KR101740294B1 - Formulations of dihydroxy octadecenoic acid and method for producing formulations - Google Patents

Abstract

Formulation and production of hydroxy fatty acid which formulate dihydroxy octadecenoic acid which has high antimicrobial activity against a wide range of phytopathogenic fungi from fruit seeds, which is a byproduct of fruit processing, using microbial biotransformation technology (A) seeding any one of peach seeds, peach seeds, plum seeds, plum seeds, separating and then pulverizing the seeds; (b) seeding the seeds extracted in step (a) (N-hexane) at a ratio of 1: 3, separating the supernatant from the supernatant and concentrating the supernatant to prepare a concentrate, (c) mixing the concentrate prepared in step (b) with hydroxy And (d) adding an auxiliary agent to the hydroxy fatty acid to formulate the composition, so that crop-tailored crop protection can be carried out.

Description

≪ Desc / Clms Page number 2 > Formulations of dihydroxy fatty acids and their preparation

The present invention relates to a method for producing a hydroxy fatty acid, and more particularly, to a method for producing a hydroxy fatty acid, which comprises using a microbial biotransformation technology to produce an eco-friendly new material hydroxy fatty acid having a high antimicrobial activity against a broad range of phytopathogenic fungi dihydroxy octadecenoic acid (hereinafter referred to as " DOD "), and a method for preparing the same.

Despite the excellent germicidal effect, currently used germicides have become a new problem because of increased tolerance of target hospital fungi and consequent increase of throughput. Therefore, there is a need to develop an environmentally friendly fungicide having a broad sterilization spectrum while exhibiting a strong sterilization effect at a low dose. In response to these demands, it is urgently required to develop a new line of disinfectant having differentiation while complementing the disadvantages of existing disinfectants.

Hydroxy fatty acid (HFA) has one or more hydroxyl groups in the central chain of common fatty acids. The hydroxy fatty acid causes the fatty acid to have a specific property such as high viscosity or reactivity by the hydroxyl group added to the fatty acid chain. The HFA is classified into a mono-, di-, and tri-hydroxy fatty acid depending on the number of hydroxyl groups to which it is attached, and an epoxy-hydroxy fatty acid or an epoxy- Oxo-hydroxy fatty acid, and the like.

Due to the unique properties of HFA, they can have a variety of physiologically active functions. As a result, they can be widely applied to various industrial fields such as new pesticides, new drugs, highly functional resins and fiber materials, biodegradable plastic materials, lubricants, .

On the other hand, 7,10-dihydroxy-8 (E) -octadecenoic acid (DOD) in HFA is easily produced by microorganisms from oleic acid and vegetable oil Hou, CT et al., J. Ind. Microbiol., 1991, 7, 123-130; Min-Jung Suh et al., Applied Micrbiology and Biotechnology 2011, 89: 1721-1727). Recently, DOD has been reported to exhibit antibacterial activity against various pathogenic bacteria (Hye-Ran Sohn, et al., Biocatalysis and Agricultural Biotechnology, 2013, 2: 85-87).

Examples of such techniques are described in documents 1 and 2 below.

For example, Patent Literature 1 below discloses a method of preparing a lyophilized microorganism culture solution by concentrating and lyophilizing a Pseudomonas aeruginosa culture solution to which vegetable oil is added, and then extracting the hydroxy fatty acid from the lyophilized microbial culture solution A method for producing a hydroxy fatty acid from a vegetable oil, comprising the steps of: 1) primary culturing Pseudomonas aeruginosa; adding a vegetable oil to the Pseudomonas aeruginosa culture to further cultivate the Pseudomonas aeruginosa culture to obtain a Pseudomonas aeruginosa culture; 2 ) Concentrating the microbial culture obtained in step 1), 3) lyophilizing the concentrated microbial culture obtained in step 2), and 4) lyophilizing the concentrated microbial culture obtained in step 2) Comprising the step of extracting a hydroxy fatty acid And a manufacturing method thereof.

Also, Patent Document 2 discloses a method for producing a hydroxy fatty acid using a Pseudomonas aeruginosa microorganism, wherein a natural vegetable oil containing oleic acid is used as a substrate, wherein the natural vegetable oil comprises olive oil, safflower seed oil Wherein the hydroxy fatty acid is selected from the group consisting of soybean oil, corn oil, sesame seed oil, perilla seed oil, grape seed oil, red pepper seed oil, canola oil, sunflower seed oil, melon seed oil, Dihydroxy-8 (E) - (4-hydroxy-8-hydroxy-8,10-dihydroxy-8,10-dihydroxy- (7,10-dihydroxy-8 (E) -octadecenoic acid).

Korean Patent Laid-Open Publication No. 2014-0106398 (published on September 3, 2014) Korean Patent Publication No. 2009-0009623 (published on Jan. 23, 2009)

However, in the conventional technology as described above, there has been a problem that it is difficult to mass produce DOD as an environment-friendly crop protection agent and produce it at low cost.

Most commercial pesticide-controlling pesticides are plant pathogenic fungi, but most of them are caused by bacteria, and no effective control method has been developed for them. In particular, plant-grown crops and fruit trees are often damaged by botanical germs, but no effective countermeasures are available.

It is urgent to develop eco-friendly crop protection agents that can significantly increase the storage stability of cultivated crops, crops and fruit trees due to the occurrence of plant diseases caused by fungi and bacteria during crop storage and storage. Although microbial pesticides have been developed for controlling a small number of plant bacterium diseases, they are not continuous and have difficulties in achieving the effect by a single treatment. Especially, it is difficult to cope with a rapid response due to pathogenic mutations of pathogenic bacteria which change in a natural environment.

In addition, in the above conventional techniques, the systematic antibacterial activity of DOD against phytopathogenic fungi has not been verified, and the problem of residual toxicity evaluation for DOD has not been disclosed at all.

In addition, in the above-described conventional techniques, the formulation of DOD is not disclosed.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made in order to solve the above problems, and it is an object of the present invention to provide a formulation of hydroxy fatty acid that optimizes DOD produced from fruit seeds and a method for producing the same.

It is another object of the present invention to provide a method for preparing a hydroxy fatty acid and a method for producing the same, in which a systematic antimicrobial activity of DOD against plant pathogenic bacteria and evaluation of residual toxicity are evaluated to prepare an environmentally friendly crop protection agent.

In order to accomplish the above object, the present invention provides a method for preparing a hydroxy fatty acid (DHA) formulation, comprising the steps of: (a) preparing a formulation of a hydroxy fatty acid from a mixture of (a) peach seed, (B) mixing the seeds extracted in the step (a) with n-hexane in a ratio of 1: 3, and dipping the seeds Separating and concentrating the supernatant to prepare a concentrate; (c) producing a hydroxy fatty acid using the concentrate prepared in the step (b); (d) adding an adjuvant to the hydroxy fatty acid to formulate And a control unit.

In the method for forming a hydroxy fatty acid according to the present invention, the adjuvant is characterized by containing polyoxyethylene sorbitan monooleate.

In order to accomplish the above object, the present invention provides a method for preparing a hydroxy fatty acid formulation, which comprises using palm oil containing oleic acid as a substrate and using Pseudomonas aeruginosa microorganism Thereby forming a hydroxy fatty acid; and injecting an auxiliary agent into the hydroxy fatty acid to formulate it.

In order to achieve the above object, the hydroxy fatty acid formulation according to the present invention comprises 0.75 g of hydroxy fatty acid as the main ingredient, 4 g of Thymol and 17 mL of Ethanol, 8 mL of Dimethyl sulfoxide as an auxiliary agent and 75 mL of Polyoxyethylene sorbitan monooleate .

INDUSTRIAL APPLICABILITY As described above, according to the formulation of the hydroxy fatty acid and the method for producing the same according to the present invention, the effect of optimizing the DOD from the fruit seed, which is a byproduct of fruit processing, at a low cost / high efficiency can be obtained.

Further, according to the formulation of the hydroxy fatty acid according to the present invention and the method for producing the same, it is also possible to achieve the effect of protecting crops customized for crops according to DOD formulation.

That is, according to the present invention, in order to establish a mass production technology of DOD, mass culture technology for using DOD as a biocidal pesticide was developed, and a continuous and stable yield was obtained in a bioreactor to establish a formulation process Development of biological pesticides using the principle of circulation of natural ecosystem using fruit water byproducts can enhance the value as biological pesticides and can be used as an environmentally friendly task to prevent pest resistance and to improve agricultural productivity dramatically And it provides an opportunity to have a direct influence on the improvement of agricultural production technology and farm profit by avoiding the existing chemical pesticide dependent agricultural management method.

In addition, microbial pesticides have been developed to dramatically increase the storage stability of cultivated crops, crops and fruit trees for plant diseases (caused by fungi and bacteria) that occur during storage after crop harvest, and to control a small number of plant bacterial diseases , It is not persistent and it is difficult to achieve the effect by single treatment. Especially, it is possible to solve the problem that it is difficult to cope with the existing protective agent such as pathogenic mutation of pathogenic bacteria changing in the natural environment.

In addition, the development of antibiotic pesticides for environmentally friendly antimicrobial materials that have broad antibacterial activity against bacteria causing bacterial plant diseases can solve the problem of difficulty in using general antibiotics as pesticides due to high production cost, It can contribute to the economic ripple effect of the development of the related industry by supplying high quality and high quality products.

1 is a graph showing DOD production from olive oil using microorganism PR3,
2 is a view showing confirmation of antimicrobial activity of DOD against representative causative bacteria of bacterial plant diseases,
FIG. 3 is a view showing confirmation of antimicrobial activity of DOD against Xanthomonas campestris, a causative organism of bacterial enteric diseases,
4 is a view for confirming the possibility of DOD production using microorganisms from vegetable oils,
5 is a process diagram for the formulation of a hydroxy fatty acid according to the present invention,
FIG. 6 is a table showing the kinds of fruit seeds and the extracted amount of ground used in the experiment of the present invention,
7 is a TLC analysis photograph for confirming DOD production using extracted fruit seed oil,
8 is a graph showing the comparison of DOD production using five kinds of fruit seed extract as a substrate,
9 is a view showing a comparison of DOD production amounts according to addition of plum seed oil concentration,
10 is a view showing comparison of DOD production amount according to addition of peach seed oil concentration,
11 is a view showing a comparison of DOD production amount according to addition of plum seed oil concentration,
12 is a graph showing the TLC analysis of the carbon source effect on DOD production in palm oil use,
13 is a graph showing the effect of carbon sources on the production of DOD when palm oil is used,
FIG. 14 is a diagram showing the TLC analysis of the influence of fructose on the production of DOD when palm oil is used,
15 is a graph showing the influence of fructose concentration on the production of DOD when palm oil is used,
16 shows TLC analysis of the effect of nitrogen source on DOD production using palm oil,
17 is a graph showing the effect of nitrogen source on the production of DOD when palm oil is used,
18 is a diagram showing the TLC analysis of the influence of urea concentration on DOD production using palm oil,
FIG. 19 is a graph showing the effect of urea concentration on DOD production using palm oil,
20 is a diagram showing the TLC analysis of effect of substrate concentration on DOD production using palm oil,
21 is a graph showing the effect of substrate concentration on DOD production in palm oil use,
Figure 22 is a drawing showing the TLC analysis of the effect of culture temperature on the production of DOD when palm oil is used,
23 is a graph showing the influence of culture temperature on DOD production when palm oil is used,
24 is a diagram showing the TLC analysis of the effect of pH on the production of DOD when using palm oil,
25 is a graph showing the influence of pH on the production of DOD when palm oil is used,
26 shows an apparatus for mass production of DOD using palm oil according to the present invention,
27 is a graph showing the comparison of DOD productivity with palm oil concentration for DOD mass production,
28 is a diagram showing antimicrobial activity of DOD against bacterial pathogens,
29 is a view showing antimicrobial activity of DOD against fungi,
30 is a diagram showing antimicrobial activity of each DOD formulation composition against bacterial pathogens,
31 is a view showing a manufacturing process of a prototype product according to the present invention.

These and other objects and novel features of the present invention will become more apparent from the description of the present specification and the accompanying drawings.

Hydroxy fatty acid (HFA) applied to the present invention is a form having a hydroxyl group in a common fatty acid and is found in a trace amount in a plant in a natural state. However, a hydroxyl group causes a fatty acid to have a high viscosity, And the like.

Recently, HFA production using microorganisms from fatty acids has been studied. Among them, Pseudomonas aeruginosa PR3 can produce 7,10-dihydroxy octadecenoic acid (DOD, Formula 1) from oleic acid with high efficiency lost.

The present inventors have developed a technology for producing DOD at a low cost by directly using vegetable oils containing oleic acid (less than 1/100 of the price of existing antibiotics), and when olive oil is used as a substrate, As a result, the DOD production efficiency was about 70%.

FIG. 1 is a graph showing DOD production using microorganism PR3 from olive oil. On the left, the results of TOD analysis of the DOD production amount according to the incubation time show that most of the olive oil in the upper layer is converted into DOD . On the right side, the sample was analyzed by GC after 72 hours. It can be seen that DOD occupies most of the entire peaks except IS (internal standard). When the actual yield for the substrate was calculated, it was found to be about 70%.

The inventors of the present invention have also found that DOD is effective against bacterial plant diseases such as potato girth rot, Clavibacter (Corynbacterium), Erwinia, Ralstonia, Xanthomonas, (See Fig. 2 and Fig. 3).

B is crude extract (1 mg), C is olive oil (1 mg), D is DOD (1 mg), B is crude extract 1 mg), and F represents DMSO. FIG. 3 is a graph showing the antimicrobial activity of DOD against Xanthomonas campestris, which is a causative agent of bacterial bacterial strains, and DOD was used to confirm the antimicrobial activity of Xanthomonas campestris, a causative agent of bacterial bacterial strains in bacterial plant diseases, using an agar dilution method. Indicating that the antimicrobial activity against bacteria is high.

The above results indicate that DOD with high antimicrobial activity from low-cost vegetable oils can be produced in large quantities cheaply using microbial metabolic engineering techniques. The high antimicrobial activity of DOD against a wide range of phytopathogenic bacteria is due to the fact that DOD The possibility of developing an effective crop protection agent for crop disease control is very high.

In addition DOD production was confirmed by P. aeruginosa PR3 strain using various vegetable oils. As a result, it was confirmed that DOD was produced in all of the used vegetable oils as shown by an arrow in FIG. Especially, DOD is produced not only in olive oil having a high content of oleic acid but also in grape seed oil, safflower seed oil, sesame oil, etc., indicating that DOD production using fruit seeds is highly possible.

FIG. 4 is a view for confirming the possibility of DOD production using microorganisms from vegetable oil.

The present invention uses refined vegetable oil, but it requires a separate cost for oil extraction and purification. Therefore, to improve the productivity of DOD, a technology for directly using the seed pulp including oil is developed to greatly reduce the production cost of DOD You can.

Hereinafter, DOD production using fruit seed oil as an embodiment of the present invention will be described with reference to FIG.

5 is a process diagram for the formulation of a hydroxy fatty acid according to the present invention.

First, fruit seed oil extraction and fatty acid analysis are explained.

Fruit seed oil was prepared by preparing seeds from seven kinds of fruits (Grape seed, Melon seed, Peach seed, Nectarine seed, Plum seed, Plum seed and Avocado seed) (S10) ).

Each fruit seed oil was prepared by mixing seeds and n-hexane at a ratio of 1: 3 (S30), immersing them for 24 hours, separating the supernatant, and concentrating (S40).

In the present invention, a dihydroxy octadecenoic acid is produced using the concentrate prepared in step S40 (S50).

The hydroxy fatty acid produced in step S50 is formulated (S60).

The percentage of extracted fruit seed oil was 22.9%, 21.5% of melon seed oil, 12% of plum seed oil, 7.7% of grape seed oil, 1.8% of peach seed oil, 1.0% of peach seed oil, The oil was identified as 0.3%.

FIG. 6 is a table showing the kinds of fruit seeds and the extracted soil mass used in the experiment of the present invention. FIG.

The fatty acid composition of seven kinds of fruit seed oil was confirmed by GC and GC / MS. The contents of the identified fatty acids are summarized in Table 1 below in terms of relative content ratios. As a result, palmitic acid, stearic acid, oleic acid and linoleic acid were identified as main components of fruit seed oils. Especially, the content of oleic acid, which is a precursor of DOD production, was 88% in the order of plum seed oil, 86% in the peach seed oil, 67% in the peach seed oil and 61% in the order of the plum seed oil. This means that it is likely to be used as a suitable substrate for DOD production.

Table 1 below shows the kinds and contents of fatty acids of the extracted fruit seed oil.

Next, we describe the production of DOD using fruit seed oil.

First, based on the results shown in Table 1, the experiment was conducted with five kinds of fruit seed oil except for the low content of avocado seed oil. For 24 hours, Pseudomonas aeruginosa (Pseudomonas aeruginosa) PR3 strain was cultured and 5 kinds of fruit seed oil were added as a substrate at 1% and cultured for 48 to 72 hours.

 The product extracted from the culture was analyzed by TLC. As a result, it was confirmed that a spot appeared at the same position as DOD when plum seed, peach seed, and plum seed oil were used as a substrate as shown in FIG. 7. The size of the spot appeared different depending on the treatment conditions, but it was estimated to be the same material when it appears at the same position.

Based on these results, the amount of DOD production was confirmed by GC analysis. As shown in FIG. 8, there was no significant difference in cell growth depending on the type of substrate added, but the amount of DOD production was significantly different depending on the type of substrate.

When plum seed oil was used as a substrate, the highest amount of DOD was produced, followed by peach seed and plum seed oil. This result is in proportion to the content of oleic acid in fruit seed extract. In particular, the content of oleic acid was higher in olive oil than in control olive oil, and the productivity was about twice higher in DOD production.

These results suggest that plum seed oil can be used effectively in DOD production.

FIG. 7 is a TLC analysis image for confirming DOD production using extracted fruit seed oil. FIG. 7 (A) shows extraction for 48 hours, FIG. 7 (B) shows extraction for 72 hours, 3-4: grape seed oil, 5-6: plum seed oil, 7-8: melon seed oil, 9-10: peach seed oil, and 11-12: plum seed oil.

Fig. 8 is a graph showing comparison of DOD production using five kinds of fruit seed extract as a substrate. Fig. 8 (A) shows cell growth and Fig. 8 (B) shows DOD yield.

Next, we compared DOD production by addition of concentration of plum seed, peach seed, and plum seed extract.

In the experiment according to the present invention, among the five fruit seed oils, plum seed oil, peach seed oil and plum seed oil were found to be most effective for DOD production. Therefore, when these three kinds of fruit seed oil were used as the substrate, the experiment was performed to compare the DOD production according to the added substrate concentration.

After culturing for 24 hours, P. aeruginosa PR3 was added to the medium at a concentration of 1.0, 2.0, 3.0% (v / v) and cultured for 48 to 72 hours. It was confirmed that cell growth and DOD production were also increased with increasing substrate concentration of plum seed oil, peach seed oil and plum seed oil.

Especially, 2% (v / v) addition of 1% (v / v) substrate resulted in about 3 times higher production of DOD than 3 kinds of fruit seed oil. The maximum yield was found to be 45% for plum seed oil, 64% for peach seed oil and 45% for plum seed oil. However, it was difficult to obtain large amounts of seeds from plums, peaches, and plums. Therefore, we optimized the productivity of olive oil and palm oil by using palm oil as an alternative substrate.

Fig. 9 is a diagram showing a comparison of DOD production amount according to the addition of plum seed oil concentration. Fig. 5 (A) shows the TLC analysis and Fig. 5 (B) shows the DOD yield.

10 is a diagram showing a comparison of DOD production amount according to addition of peach seed oil concentration, wherein FIG. 10 (A) shows the TLC analysis and FIG. 10 (B) shows the DOD yield.

11 is a diagram showing a comparison of the DOD production amount according to addition of the concentration of plum seed oil. FIG. 11 (A) shows the TLC analysis and FIG. 11 (B) shows the DOD production amount.

Next, the optimal conditions of DOD production using palm oil, which is another embodiment of the present invention, will be described.

First, the effect of carbon source on the production of DOD when palm oil was used was examined.

In the grape seed oil, which was expected to be low in DOD productivity, the experiment was carried out to replace the palm oil having an oleic acid content of about 39% to obtain the optimal conditions for DOD production.

The composition of the medium plays an important role in DOD production. Therefore, the effects of carbon source and concentration, nitrogen source and concentration, and substrate were investigated to determine optimum conditions for DOD production.

To investigate the effect of carbon sources on DOD production, nine carbon sources were tested at standard concentrations of 0.4% (v / v).

12 (A) shows extraction of 48 hours, FIG. 12 (B) shows extraction of 72 hours, and lane 1-2: Glucose , 3-4: Galactose, 5-6: Fructose, 7-8: Lactose, 9-10: Maltose, 11-12: Sucrose, 13-14: Xylose, 15-16: Glycerol, 17-18: Whey powder .

FIG. 13 is a graph showing the effect of carbon sources on the production of DOD when palm oil is used, in which FIG. 13 (A) shows cell growth and FIG. 13 (B) shows DOD yield.

As can be seen from Fig. 13, the microorganisms grew well regardless of the type of carbon source, but showed a large difference in DOD production. Most of the DOD was produced in the medium containing fructose, and the DOD was hardly produced in the xylose and galactose medium.

Next, the effect of fructose concentration on the production of DOD when palm oil was used was examined.

In the above experiment, it was found that fructose was the most effective carbon source in DOD production. Therefore, an experiment was conducted to investigate the effect of fructose concentration on DOD production.

As shown in FIG. 14, microbial growth by fructose concentration did not show any significant difference. However, the amount of DOD increased with increasing fructose concentration and reached the maximum at 0.5% (v / v). And the DOD production was decreased at higher concentration than 0.5% (v / v). Based on these results, the concentration of fructose was fixed to 0.5% (v / v) and the following experiment was conducted.

14 (A) shows extraction of 48 hours and FIG. 14 (B) shows extraction of 72 hours. Fig. 14 (A) 2: Control, 3-4: 0.1% (v / v), 5-6: 0.3%, 7-8: 0.5%, 9-10: 0.7%, and 11-12: 1.0%.

FIG. 15 is a graph showing the influence of fructose on the production of DOD when palm oil is used. FIG. 15 (A) shows cell growth and FIG. 15 (B) shows DOD production.

In addition, the effect of nitrogen source on the production of DOD when palm oil was used was examined.

Several organic and inorganic nitrogen sources were used to study the effects on DOD production. The control SM6 medium contains 0.1% (v / v) diammonium hydrogen phosphate and 0.1% (v / v) yeast extract. Yeast extract, malt extract, peptone, tryptone, glutamine and urea, and inorganic nitrates such as ammonium nitrate, , Ammonium sulfate, and ammonium phosphate were used. Each of the nitrogen sources was quantitatively assayed at a concentration of 1.13 g / L (about 0.1%, w / v) equivalent to that of the standard medium.

FIG. 16 shows TLC analysis of the effect of nitrogen source on the production of DOD in palm oil using lane 1-2: Control, 3-4: Yeast extract, 5-6: Glutamine, 7-8: Malt extract, 9-10: Tryptone, _{11-12: Urea, 13-14: NH 4} NO 3, 15-16: (NH 4) 2 SO 4, 17-18: (NH 4) 2 HPO 4, 19-20: shows Peptone.

17 is a graph showing the effect of nitrogen source on the production of DOD when palm oil is used, in which FIG. 17 (A) shows cell growth and FIG. 17 (B) shows DOD yield.

As shown in Fig. 17, the microorganisms show high cell growth in the nitrogen source except malt extract, tryptone, and peptone. The amount of DOD production was found to be 39.6, 13.8, and 25.2 mg, respectively, per 50 ml of medium containing urea, ammonium phosphate and ammonium nitrate. However, the DOD yield was much lower than the DOD yield of 61.5 mg / 50 ml in the control medium using mixed nitrogen source Respectively.

These results show that the use of mixed nitrogen source medium is more effective for DOD production than single nitrogen source medium at the same concentration. However, due to its usefulness and low cost, urea was regarded as the top nitrogen source for DOD production, and the effect of the concentration of urea on DOD production was tested.

Next, we examined the effects of urea concentration on cell growth when palm oil was used.

The C / N ratio affects the accumulation of metabolites as a sensitive parameter in the fermentation process. High C / N ratios limit bacterial growth and increase accumulation of metabolites. Based on the previous experiment, the effect of the concentration of urea on DOD production was investigated.

18 shows the TLC analysis on the effect of urea concentration on the production of DOD when palm oil is used, FIG. 18 (A) shows extraction for 48 hours, FIG. 18 (B) shows extraction for 72 hours, lane 1-2: 3-4: 10 mM, 5-6: 50 mM, 7-8: 100 mM, 9-10: 150 mM, and 11-12: 200 mM.

19 is a graph showing the effect of urea concentration on the production of DOD when palm oil is used, in which FIG. 19 (A) shows cell growth and FIG. 19 (B) shows DOD production.

As shown in FIG. 19, the microorganism increased with increasing urea concentration and decreased rapidly at 100 mM or more. However, the DOD yield was the highest at 10 mM, the lowest concentration, and the DOD yield decreased with increasing urea concentration. Since the DOD productivity was higher in the medium with mixed nitrogen source than in the case of using the single nitrogen source element, the following experiment was continued using a mixed medium of diammonium hydrogen phosphate and yeast extract.

In addition, the effect of substrate concentration on the DOD production was examined by using palm oil.

DOD produced from oleic acid and triolein by P. aeruginosa PR3 appears to be consumed again by microorganisms over time. These results indicate that the DOD yield can be increased as a sufficient substrate is supplied. Therefore, P. aeruginosa Experiments were conducted to find the optimal substrate concentration for effective DOD production from palm oil using PR3.

20 (A) shows extraction of 48 hours, FIG. 20 (B) shows extraction of 72 hours, and lane 1-2: 0.6%. FIG. 20 shows the TLC analysis on the effect of substrate concentration on the production of DOD, , 3-4: 1.0%, 5-6: 1.4%, 7-8: 2.0%, and 9-10: 3.0%.

FIG. 21 is a graph showing the influence of the substrate concentration on the production of DOD when palm oil is used, in which FIG. 21 (A) shows cell growth and FIG. 21 (B) shows DOD yield.

As shown in FIG. 21, it was confirmed that cell growth also increased with increasing substrate concentration. It was found that as the substrate concentration increased from 0.6 to 1.4% (v / v), the DOD yield increased, while at the higher concentration, the same yield was maintained. The optimum substrate concentration was 1.4% (v / v), which was about 1.5 times higher than that in the previous experiment when added at 1.0% (v / v). These results indicate that the concentration of specific substrate is higher than that of P. aeruginosa It is required for effective DOD production from palm oil by PR3.

On the other hand, the effect of culture temperature on DOD production was examined by using palm oil.

The same concentration of P. aeruginosa cells were cultured and the optimum culture temperature for DOD production was examined at 20 ~ 27 ℃. All culture conditions except incubation temperature were the same under standard conditions.

22 shows the TLC analysis on the influence of the culture temperature on the production of DOD on the palm oil use, in which FIG. 22 (A) shows extraction for 48 hours, FIG. 22 (B) shows extraction for 72 hours, 3-4: 25 ° C, 5-6: 27 ° C, 7-8: 30 ° C, 9-10: 35 ° C, and 11-12: 40 ° C.

23 is a graph showing the effect of culture temperature on the production of DOD when palm oil is used, in which FIG. 23 (A) shows cell growth and FIG. 23 (B) shows DOD yield.

As shown in FIG. 23, cell growth tended to increase with increasing temperature. However, DOD production increased gradually to 27 ℃, but decreased from 30 ℃. DOD was not produced at above 40 ℃. When olive oil was used as a substrate, DOD was produced at 20 ~ 30 ℃. When cultured at 27 ℃, the yield of DOD was about 2.8 g / L, yielding 71%.

Based on these results, it was confirmed that the optimum DOD production temperature using palm oil was 27 ℃.

In addition, the effect of pH on the production of DOD when palm oil was used was examined.

The effect of initial pH of the medium on the DOD production was examined in the range of pH 3 ~ 10. Overall DOD productivity was good when alkaline, especially DOD production was maximum between pH 8 and 9. In addition, it was confirmed that DOD was hardly produced at pH below 6, which is an acidic pH. This shows that DOD productivity is higher in alkaline environments than in acidic environments. However, cell growth was the highest at pH 7, and decreased above that. showed about 50% cell growth at pH 6 at its maximum at pH 7, but little DOD was produced. This is consistent with the effect of pH on the production of DOD from olive oil as a substrate.

The yield of DOD was maximized to about 1.7 g / L at pH 8, and the experiment was continued by fixing pH 8 to the pH of the initial medium.

24 (A) shows extraction for 48 hours, FIG. 24 (B) shows extraction for 72 hours, and lane 1-2: pH 5 and 3 -4: pH6, 5-6: pH7, 7-8: pH8, 9-10: pH10, 11-12:

25 is a graph showing influences of pH on production of DOD when palm oil is used, in which FIG. 25 (A) shows cell growth and FIG. 25 (B) shows DOD yield.

Next, mass production of DOD using palm oil will be described according to the present invention.

Based on the experimental results described above, DOD mass production using palm oil was carried out.

(V / v) and 1.5% (v / v) of palm oil, respectively, using a 5 L incubator.

26 shows a device for mass production of DOD using palm oil according to the present invention and shows a process of mass production of DOD using a 5 L incubator.

27 is a graph showing a comparison of DOD productivity according to the palm oil concentration for mass production of DOD. Fig. 27 (A) shows the addition of 1.0% (v / v) palm oil, (v / v) addition.

As shown in FIG. 27, the DOD production gradually increased with time, and the yield was maximized at 72 hours and gradually decreased thereafter. The optimum DOD production time was 72 hrs. The productivity of DOD was 1.95g / L and 3.35g / L, respectively, about 1.7 times higher than that of 1% (v / v) And showed a yield of 57%. In the previous experiment, when the olive oil was used as a substrate and the d-batch was performed, the DOD yield was 12 g / L, which was about 3.5 times higher than the batch result. Based on these results, it can be expected that the production yield of the DOD is about 80% or more when the palm oil is used as the substrate and the DOD is produced by the batch arrangement.

Hereinafter, the process (S60) of purifying DOD prepared as described above and formulating it will be described.

First, the DOD antimicrobial activity test was performed on the causative bacteria of each crop.

The DOD produced in the above step S50 was purified (S60), and the antimicrobial activity test for the causative microorganism of each crop was carried out. As shown in Table 2, 8 kinds of bacterial pathogens and 6 kinds of fungal pathogens were tested for antimicrobial activity.

Table 2 shows the kinds of plant pathogens for the DOD antibacterial activity test.

The antimicrobial activity of DOD against bacterial pathogens was also examined.

Bacteria were assayed for antimicrobial activity by determining the size of the clear zone using plate diffusion assay on agar medium. DOD concentration was dissolved in 20 μl of DMSO in the range of 0.5 to 2.0 mg. Penicillin and gentamicin were used as positive controls.

As shown in Fig. 28, Pseudomonas syringae pv. Syringae, Ralstonia solanacearum, Xanthomonas campestris KACC 10490 and Xanthomonasaxonopodispv. citriKACC 10443, Clavibacter michiganensis subsp. showed antimicrobial activity against DOD in michiganensis. However, no antimicrobial activity against DOD was observed in the causative bacteria of spotted, wilted, or cloudless disease.

FIG. 28 is a photograph showing the antibacterial activity of DOD against bacterial pathogens. (A) Pseudomonas syringae pv. Syringae, (B) Ralstonia solanacearum, (c) Xanthomonas campestris KACC 10490, (D) Pseudomonas syringae pv. Sesami, (E) Erwinia sp, (F) Xanthomonas axonopodis pv. citri KACC 10443, (G) Clavibacter michiganensis subsp. michiganensis corrig, (H) Pseudomonas syringae pv. actinidiae, < / RTI > 1: DOD-MG 0.5 mg, 2: DOD 0.5 mg, 3: DOD 1.0 mg, 4: DOD 1.5 mg, 5: DOD 2.0 mg, 6: Penicillin 10,, 7: Gentamicin 10,, .

Based on these results, an experiment was conducted to measure the minimum treatment concentration of DOD for the causative organisms of botanical germs. The MIC (minimum inhibitory concentration) value was measured by treating DOD at a concentration ranging from 1.95 to 500 μg / ml using a liquid medium.

As shown in Table 3, MIC ₅₀ The value of Clavibacter michiganensis subsp. michiganensis was the lowest at 125 ㎍ / ㎖, followed by Pseudomonas syringae pv. Syringae, Ralstonia solanacearum, Xanthomonas campestris KACC 10490 and Xanthomonas axonopodis pv. citri KACC 10443 showed a low value of 250 / / ml.

Based on these results, it was confirmed that DOD treatment at a concentration higher than 125-500 ㎍ / mL, which is the range of MIC ₅₀ value, inhibits about 70 ~ 90% of bacterial pathogens. Also, And it showed strong antimicrobial activity against pathogenic bacteria.

Table 3 is a table showing the minimum inhibitory concentrations of DOD against bacterial pathogens.

These results suggest that DOD has a strong antimicrobial activity against pathogenic bacteria and pathogenic bacteria.

Next, the antimicrobial activity of DOD against fungi was examined.

The antimicrobial activity of the fungi was determined by measuring the size of the zona pellucida using plate diffusion assay on agar medium. DOD was dissolved in 10 μl of DMSO at a concentration ranging from 1 mg to 5 mg, and ketoconazole (Ketoconazole) was used as a positive control.

As shown in FIG. 29, there was no antimicrobial activity against DOD for Rhizoctonia solani AG-1 (IA) KACC 40101, Corynespora cassiicola KACC 40964, Colletotrichum gloeosporioides KACC 40690 and Botrytis cinerea KACC 40574. However, for the Botryosphaeria dothidea KACC 45481, a zona pellucida, the zona pellucida could be measured and the antimicrobial activity against DOD was confirmed.

(A) Rhizoctonia solani AG-1 (IA) KACC 40101, (B) Corynespora cassiicola KACC 40964, (C) Colletotrichum gloeosporioides KACC 40690, (D) Colletotrichum acutatum KACC 40805 , (E) Botryosphaeria dothidea KACC 45481 (F) Botrytis cinerea KACC 40574; 1: 25 μg of ketoconazole, 2: 1 mg of DOD, 3 mg of DOD, 4 mg of DOD, and 5 μl of DMSO.

Next, selection of supplements for DOD formulation will be described.

In order to improve the stability and efficacy of the produced DOD, an experiment was conducted to select an adjuvant.

Among the non-ionic surfactants used in general agriculture, the adjuvants were examined for their physical properties for five surfactants with high dispersibility and proven stability (Table 4). Five types of surfactants are listed in the Inert Ingredients List 3 (approved by the US Environmental Protection Agency (EPA) as specified in the Enforcement Regulations of the Act on the Promotion of Environmentally Friendly Farming and Fisheries and the Support for Organic Foods) 4, only the substances that can be used as auxiliary agents in the production of organic agricultural materials were selected.

Table 4 shows the adjuvants used in the tests according to the invention.

Diffusion experiments, adhesion experiments, drying time experiments and vesiculation experiments were carried out. The diffusion experiments were performed by diluting each adjuvant with a dilution factor of 2,000 on a flat table, dropping 3 times by 20, and spreading area after 4 minutes The test was carried out by immersing the pepper leaves in a beaker for 5 seconds and placing them on the test table. The drying time experiment was performed by diluting the supplements to 2,000 times, The pepper leaves were immersed for 5 seconds, placed on a test table, and the time when the test agent was dried by 90% or more was measured. The suppositories of each of the suppository tests were diluted 2,000 times, and then the suppositories were shaken 20 times in a constant volume flask, and 10 minutes after the beakers were dispensed. In order to verify the plant stability of each adjuvant, the degree of weakness was confirmed by spraying tomato seedlings at a dilution of 2,000 times, 1,000 times, 500 times, and 250 times.

Diffusion tests were performed on the test table with each adjuvant. As a result, the best adjuvant was polyoxyethylene dodecyl monoether, followed by polyoxyethylene sorbitan monooleate, , Polyoxyethylene alkylamine ether, polyoxyethylene (1,1,3,3-tetramethylbutyl) phenyl ether and sodium bis (2-ethylhexyl) sulfosuccinate.

Table 5 shows the degree of diffusion for each adjuvant.

As a result, the most adherent agent was polyoxyethylene dodecyl monoether, followed by polyoxyethylene sorbitan monooleate, polyoxyethylene (1,1,3,3-tetramethylbutyl) phenyl ether, and polyoxyethylene alkylamine ether showed similar results, and the adhesion of sodium bis (2-ethylhexyl) sulfosuccinate was the poorest.

Table 6 shows the degree of adhesion of the adjuvant applied to the present invention.

As a result of the drying time test, it was found that polyoxyethylene sorbitan monooleate was the most rapid drying agent for the pepper leaf, followed by polyoxyethylene dodecyl monoether, polyoxyethylene (1,1,3,3-tetramethylbutyl) phenyl ether, bis (2-ethylhexyl) sulfosuccinate.

Table 7 shows the drying time of the auxiliary agent applied to the present invention.

As a result of the puffing test, polyoxyethylene dodecyl monoether, polyoxyethylene sorbitan monooleate, and polyoxyethylene (1,1,3,3-tetramethylbutyl) phenyl ether were the best suppositories. Sodium bis (2-ethylhexyl) sulfosuccinate, Polyoxyethyelene alkyl amine ether showed the worst vesicle force.

Table 8 shows the squeezing force of the adjuvant applied to the present invention.

As a result of the experiment, the degree of weakness of the adjuvant was found to be weak in Polyoxyethyelene alkyl amine ether only at a dilution ratio of 1,000 times, which is commonly used in a farmhouse. Polyoxyethylene dodecyl monoether and sodium bis (2-ethylhexyl) Sulfosuccinate showed weak mild symptoms.

Therefore, it was concluded that polyoxyethylene sorbitan monooleate (polyoxyethylene sorbitan monooleate), which is superior in stability to polyoxyethylene dodecyl monoether, was selected as an adjuvant.

Table 9 shows the degree of weakness according to the adjuvant applied to the present invention.

Next, the optimal formulation for DOD formulation was experimented according to the present invention.

Based on the conclusions from the experiments described above, DOD preparation samples were prepared with the composition of Table 10, which is the optimum mixing condition, and the antimicrobial activity against plant pathogenic bacteria was measured.

Table 10 shows the optimal formulation of the DOD formulation according to the present invention.

In order to confirm the stability of the prototype product, physical changes and antimicrobial activity against plant pathogens were measured by aging test at 54 ± 2 ° C for 14 days and 0 ± 2 ° C for 7 days.

The aging change was carried out according to the 'criteria and method of aging change test (common to microbial pesticides and biochemical pesticides)' of the 'Examination methods for registration of biological pesticides and registration application documents' notified by Rural Development Administration.

Changes in physical properties were determined by visual inspection at dilution. Antimicrobial activity against plant pathogenic bacteria was evaluated by Pseudomonas syringae pv. Syringae, and Ralstonia solanacearum were determined by using plate diffusion assay on agar medium. Gentamicin was used as a positive control.

As shown in Table 10, Pseudomonas syringae pv. The antimicrobial activity against Syringae and Ralstonia solanacearum was Pseudomonas syringae pv. G-3, G-4, G-5, T-2, and G-4 for Ralstonia solanacearum. T-3, and T-4.

30 shows antimicrobial activity of each DOD preparation composition against bacterial pathogens. (A) Pseudomonas syringae pv. Syringae, (B) Ralstonia solanacearum, and A-1, B-1 Disc No. 1. 1: 10 μg / 10 μl of Gentamicin, 2 μl of G-1 × 10 dilution, 10 μl of 3: G-2 × 10 dilution, 10 μl of 4: 10 μl of 6: G-5 × 10 dilution, and 10 μl of 7: DMSO. 1: 10 μg / 10 μl of gentamicin, 2 μl of T-1 × 10 dilution, 10 μl of 3: T-2 × 10 dilution, 10 μl of 4: 10 μl of 6: T-5 × 10 dilution, and 10 μl of 7: DMSO.

As a result of the heating stability test (14 days at 54 ± 2 ° C), there was no change in physical properties in all the compositions, but the antimicrobial activity showed excellent compositions of G-5, T-4 and T-5.

Table 11 shows the plate clearance (mm) on a plate by DOD formulation composition for bacterial pathogens.

As a result of the heating stability test (14 days at 54 ± 2 ° C), there was no change in physical properties in all the compositions, but the antimicrobial activity showed excellent G-4 and T-4 composition.

Table 12 shows the heating stability test by composition.

G-2, G-3, G-4, G-5, and G-5 were found to have a layer separation at the composition containing geraniol as a result of low temperature stability test (7 days at 0 ± 2 ° C) T-3, T-4 and T-5.

Table 13 shows the low temperature stability test by composition.

From the above results, it was found that the optimal formulation for DOD formulation was suitable for the T-4 composition in terms of effectiveness, stability, and economy. In addition, it is shown that the stable period of 14 days at 54 ± 2 ℃ over the aging change standard indicates that the efficacy is guaranteed for 1 year, so it is also possible to set the guarantee period of the prototype to 1 year or more.

Table 14 shows the optimum composition of the DOD preparation.

As a result of the above experiment, a prototype product was manufactured through the manufacturing process of FIG. 31 as an optimal combination composition of the DOD preparation, and a pot experiment for pepper foot wilt was performed. 31 shows a manufacturing process of a prototype product according to the present invention.

A 10 cm diameter port was filled with seedlings and seedlings were planted to induce the onset of Ralstonia solanacearum suspension, which is a causative agent of foot rot disease, at 30 days (10 ⁷ cfu / mL) at 4th leaf stage, and the onset inhibition effect was observed for 4 weeks .

DOD preparation was treated once, 7 days before pathogen treatment, and 7 times 10 times / 3.3m ² , 5 times at 7 days after treatment.

Ralstonia solanacearum was observed on average 8 days after treatment, and each treatment was repeated three times for 9 weeks to obtain the mean value. In order to determine whether the prototype was weak or not at the site, the presence or absence of the weakness was observed every 7 days after the treatment. The evaluation of the weakness was carried out according to the 'criteria and method of the weak test' of the 'Pesticide registration test standards and methods' notified by Rural Development Administration. Residual toxicity tests were also conducted to confirm the stability of the treated medicines.

In the final study, the incidence of incontinence of pepper foot blight was 92.6%, but the incidence rate of 25.9% and the control value of 73.3% in the quantitative treatment (10mL / 3.3m ² ) were shown. In the case of the dose (20 mL / 3.3 m ² ), the incidence rate was 18.5% and the control value was 81.3%. In addition, no toxicity was detected in all treatments for 5 treatments, and toxicity was not detected as a result of the residual toxicity test, which confirmed the stability of the DOD preparation.

Table 15 shows the control of pepper foot wilt disease according to the number of treatments of the prototype.

Table 16 shows the occurrence of the weakness after the treatment of the prototype.

Although the present invention has been described in detail with reference to the above embodiments, it is needless to say that the present invention is not limited to the above-described embodiments, and various modifications may be made without departing from the spirit of the present invention.

By using the hydroxy fatty acid formulation and the manufacturing method according to the present invention, it is possible to optimize DOD from Fruit Seed which is a byproduct of fruit processing at a low cost / high efficiency and formulate it.

Claims (4)

A method of formulating a dihydroxy octadecenoic acid,
(a) preparing seeds of any one of peach seed, peach seed, plum seed and plum seed, separating and then pulverizing and extracting seeds,
(b) mixing the seeds extracted in the step (a) with n-hexane in a volume ratio of 1: 3, and separating the supernatant and concentrating the concentrate to prepare a concentrate,
(c) producing a hydroxy fatty acid using the concentrate prepared in the step (b)
(d) adding an adjuvant to the hydroxy fatty acid to formulate it,
Wherein said step (c) comprises the step of contacting Pseudomonas aeruginosa The PR3 strain is cultured, and the concentration of the substrate, which is the concentrate, is added to the medium at a ratio of 2.0% (v / v) and cultured for 48 to 72 hours.
In step (d), 4 g of Thymol and 17 ml of ethanol are added to 0.75 g of hydroxy fatty acid, and 8 ml of dimethyl sulfoxide and 75 ml of polyoxyethylene sorbitan monooleate are added as the adjuvant, thereby forming a DOD (dihydroxy octadecenoic acid) formulation. delete A step of using a palm oil containing oleic acid as a substrate and a Pseudomonas aeruginosa microorganism to form a hydroxy fatty acid;
A step of injecting an adjuvant into the hydroxy fatty acid to formulate it,
Wherein the formulation is carried out by adding 4 g of Thymol and 17 mL of ethanol to 0.75 g of hydroxy fatty acid, and adding 8 mL of dimethyl sulfoxide and 75 mL of polyoxyethylene sorbitan monooleate as the adjuvant to the DOD (dihydroxy octadecenoic acid) formulation. delete

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