living microorganisms that can be easily degraded by microorganisms and have minimal effects on humans or livestock. Therefore, research on the production of biological surfactants by microorganisms is a situation that needs to be applied, and it is necessary to confirm the efficiency by applying to actual jujube farming.

Korean Patent Publication No. 10-1997-0006157 (published on April 24, 1997) discloses a new strain, Gram-negative and oxidase-negative, capable of producing surfactants, Acinetobacter Calcoaceticus ( Acinetobacter). calcoacetobacter ) is disclosed a method for separating and recovering a new biosurfactant.

According to Korean Patent Publication No. 10-1997-0006157, ethyl alcohol 0.5 to 5% (v / v) as a carbon source, urea 0.1 to 1.0% (w / v) as a nitrogen source, magnesium sulfate (MgSO ₄ · 7H ₂ O) as a trace element ) 0.01 to 0.1% (w / v) was dissolved in 0.15M phosphate buffer {K ₂ HPO 41.69% (w / v) + KH ₂ PO ₄ 0.726% (w / v)} at 37 ° C for 1 day in the culture medium. After incubation to prepare the inoculum bacteria inoculated at 1% (v / v) of the culture volume in a batch (Batch type) 5L incubator temperature of 30 ℃, stirring speed 300 to 400 rpm, aeration rate 2 to 5 Culture was performed by L / min.

In addition, while the exponential growth of the cells, the surfactant is present in the state attached to the surface of the cell is gradually secreted out of the cell during the rest of the cell growth stops. Therefore, in order to obtain all the produced biological surfactants, the ammonium sulfate precipitation separation method is used for the former to separate not only the surfactants released into the cell but also the surfactants attached to the cell membrane. For example, 8-30 mM EDTA is used to separate the surface active substances attached to the cell membranes while destroying the stability of the cell membranes, and then the surfactants are separated and recovered using the ammonium sulfate precipitation separation method.

In the present invention, to isolate the microorganisms that produce environmentally friendly and biodegradable biosurfactant as a pesticide supplement to increase the efficacy of the pesticide component used in the process of removing various pathogens causing jujube disease, and to determine the efficacy .

The present invention Pseudomonas Erujinosa having a surfactant producing ability (Pseudomanas aeruginosa) Ab7 (KCTC12008BP).

In addition, the present invention is a method for producing a culture medium having a surfactant activity by culturing microorganisms, Pseudomanas Erujinosa ( Pseudomanas) aeruginosa ) Ab7 (KCTC12008BP) provides a method for producing a culture medium having a surfactant activity, characterized in that using. In this case, the culture is preferably carried out at a temperature of 36 ° C. in a King B medium containing 55 mM fructose or TY medium containing 1% glycerin and 55 mM fructose. Incubate daily.

In the present invention, to find a microorganism having a biosurfactant production ability in soil, etc. in Korea, Pseudomanas aeruginosa (Pseudomanas aeruginosa ) Ab7 strain to produce a surfactant was isolated, and confirmed the characteristics and effects of the biosurfactant they produce .

On the other hand, in the present invention was established the optimal culture conditions for the production of surfactant, by using a variety of controlled media and culture conditions can be significantly reduced manufacturing costs by using cheap raw materials instead of expensive media.

It was confirmed that the Ab7 strain of the present invention produces a surfactant.

When the culture medium of the Ab7 strain isolated from the present invention was concentrated and treated with mold and staphylococcus aureus, the antibacterial effect could be confirmed.

As a result of concentrating the culture solution containing the surfactant of the present invention and spraying on the jujube fruit, it was observed that the corruption of the fruit is suppressed even if stored for 3 to 4 weeks.

1 is a graph showing the surface tension according to the type and dilution rate of Pseudomonas strains in King B medium.
Figure 2 is a graph showing the surface tension of the Ab7 strain culture medium according to the addition of sugars to King B medium.
Figure 3 is a graph showing the surface tension of the Ab7 culture solution according to the incubation time and dilution ratio of Ab7 strain in King B medium.
Figure 4 is a graph showing the surface tension of the Ab7 strain culture medium according to the addition of glycerin or fructose to TY medium and King B medium.
Figure 5 is a graph showing the surface tension of the Ab7 culture solution according to the dilution rate of the culture temperature and Ab7 strain in King B medium.
Figure 6 is a graph showing the surface tension of Ab7 culture medium according to the type of nitrogen source in King B medium.
Figure 7 is a graph showing the surface tension of the Ab7 culture solution according to the heat treatment and dilution ratio of Ab7 strain.
8 is a diagram showing the antimicrobial effect of the Ab7 culture on the gray mold.
9 is a view showing the antimicrobial effect of the hydrophobic portion (left) and the hydrophilic portion (right) of the Ab7 culture solution extracted with butanol against staphylococci.
Fig. 10 is a diagram showing three weeks after the jujube fruit (left) and the control jujube fruit (right) to which the Ab7 culture solution was applied.

Hereinafter, the present invention will be described in more detail with reference to the following Examples and Experimental Examples. However, the scope of the present invention is not limited to the following embodiments, and includes modifications of equivalent technical ideas.

[ Example  1] Strain Selection

(1) Isolation of Strains and Preparation of Media

In the present invention, to isolate the strain Pseudomonas ( Pseudomonas ) as a surfactant producing strain of rhamnolipid series in the soil.

King B medium and TY medium were used to culture Pseudomonas genus strains.

King B medium is 2 g of proteose peptone, 1 g of glycerin, K ₂ HPO ₄ 7H ₂ 0 0.15 g, MgSO ₄ 0.15 g of 7H ₂ O, 100 ml of distilled water (pH 7.2), and agar (agar) was added 1.5% to form a flat medium.

In addition, TY medium is 0.8 g of tryptone, 0.5 g of yeast extract (yeast extract), 0.25 g of NaCl, 100 ml of distilled water, agar (1.5%) was added to make a flat medium.

(2) Selection of strain which produces excellent surfactant

In the present invention, to find a strain that efficiently produces a surfactant, first selected chemical surfactants commonly used in the periphery dilution in distilled water 1 times, 1/10 times, 1/100 times, 1 / 1,000 times and the surface Tension was measured (see Table 1).

As a result, Triton-X100 showed the lowest surface tension, which was the best as a surfactant. Water soap and Tween 80 showed higher surface tension with dilution, which did not appear to be superior to Triton-X100 as a surfactant.

Surface tension of 10% chemical surfactant with dilution factor
Dilution factor 10%
Tween 80 10%
SDS 10%
Triton X-100 10%
Kitchen soap × 1 44.3 37.5 36.6 35.7 × 1/10 48.3 39.3 36.8 39.0 × 1/100 49.7 42.2 37.3 43.2 × 1/1000 51.5 46.5 37.5 53.5

On the other hand, it was separated and Pseudomonas (Pseudomonas) bacterium belonging to the genus exhibiting fluorescence in the King B plate medium from the soil collected from Chungbuk Cheongwon-gun, were investigated whether they produce a surface active agent.

As a result, the Ab7 strain showing the lowest surface tension among Pseudomonas genus strains producing rhamnolipid-based surfactants was finally selected as a strain used in the present invention (see FIG. 1).

Selected Pseudomanas Pseudomanas aeruginosa ) Ab7 strain was assigned to Korean Collection for Type Cultures (KCTC) on August 24, 2011, and received accession number KCTC12008BP.

[ Example  2] Strain Identification

(One) Ab7  To measure the biochemical properties of the strain API test

In the present invention, to measure the biochemical properties of Pseudomonas strain API 20NE kit was used. Examined the biochemical properties of the strain Ab7 result, it was confirmed by industrial Rouge (P. aeruginosa), Pseudomonas with a probability of 99.9% (see Table 2).

Biological Characteristics of Pseudomonas Ab7 Strains
Biochemical tests Pseudomonas aeruginosa Pseudomonas fluorescens Pseudomonas putida Pseudomonas
Ab7 nitrate reduction + +/- - + İndole production - - - - glucose acidification
-
-
-
- arginine dehydrolase + + + + urease +/- - - + esculin hydrolysis - - - - gelatin hydrolysis + +/- - + β-galactosidase - - - - Assimilation of
glucose
+
+
+
+ arabinose - + +/- - mannose - + +/- - mannitol + + - + N-acetyl-glucosamine + + - - maltose - - - - gluconate + + + + caprate + + + + adipate + - - + malate + + + + citrate + + + + phenyl acetate - - - - cytochrome oxidase + + + +

(2) Ab7  16S of strain rRNA  Gene Sequencing Analysis

In the present invention, 16S rRNA gene sequence was analyzed for more detailed strain identification. The purely isolated AB7 strain was inoculated in NA (Nutrient broth agar) medium, incubated at 28 ° C. for 2 days, and DNA was extracted by modifying the benzyl chloride method.

In order to amplify PCR (Polymerase Chain Reaction) of 16S rRNA gene, 5'-AGAGTTTGATCCTGGCTCAG-3 'forward and 5'-GGTTACCTTGTTACGACTT-3' were used as primers. PCR was performed for 2 minutes at 95 ° C. Initial dissociation was performed 30 times three times: dissociation (94 ° C, 30 seconds), hybridization (58 ° C, 30 seconds), and polymerization (72 ° C, 45 seconds). (72 ° C., 5 min) was performed.

The amplified 16S rDNA was purified using the PCR Product Purification Kit, and the PCR purified product was analyzed using the Genetic Analyzer 310A. The sequence was homologous from the data of DDBJ / NCBI / Genebank and Ribosomal Database Project II (RDP II). A search was performed.

The 16S rRNA gene sequence of the Ab7 strain was analyzed and found to be 1,406 bases, 99% identical to that of the P. aeruginosa S25 strain.

Therefore, from the results of the above API test and 16S rRNA gene sequence analysis, it was clearly identified that the Ab7 strain isolated from the present invention is P. aeruginosa .

[ Example  3] Optimum Production Conditions for Surfactants

Pseudomonas aeruginosa isolated from the present invention ( P. aeruginosa ) Ab7 strains to investigate the optimum conditions for the production of surfactant. Surfactant Ab7 strains were cultured by adding various additives to King B medium and TY medium for efficient production, and to compare the culture time, culture composition, culture temperature, and nitrogen source.

(One) King  According to B medium composition Ab7  Surfactant of Strains Production capacity  compare

Were cultured by the addition of a disaccharide including monosaccharides and lactose (lactose) including glucose (glucose) in the King B medium for industrial Rouge (P. aeruginosa) efficient production of surfactant Ab7 Pseudomonas strains by 55 mM each of the surfactant-producing ability Was compared.

Experimental results showed that the addition of monosaccharides was more effective for the production of surfactants than the addition of disaccharides, and the surface tension was lower in the samples diluted 1 / 100-fold with 55 mM fructose. It can be seen that a lot of production (see Figure 2).

In addition, when the Ab7 strain was incubated for 1 to 5 days in the medium to which fructose was added to King B medium, the surfactant production capacity was compared, and it was found that the most active production capacity was shown on the 2nd day of culture ( 3).

(2) TY  According to the composition of the badge Ab7  Surfactant of Strains Production capacity  compare

Unlike in King B medium, TY medium does not contain glycerin (glycerin), and thus, surfactant production ability was compared by adding 1% of glycerin (glycerin) or 55 mM of fructose (fructose).

As a result, a large amount of surfactant was produced in TY medium containing glycerin and fructose. However, fructose (fructose) showed a lower value than that in King B medium added 55 mM (see Figure 4).

(3) King  Depending on the temperature in B medium Ab7  Surfactant of Strains Production capacity  compare

While culturing the Ab7 strain in King B medium to which fructose was added, the culture temperature of the Ab7 strain was compared by varying the culture temperature to 28 ° C, 32 ° C, 36 ° C, and 38 ° C.

Experimental results showed the most efficient surfactant production at 36 ° C. than at low temperature, rather inhibited at 38 ° C. (see FIG. 5).

(4) King  Depending on the type of nitrogen source in B medium Ab7  Surfactant of Strains Production capacity  compare

Proteose peptone, which is mainly used as a nitrogen source in King B medium, is a relatively expensive reagent, so it is replaced with an inexpensive raw material such as soybean meal or skim milk, and it is a surfactant of Ab7 strain. The production capacity was compared.

As a result of the experiment, when skim milk was cultured using skim milk, it showed efficient surfactant production ability, and it was confirmed that skim milk was an excellent substitute for proteose peptone (see FIG. 6). ).

[ Example  4] Thermal Stability and Characterization of Surfactants

(One) Ab  Thermal Stability Study of 7 Strains

In order to examine the thermal stability of the surfactant produced by the Ab7 strain, the heat resistance of the Ab7 strain culture was investigated by examining the change in surface tension.

After 10 minutes at 60 ℃, 80 ℃, and 100 ℃, the surface tension was measured. As a result of heat treatment at 60 ℃, 80 ℃, there was almost no change. The tension was as high as 5 dyne / cm, indicating some loss (see FIG. 7).

Therefore, since a surfactant is Ab7 strain produced it is stable for up to 80 ℃, subsequently, even if the heating for the purpose of eliminating the labor Rouge (P. aeruginosa) Ab7 strain Pseudomonas surfactants producing strain surfactant to be little loss occurs Judging.

(2) Ab7  Characterization of Surfactants Produced by Strains

The culture solution of Ab7 sample was extracted with butanol and concentrated separately to hydrophilic and hydrophobic portions.

As a result, almost all surfactants could be separated and concentrated in the hydrophobic portion extracted by butanol (see Table 3).

Surface Tension of Hydrophobic and Hydrophilic Sections of Ab7 Strain Cultures Extracted with Butanol × 1 × 1/10 × 1/100 Ab7 (× 1) 34 37.6 52 Ab7-butanol hydrophobic 36.3 37 51.3 Ab7-butanol hydrophilic 59.6 69 75.5

[ Example  5] Confirm antimicrobial effect and actual application

The culture medium containing the surfactant produced by the Ab7 strain was concentrated and applied to the target bacteria. The target bacteria were plated on a flat plate medium and a circular disc filter was used to check growth inhibition.

In addition, as an initial experiment for applying to actual crops, the experimentally produced and concentrated biological surfactant was applied to the harvested jujube, and the jug was hydrated and stored in a container so as not to dry at room temperature, and the jujube was damaged. We tried to observe after 3-4 weeks.

(One) Ab7  Confirmation of Growth Inhibition of Surfactants Produced by Strains

To see the antimicrobial effect of surfactants produced by Ab7 strain,Botrytis hyacinthi), And the concentrated Ab7 culture solution was dropped by a paper filter and then incubated. As a result, it was found that the growth of gray mold was suppressed (see FIG. 8).

Also, Staphylococcus sp. LS2 was used as a target bacterium, and the hydrophobic and hydrophilic portions of the butanol extract of Ab7 culture were dropped by paper disc and then cultured. It was found that the growth of Staphylococcus aureus was inhibited (see FIG. 9).

This means that the hydrophobic portion and the non-hydrophilic portion with surfactant activity are separate substances, and both show antibacterial effects against Staphylococcus aureus.

(2) Ab7  Observation of corruption by actually applying the surfactant produced by the strain

The experimental group treated with the Ab7 sample and the jujube-inoculated jujube were inoculated over 4 weeks. As a result, the jujube fruit (left in FIG. 10) sprayed with Ab7 culture was relatively intact (see FIG. 10).

Korea Biotechnology Research Institute KCTC12008BP 20110824

Claims (3)

delete Pseudomanas aeruginosa Ab7 (KCTC12008BP) was added to TY medium containing 1% glycerin and 55 mM fructose, or in King B medium containing 55 mM fructose. 2 days incubation at a temperature of ℃ to prepare a culture medium having a surfactant activity,
The culture medium having the surface active ability has a growth inhibitory ability against gray mold and Staphylococcus aureus, the production method of the agrochemical supplements culture medium, characterized in that to suppress the corruption of jujube.

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