KR101743553B1 - Production of dihydroxy fatty acid and monorhamnolipid from vegetable oil by Pseudomonas aeruginosa KACC 10186 - Google Patents

Abstract

The present invention relates to a process for the co-production of dihydroxy fatty acids and monoramphosphides from vegetable oils by the strain Pseudomonas aeruginosa KACC 10186. Pseudomonas aeruginosa KACC 10186 strain co-produces dihydroxy fatty acid and monoramnolipid so that it is useful for mass production of industrially utilizable dihydroxy fatty acid without the addition of a separate surfactant .

Description

Production of dihydroxy fatty acid and monoramnolipid from vegetable oil by Pseudomonas aeruginosa KACC 10186 {Production of dihydroxy fatty acid and monorhamnolipid from vegetable oil by Pseudomonas aeruginosa KACC 10186}

The present invention relates to a process for the co-production of dihydroxy fatty acids and monoramnolipids from vegetable oils by Pseudomonas aeruginosa KACC 10186.

Hydroxy fatty acid (HFA) is a form that has at least one hydroxyl group in the central chain of a common fatty acid. The hydroxyl group added to the fatty acid chain gives the fatty acid a unique property such as high viscosity and reactivity (Chemical and Biological conversion of soybean oil for industrial products, in Fats for the Future, edited by RC Cambie, Ellis Horwood Ltd Press, Chichester, pp: 301-317). Hydroxy fatty acids are classified into mono-, di-, and tri- (hydroxy) fatty acids depending on the number of hydroxyl groups to which they are attached and include a separate structure in addition to the hydroxyl group Or an epoxy-hydroxy fatty acid or an oxo-hydroxy fatty acid.

Due to the unique properties of hydroxy fatty acids, they can have a variety of physiologically active functions and as a result they can be applied to a wide range of industries including agrochemicals, pharmaceuticals, high-functional resins and fiber materials, biodegradable plastic materials, lubricants, .

Although various functionalities of hydroxy fatty acids have been known to date, it is known that hydroxy fatty acids present in nature are present only in trace amounts in plants. Therefore, research was attempted to produce hydroxy fatty acids using microorganisms.

It is known that Pseudomonas aeruginosa PR3 can produce mono-, di-, and tri-hydroxy fatty acids from various fatty acid substrates and vegetable oils (Kim, H. Gardner, HW and Hou, CT 10 ) -Hydroxy-8 (E) -octadecenoic Acid, an Intermediate in the Conversion of Oleic Acid to 7,10-Dihydroxy-8 (E) -octadecenoic Acid, J. Am. Oil. -99, Kim, H. Kuo, TM and Hou, CT Production of 10,12-Dihydroxy-8 (E) -octadecenoic Acid, an Intermediate in Conversion of Ricinoleic Acid to 7,10,12-Trihydroxy- ) -octadecenoic Acid by Pseudomonas aeruginosa PR3. J. Ind. Microbiol. Biotechnol. 1999, 24, 167-172, Kim, H. Gardner, HW and Hou, VC T. Production of Isomeric (9,10,13) (10E) -octadecenoic Acid from Linoleic Acid by Pseudomonas aeruginosa PR3. J. Ind. Microbiol. Biotechnol., 2000, 25, 109-115). In particular, a study on the production of 7,10-dihydroxy-8 (E) -octadecenoic acid (7,0-Dihydroxy-8 (E) -octadecenoic acid) as a dihydroxy fatty acid by Pseudomonas aeruginosa PR3 Production of 7,10-dihydroxy-8 (E) -octadecenoic acid from olive was carried out in a lot of ways (Min-Jung Suh, Ka-Yeon Baek, Beom-Soo Kim, Ching T. Hou and Hak- oil by Pseudomonas aeruginosa PR3 Applied Micrbiology and Biotechnology 2011, 89 (6):. 1721-1727, Jae-Han Bae, Min-Jeong Suh, Na-Young Lee, Ching T. Hou, and Hak-Ryul Kim (2010.12). Production of a Value-added Hydroxy Fatty Acid, 7,10-dihydroxy-8 (E) -octadecenoic Acid, from High Oleic Safflower Oil by Pseudomonas aeruginosa PR3, Biotechnology and Bioprocess Engineering 2010.12 15 (6): 953-958) 7,10-Dihydroxy-8 (E) -octadecenoic Acid is known to have a high antimicrobial activity against various pathogenic bacteria (Hye-Ran Sohn , Jae-Han Bae, Ching T. Hou, Hak-Ryul Kim (2013. 08) Antibacterial activity of a 7,10-dihydroxy-8 (E) -octadecenoic acid against plant pathogenic bacteria Enzyme Microb Technol 2013. 53: 152-153, Hye-Ran Sohn , Ka-Yeon Back, Ching T. Hou, and Hak-Ryul Kim (2013. 01) Antibacterial Activity of a 7,10-dihydroxy-8 (E) -octadecenoic Acid against Food-born Pathogenic Bacteria. Biocatalysis and Agricultural Biotechnology, 2013, 2 (1): 85-87). As a microorganism producing dihydroxy fatty acid, some strains other than Pseudomonas aeruginosa PR3 strain are known, but their productivity is reported to be very low (De Andres, C., Mercade, E., Guinea, J. and Manresa, A. 1994) 7,10-Dihydroxy-8 E -Octadecenoic Acid Produced by Pseudomonas sp. 42A2: Evaluation of Different Cultural Parameters of the Fermentation, World J. Microbiol. Biotechnol. 10 , 106-109).

As described above, since the strain producing dihydroxy fatty acid is very limited, the development of a new strain producing dihydroxy fatty acid needs to be continuously carried out.

Surfactants, on the other hand, are amphipathic substances that can reduce surface tension and reduce interfacial tension between two different phases to promote emulsion dispersion. They are utilized in various fields such as medicines, foods, cosmetics, pesticides, detergents and the like by taking advantage of their excellent washing power, emulsifying power and dispersing power. However, surfactants currently practically used are chemically synthesized using petrochemicals as their raw materials. However, they have a disadvantage that they are mostly toxic and not readily biodegraded in nature (Jitendra D. Desai and Ibrahim M. Banat, Microbiological Production of Surfactants and Their Commercial Potential, Microbiology and Molecular Biology Reviews , 1997, 61 (1), 47-64).

On the other hand, biosurfactant is a surfactant obtained from biological materials such as microorganisms, animals and plants, and has been attracting attention as a material capable of replacing a chemical synthetic surfactant because of its low toxicity and easy decomposition in the natural world. In particular, the production of biosurfactants by microorganisms is easier to handle than other animals and plants, and various kinds of surfactants can be produced depending on the species. As the growth speed is fast, mass production is possible in a short time, and thus it is getting more attention. The hydrophobic part of the microbial surfactant is composed of saturated fatty acid, unsaturated fatty acid, fatty acid alcohol and the like, and the hydrophilic part is composed of carbohydrate, amino acid, peptide, protein, phosphate group and the like. Depending on the type of surfactant, microbial surfactants are classified into glycolipids, lipopeptides, lipoproteins, fatty acids, phospholipids, and polymeric surfactants. Among the microbial surfactants, sophorolipid is a glycolipid surfactant with various advantages such as the use of inexpensive substrate, easy separation and low separation of products, relatively high production yield, high interfacial activity and antibacterial activity Which is suitable for industrialization through mass production. Rumor pods in sophorolipids have Rum nos in the hydrophilic part and fatty acid derivatives in the hydrophobic part. Depending on the number of Rumnos molecules connected, it is divided into monorhamnolipid and dirhamnolipid, and chain lengths of fatty acid derivatives are also varied. Generally, when the ranoripalipide is produced by microorganisms, it is produced in a mixture of monorhamnolipid and dirhamnolipid having various chain lengths (M. Benincasa, J. Contiero, MA Manresa, IO Moraes Rhamnolipid production by Pseudomonas aeruginosa LBI growing on soapstock as the sole carbon source. Journal of Food Engineering 2002, 54, 283-288, E. Deziel, F. Lepine, S. Milot, R. Villemur, Mass spectrometry monitoring of rhamnolipids from a growing culture of Pseudomonas aeruginosa strain 57RP. Biochimica et Biophysica Acta 2000, 1485, 145-152, Yu-Hong Wei, Chien-Liang Chou, Jo-Shu Chang. Rhamnolipid production by indigenous Pseudomonas aeruginosa J4 originating from petrochemical wastewater. Biochemical Engineering Journal, 2005, 27, 146-154). However, such a mixture of rhamnolipids requires a separate purification process to obtain the required rhamnolipid from the mixture, which is disadvantageous for mass production. For this reason, when microorganisms produce any one of various rhamnolipids as a main product, they have favorable conditions for mass production.

Since dihydroxy fatty acid is not a hydrophilic substance, it is necessary to use a separate surfactant to industrially use dihydroxy fatty acid. Also, since monoramnolipid can be used as a biosurfactant as described above, the use of a microorganism capable of producing monoramnolipid and dihydroxy fatty acid at the same time makes it possible to use a dihydroxy fatty acid produced from a microorganism without addition of an additional surfactant It will be very advantageous for practical use.

Thus, the present inventors have completed the present invention by newly confirming that Pseudomonas aeruginosa KACC 10186 strain produces dihydroxy fatty acid and rhamnolipid simultaneously.

It is an object of the present invention to provide a method for co-producing a dihydroxy fatty acid and a monoramnolipid from a vegetable oil using Pseudomonas aeruginosa KACC 10186 strain.

In order to achieve the above object, the present invention provides a method of co-producing a hydroxy fatty acid and a monoramnolipid from a vegetable oil, comprising culturing Pseudomonas aeruginosa KACC 10186.

The Pseudomonas aeruginosa strain KACC 10186 of the present invention co-produces a dihydroxy fatty acid and a monoramnolipid from a vegetable oil, so that the industrially utilizable dihydroxy fatty acid can be mass- Can be advantageously used for production.

1 shows a TLC analysis result of a product produced from Pseudomonas aeruginosa KACC 10186 (lane 1: monoramnolipid standard, lane 2: DOD standard, lane 3: crude extract of product).
Figure 2 is a graph showing the GC analysis results of the product produced from Pseudomonas aeruginosa KACC 10186;
3 is a graph showing the results of GC / MS analysis of the DOD peak among the products produced from Pseudomonas aeruginosa KACC 10186;
FIG. 4 is a graph showing the results of GC analysis of spots corresponding to monoramnolipids among products produced from Pseudomonas aeruginosa KACC 10186
FIG. 5 is a graph showing the results of LC / MS analysis of spots corresponding to monoramnolipids among products produced from Pseudomonas aeruginosa KACC 10186
6 is a graph showing the result of MS / MS analysis of a peak of a mass of 503.3 corresponding to monoramnolipid to confirm the structure of the product.
Figure 7 shows the structure of DOD and monoramnolipid in the products produced from Pseudomonas aeruginosa KACC 10186
FIG. 8 is a graph showing the results of TLC analysis of products produced from Pseudomonas aeruginosa PR3 and Pseudomonas aeruginosa KACC 10186. FIG. Lane 1: DOD standard material, lane 2: product of Pseudomonas aeruginosa PR3, lane 3: product cultivated only substrate without microorganism, lane 4-5: product from Pseudomonas aeruginosa KACC 10186.
Figure 9 shows the effect of culture time on DOD and monoramnolipid production by Pseudomonas aeruginosa KACC 10186.
Figure 10 shows the effect of culture temperature on DOD and monoramnolipid production from Pseudomonas aeruginosa KACC 10186.

The present invention provides a method for co-production of a hydroxy fatty acid and a monoramnolipid comprising culturing a Pseudomonas aeruginosa strain KACC 10186.

The Pseudomonas aeruginosa strain KACC 10186 has been deposited with the Korean Agricultural Microbiology Research Center (Accession No. KACC 10186).

The Pseudomonas aeruginosa KACC 10186 strain simultaneously produces a dihydroxy fatty acid and a monoramnolipid.

The dihydroxy fatty acid may be any hydroxy fatty acid having two hydroxyl groups in the central chain of the fatty acid. Preferably, the dihydroxy fatty acid is a fatty acid having 18 carbon atoms, Dihydroxy-8 (E) -octadecenoic acid (7,10-dihydroxy-8-octadecenoic acid) having a hydroxyl group and one double bond between carbon number 8 and carbon number 9, 8 (E) -octadecenoic acid, DOD).

The monoramnolipid may be any rhamnolipid having a fatty acid linked to one molecule of rhamnose, but preferably two molecules of fatty acid having 10 carbon atoms are connected in series to one molecule of rhamnose. Lt; / RTI >

The substrate used to co-produce the Pseudomonas aeruginosa KACC 10186 strain with dihydroxy fatty acid and monoramnolipid is a vegetable oil and is preferably selected from the group consisting of olive oil, safflower seed oil, soybean oil, corn oil, sesame seed oil, But are not limited to, seed oil, grape seed oil, red pepper seed oil, canola oil, sunflower seed oil, melon seed oil, rapeseed oil, The vegetable oil can be obtained by any method such as a solvent extraction method or a pressure extraction method, which has been conventionally used for extracting oil from seeds or fruits of natural plants, and can also be obtained commercially.

In the culturing step, the culturing temperature is preferably from 10 캜 to 45 캜, more preferably from 20 캜 to 40 캜, but is not limited thereto.

In the culturing step, the incubation time means the incubation time after the addition of the substrate, preferably 24 hours to 240 hours, more preferably 48 hours to 120 hours.

The pH of the culture medium in which the strain is cultured in the culturing step is preferably 4.0 to 10.0, more preferably 6.5 to 8.5, but is not limited thereto.

The carbon source of the strain is selected from the group consisting of glucose, galactose, lactose, glycerol, xylose, sucrose, maltose and fructose. And is preferably selected from the group consisting of glycerol, glucose, galactose, lactose, fructose, and mixtures thereof, but is not limited thereto.

The nitrogen source of the strain may be selected from the group consisting of yeast extract, malt extract, glutamine, ammonium nitrate (NH ₄ NO ₃ ), peptone, tryptone, ammonium chloride, ammonium sulfate, Ammonium phosphate, urea, and the like, but are not limited thereto.

Hereinafter, the present invention will be described in detail with reference to examples. However, these are for the purpose of illustrating the present invention in more detail, and the scope of the present invention is not limited thereto.

Example 1 Culture of Pseudomonas aeruginosa KACC 10186 strain

Pseudomonas aeruginosa KACC 10186 strain was used to co-produce a dihydroxy fatty acid and a monoramnolipid with a vegetable oil as a substrate and cultured under the following conditions.

More particularly, the Pseudomonas air Rouge labor KACC 10186 the YPB medium strains (0.7% glycerol, 0.1% beef extract (beef extract), 0.2% yeast extract, 0.5% peptone, 0.5% NaCl, 0.02% MgSO 4) 27 ℃ conditions Lt; / RTI > The composition of the medium was replaced with the necessary carbon source instead of glycerol if necessary, and the yeast extract and the peptone were replaced with the necessary nitrogen source. The pH of the medium was maintained at 7.5, and the strain was subcultured every 2-3 days. 1% of vegetable oil substrate was added to KACC 10186 culture medium for 24 hours in YPB medium, incubated for the necessary time, and 6N HCl was added to lower the pH of the medium to 2.0 or less. Immediately after the completion of the incubation, the same amount of ethyl acetate was added to the extract, and the extract was concentrated on a rotary evaporator. The culture solution of the Pseudomonas aeruginosa KACC 10186 strain obtained through the above process contains a hydroxy fatty acid, especially 7,10-dihydroxy-8 (E) -octadecase, which is an antibacterial activity material, (7,10-dihydroxy-8 (E) -octadecenoic acid, DOD) and monoramnolipid.

Example 2. Verification of co-production ability of hydroxy fatty acid and monoramnolipid from Pseudomonas aeruginosa KACC 10186 strain

2.1 Thin layer chromatography analysis

Pseudomonas aeruginosa KACC 10186 strain was cultivated in YPB medium (0.7% glycerol, 0.1% beef extract, 0.2% yeast extract, 0.5% peptone, 0.5% NaCl, 0.02% MgSO ₄ ) at 27 ° C and 200 rpm for 24 hours Lt; / RTI > Olive oil was added to the culture of the obtained strain at a concentration of 1% of the culture medium, followed by culturing for 3 days, followed by 6N HCl to lower the pH of the culture medium to 2.0 or less. Immediately after the incubation, the same amount of ethyl acetate was extracted and the extract was concentrated on a rotary evaporator. Which was analyzed by thin layer chromatography (TLC). Thin layer chromatography (TLC) was performed on a glass substrate coated with silica gel. The solvent for the separation of the products was toluene (Toluene): Dioxan: Acetic acid (79: 14: 7 , v / v / v) was used. After the product was separated, the product was confirmed by spotting the product by spraying 50% solution of sulfuric acid on the substrate and heating it at 100 ° C for 10 minutes or more. The results are shown in Fig.

As shown in Fig. 1, the spot production of (1) and (2) was confirmed in the culture extract (lane 3), and the spot of (1) E) -octadecenoic acid (DOD) standard sample, and the spot of (2) appeared at the same position as the monoramnolipid standard sample of lane 1, so (1) , And the (2) spot was determined to be monoramnolipid.

2.2 Gas Chromatographic Analysis

In order to confirm the structure of the material of the spots identified in Example 2.1, the same sample used in lane 3 of FIG. 1 was analyzed by gas chromatography. More specifically, 1 ml of diazomethane was added to 10 mg of the sample to be analyzed, and the mixture was allowed to stand at room temperature for 5 minutes. Diazomethane was removed using nitrogen gas, and 1 ml of TMSI + pyridine (1: 4, v / v) was added and left for 40 minutes. After the residual solvent was removed by using nitrogen gas, 200 μl of a solution for gas chromatography (Dichrolomethane: Methanol = 95: 5, v / v) was added and 1 μl of the final sample was injected into a gas chromatograph. The column used was a hydrophobic column, and the analysis was performed at a temperature ranging from 100 ° C to 300 ° C, and the analysis time was within 1 hour. As an internal standard, heptadecanoic acid (C17: 0) was used. The results are shown in Fig.

As shown in Fig. 2, in the extract produced by the Pseudomonas aeruginosa strain KACC 10186 using olive oil as a substrate, it is possible to confirm the peak (1) appearing at the same time as the DOD and the peak (2) appearing at another position have. (1) peak appeared in the same time zone as DOD, so it was judged to be DOD.

2.3 GC / MS analysis for DOD identification

The structure of the peak material (1) obtained in Example 2.2 was confirmed by GC / MS. The GC / MS was carried out according to the gas chromatography method of Example 2.2, the column having a length of 30 m or more, and a mass analyzer was installed in a detector of the separated molecules. The results are shown in Fig.

As shown in FIG. 3, the structure of the hydroxy fatty acid produced by the KACC 10186 strain according to the present invention has a total of two hydroxyl groups, one in each of the 7-carbon and 10-carbon on the fatty acid chain having 18 carbon atoms, And one double bond between carbon number 9 and carbon number 9, confirming the same structure as the known DOD.

2.4 Identification of monoramnolipid

To confirm the structure of the peak (2) by thin layer chromatography of Example 2.1 and the structure of the peak of (2) by gas chromatography of Example 2.2, the spot (2) of Example 2.1 was separated and purified And analyzed by gas chromatography. The analytical method was carried out in accordance with the gas chromatography method of Example 2.2, and the results are shown in FIG. As shown in the figure, there was a peak at 33.9 minutes in addition to the internal standard. The purity of the peak was 95% except for the internal standard.

The separated and purified samples were analyzed by liquid chromatography / mass spectrometry and compared with the monoramnolipid standard samples. As shown in Fig. 5, the mass of the purified sample (2. Sample sample) is mainly shown at 503.3 position, which is the same as that of the monoramnolipid standard sample (3. STD sample) It was judged to be monoramnolipid. The structure of the material at this position was confirmed by MS / MS and is shown in Fig.

As shown in Fig. 6, the ranoriphipide produced by the Pseudomonas aeruginosa strain KACC 10186 of the present invention is a monoramnolipid in which one molecule of Rumnose and two molecules of fatty acid having 10 carbon atoms are connected in series, as a standard sample It was confirmed that it was the same material as the monoramnolipid used.

Through the above experiments, it was confirmed that the substances produced by the Pseudomonas aeruginosa KACC 10186 strain of the present invention were DOD and monoramnolipid. The Pseudomonas aeruginosa KACC 10186 strain co-produced DOD and monoramnolipid from vegetable oil Respectively. The structure of DOD and monoramnolipid is shown in Fig.

Example 3: Verification of differences between substances produced from Pseudomonas aeruginosa KACC 10186 and Pseudomonas aeruginosa PR3 strain

In order to confirm the product difference between Pseudomonas aeruginosa KACC 10186 strain of the present invention and Pseudomonas aeruginosa PR3 strain, which is well known as a DOD production strain, two strains were used in accordance with the method of Example 1 and Example 2.1, The crude extract of the produced product was analyzed by thin layer chromatography and the results are shown in FIG. As shown in FIG. 8, Pseudomonas aeruginosa PR3 (No. 2 lane) showed only DOD spot (lane 1), whereas Pseudomonas aeruginosa KACC 10186 strain (No. 4 and No. 5 lane) And monoramnolipid spots, the Pseudomonas aeruginosa KACC 10186 strain of the present invention produces a substance different from the Pseudomonas aeruginosa PR3 strain.

Example 4. Effect of culture conditions on DOD and monoramnolipid co-production of Pseudomonas aeruginosa KACC 10186

To examine the effects of the KACC 10186 strain of the present invention on the co-production of DOD and monoramnolipid, the changes in the production amounts of DOD and monoramnolipid according to the culture time were analyzed. The medium conditions and culture conditions were the same as those of Example 1 and Example 2.1, and samples were collected every 24 hours as the incubation time elapsed. After the products were obtained, they were analyzed by gas chromatography The results are shown in FIG. As shown in FIG. 9, it was found that the ratio of the amount of DOD and the amount of monoramnolipid produced varied with the incubation time. In the case of DOD, the amount of DOD was increased at the early stage of culture but decreased as the incubation time was hardened, and that of monoramnolipid was continuously increased with the lapse of incubation time.

In order to examine the effect of the KACC 10186 strain of the present invention on the co-production of DOD and monoramnolipid, the changes in the production amounts of DOD and monoramnolipid according to the culture temperature were analyzed. Other conditions except for the incubation temperature were as described in Example 1 and Example 2.1, and the incubation temperature was changed to 20, 25, and 27 ° C. After 72 hours from the addition of the substrate, the sample was collected, The gas chromatographic analysis was carried out according to the method of Example 2.2 above, and the results are shown in FIG. As shown in FIG. 10, it was confirmed that the ratio of the amount of DOD and the amount of monoramnolipid produced varied depending on the culture temperature. DOD was produced at relatively low temperature and decreased as the incubation temperature increased. However, it was found that the production amount of monoramnolipid was lowered when the incubation temperature was lower, and the amount of monolamnolipid was increased continuously as the incubation temperature was increased there was.

From the above results, it was confirmed that the ratio of the amount of DOD and monoramnolipid produced can be changed according to the change of the culture conditions during the co-production of DOD and monoramnolipid using the KACC 10186 strain of the present invention. And the ratio of the two products can be appropriately adjusted according to need, so that it can be used industrially.

Claims (9)

Pseudomonas air Rouge labor (Pseudomonas aeruginosa) KACC 10186 culturing strains; Pseudomonas air Rouge labor (Pseudomonas aeruginosa) 7,10- dihydroxy -8 by KACC 10186 strains containing the (E) - octanoic acid dese (7 , 10-dihydroxy-8 (E) -octadecenoic acid) and monorhamnolipid.
delete delete The production method according to claim 1, wherein the monoramnolipid is a structure in which two molecules of fatty acid having 10 carbon atoms are connected in series to one Rumnose molecule.
The production method according to claim 1, wherein the strain uses vegetable oil as a substrate.
The vegetable oil according to claim 5, wherein the vegetable oil is selected from the group consisting of olive oil, safflower seed oil, 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, Lt; RTI ID = 0.0 > 1, < / RTI >
The production method according to claim 1, wherein the culturing temperature of the step of culturing the strain is 10 ° C to 45 ° C.
The production method according to claim 1, wherein the culturing time of culturing the strain is 24 hours to 240 hours.
The method according to claim 1, wherein the carbon source of the strain is selected from the group consisting of glucose, galactose, lactose, glycerol, xylose, sucrose, maltose, < / RTI > fructose.

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