KR20050076124A - Sophorolipid production from soybean dark oil processing by-product - Google Patents

Abstract

The present invention relates to a method for producing sophorolipid using soybean dark oil (SDO), and more particularly, after culturing candida yeast in medium containing SDO, hexane (hexane) in the culture solution. And eluting the phospholipids by treating with ethyl acetate, and then treating hexane to remove residual oil. According to the present invention, SDO, which is mostly discarded as it is, can be recycled inexpensively in the production of a microbial surfactant.

Description

Sophorolipid production from soybean dark oil processing by-product}

The present invention relates to a method for producing vesicle lipids using soybean dark oil (SDO) and a surfactant comprising vesicle lipids produced by the method.

Surfactants are substances that adsorb to the interface in dilute solutions to reduce their surface tensions.The global surfactant market reached $ 2 billion in 1988 and about $ 9.4 billion in 1994, growing more than 400% in just six years. The demand is increasing year by year. However, most of these have been reported as chemical synthetic surfactants that are not biodegradable by themselves and cause environmental pollution. Thus, interest in microbial surfactants with low toxicity and high biodegradability, environmental compatibility and high selectivity, and specific activity at extreme temperatures, pH and salts (Maier, et al. , Appl. Microbiol.Biotechnology , 54: 625-633, 2000).

Microbial surfactant means a biodegradable surfactant produced by microorganisms such as yeast, mold, bacteria, etc., and is produced extracellularly or intracellularly depending on the strain. Microbial surfactants are non-toxic and easy to biodegrade compared to chemical synthetic surfactants, and thus there is an advantage that they are not secondary pollution sources. Therefore, it can be widely applied in various fields such as cosmetics, pharmaceuticals (Klekner and Kosaric, 1993), food, detergents, pulp and paper, secondary recovery of crude oil (Kosaric and Gray, 1987), environmental purification.

In producing biological surfactants from microorganisms, the main economic factors are the cost, separation and productivity of the raw materials. In converting 50% of the raw material directly into the microbial surfactant, the cost of the raw material amounts to 40 to 50% of the total cost of producing the microbial surfactant. Therefore, developing cheaper raw materials becomes a factor that can reduce the overall production cost. To date, carbohydrates (Klekner, et al., Biotechnol. Letter , 13: 345-348, 1991), vegetable oils (Zhou, et al., J. Am. Oil. Soc. 69: 89-91, 1992), Animal fat (Deshpandle and Daniels, Biores. Technol. 54: 143-150, 1995), n-alkanes (Davila, et al., J. Ind. Microbiol. 13: 249-257, 1994) and single cell oil (Daniel et al., Biotechnol. Lett. 20: 1153-1156, 1998). There have been studies to produce vesicle lipids using various raw materials.

Sophorolipid is a representative biosurfactant in the form of glycolipid produced in Candida bombicola . Other yeast of, in particular Candida reported Li N-Sys (Candida bogoriensis), Candida magnolia in (Candida magnolie), Candida thereof pen giseri (Candida gropengisseri), Candida Bahia coke produced in relatively large amount in the resulting culture by the (Candida apicola) Has been reported (R. Hommel, Biodegradation , 1: 107, 1991).

Sophorolipid is usually produced by supplying non-aqueous carbon sources such as hydrocarbons and fats and oils, and is mainly produced in late cultivation (top phase) when the increase in cell mass is stopped, but among the above yeast, it is possible to directly produce phospholipide from sugars. There is also. In the cultivation of Candida Bombbiola ATCC 22214, about 70 g of sorbolipids per liter of culture medium can be obtained by appropriately combining a sugar such as glucose and a natural oil such as sunflower oil as a carbon source (Cooper, et al., Applied and Environmental Microbiology , 47: 173, 1984). In addition, vesicle lipids can be obtained by using the resting cells of the yeast. By reacting the cells obtained after culturing in a medium prepared by glucose, yeast extract, urea, etc. with a solution containing several carbon sources, about 18 g of phospholipid can be produced per liter of reaction solution (Gobbert , Biotechnology Letters , 6: 225, 1984). However, the production of such phospholipids has a problem of high cost and low yield.

Meanwhile, SDO (soybean dark oil), which is a kind of vegetable oil by-product, is a black non-edible oil. SDOs consist of fatty acids (65-75%) and triglycerides (20-30%), but most are discarded as-is, with only a small amount being recycled for industrial fatty acid production.

Thus, the present inventors completed the present invention by culturing candida yeast bacteria in a medium containing SDO, and then extracting the vesicle lipids from the culture solution.

It is an object of the present invention to provide a method for efficiently producing vesicle lipids from soybean dark oil (SDO).

In addition, another object of the present invention is to provide a surfactant containing the phospholipid.

In order to achieve the above object, the present invention comprises the steps of (1) culturing the yeast bacteria of the genus Candida in a medium containing soybean dark oil (SDO), and (2) treating the yeast culture medium with ethyl acetate to obtain vesicle lipids After extracting, treating the hexane provides a method for producing phosphorelipid comprising the step of removing residual oil.

In addition, the present invention provides an antimicrobial activity of vesicle lipids produced by the above production method.

In addition, the present invention provides a surfactant comprising the above phospholipid.

Hereinafter, the present invention will be described in detail.

In order to achieve the above object, the present invention comprises the steps of (1) culturing the yeast strain of Candida genus in a medium containing SDO, and (2) extracting the vesicle lipid by treating the yeast culture medium of step 1 with ethyl acetate, It provides a method for producing phospholipids comprising the step of removing residual oil by treating.

Step 1 of the present invention is a step of culturing Candida yeast in a medium containing SDO. In this case, the SDO is contained in the medium 0.5 to 20.0 parts by weight (w / v), preferably contained in 5.0 to 15.0 parts by weight (w / v). Sophorolipid production in yeast is strongly influenced by the lipid substrate used as secondary substrate (Hu and Ju, Enzyme Microbial Technol ., 83: 189-198, 2000). In the present invention, SDO was used as the secondary substrate, and since the price of SDO is less than half the price of other vegetable oils, productivity can be improved.

Candida in yeast of step 1 in the present invention is one selected from the group consisting of Candida spring rain coke (Candida bombicola), Candida Bahia coke (Candida apicola), and Candida petroleum (Candida petrollium), Candida in the preferred embodiment of the present invention Bombibola was used.

Step 2 of the present invention is a step of removing the residual oil by treating the yeast culture medium of step 1 with ethyl acetate to extract the phospholipid, and then treated with hexane. At this time, in order to purely separate the phospholipid of the present invention, ethyl acetate of step 2 is treated with 100 to 200% by volume (v / v) with respect to the yeast culture medium of step 1, and hexane is 10 to 50% by volume (v / v).

In addition, the present invention provides an antimicrobial activity of vesicle lipids produced by the above production method.

Sopolori lipid has a general structure as follows, there are largely the lactone type such as (1) and the acid type such as (2). Wherein R ₁ and R ₂ are H or COCH ₃ and R ₃ is H or CH ₃ . R ₄ is a saturated or unsaturated hydrocarbon group having 12 to 16 carbon atoms when R ₃ is H, and a saturated or unsaturated hydrocarbon group having 11 to 15 carbon atoms when R ₃ is CH ₃ .

<General Structural Formula>

Lactone forms are cosmetic products (Mager, et al., Appl. Microbiol. Biotechnol. 54: 625-633, 1987) and skin fibers such as antidandruff, bacteriostatic agents, and deodorants . It is used as a factor (WO No. 9,964,279) that stimulates metabolism of skin fibroblasts. Acid vesicle lipids are particularly used as skin therapeutic agents, such as factors for fibrinolysis, healing, keratin, bleaching and macrophage activity (US Pat. No. 5,981,497). The lactone type exhibits at least four to twenty times the antimicrobial effect of Gram-positive bacteria, which is similar to structural biological cell membranes.

The present inventors have focused on the fact that the fatty acid structure of SDO is similar to vegetable oils such as soybean oil or corn oil, and used it as a raw material for producing vesicle lipids. Sophorolipid produced using SDO in the present invention corresponds to the lactone type, and has antimicrobial activity. Lactone-type structural formula generally shows a ring-shaped structure as in the general structural formula (1), as a result of confirming through a preferred embodiment of the present invention, the phospholipid produced using SDO Bacillus subtilis ( Bacillus subtilis) ) And Propionibacterium acne showed cell growth inhibitory effect.

The present invention provides a surfactant comprising the above vesicle lipid.

Sophorolipid has the highest production of microbial surfactants reported as glycolipid-based surfactants produced in yeast. In the case of phospholipid, the production cost is about twice that of sodium dodecyl sulfate (SDS) and linear alkylbenzene sulphonate (LAS), which are chemical synthetic surfactants, but the SDO used in the present invention is inexpensive as a raw material. Therefore, the phospholipids using SDO are significantly industrially useful because their production cost is significantly reduced. In addition, SDO can be recycled in the production of vesicle lipids without being disposed of as it is, and is suitable for environmental protection due to its high biodegradation activity.

Surfactant-containing surfactant produced by the present invention can be applied to anti-dandruff, bacterial growth inhibitors, deodorants, antibacterial cleaning agents such as cosmetics, soap or shampoo.

In a preferred embodiment of the present invention, SDO was used to produce sophorolipids from Candida bombicola ATCC 22214. Candida bom cola was fed incubated for 7 days to obtain 90 g / L of vesicle lipids (see Table 1 ), and the fatty acid composition was mainly linoleic acid and oleic acid ( Table 1 ). 2 ).

The critical micelle concentration (CMC), minimum surface tension, and molar solubility ratio (MSR) of hydrocarbons produced by the present invention in aqueous solution are chemical synthesis interfaces. Results equivalent to those of the activator are shown (see Table 3 ). In addition, the dispersing power of phophorolipids is rather high than chemically synthesized surfactants such as SDS (see Table 4 ).

In addition, the phospholipids produced by the present invention have antimicrobial activity against Propionibacterium acne and Bacillus subtilis (see Table 5 ). In addition, even if the antimicrobial activity of Sopolori lipid against Propionibacterium acne compared with commercial antimicrobial agents, it shows an excellent cell growth inhibitory effect (see Table 6 ).

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Strain Culture and Isolation of Sophorolipid

<1-1> Strains and Cultivation of Strains

Yeast Candida bombicola ATCC 22214 (ATCC) was passaged in YM agar slant medium.

5% (v / v) of the culture supernatant was inoculated into a 2.5 L jar fermenter (Kobiotech, Korea) using as an inoculum. At this time, the basic medium for culture is 100 g of glucose per 1 liter of distilled water, 5 g of yeast extract, 1 g of KH ₂ PO ₄ , 0.5 g of MgSO ₄ · 7H ₂ O, 0.1 g of CaCl ₂ · 2H ₂ O, 0.1 g of NaCl, 0.7 g of peptone, and 10% (w / v) of soybean dark oil (SDO), corn oil or soybean oil. Fermenter operation conditions were 1 L of total volume, temperature 30 ° C., pH 3.5, stirring speed 550 rpm, aeration rate 1 vvm, and incubation period of 7 days. Thereafter, the production (g / L) of vesicle lipids by Candida bombi cola was examined as the culture day progressed ( FIG. 2 ). That is, dry cell weight (△), phospholipid (■), and oil supplied when fed-batch with 100 g / l initial glucose and 100 g / l SDO. Changes in the amount of feeding oil (▼) and residual oil (○) were observed.

<1-2> Separation of Sopolori Lipid

Culture broth was extracted three times with the same volume of ethyl acetate. In order to obtain the phospholipid stock solution, the ethyl acetate layer was evaporated with a rotary vacuum evaporator (Eyela, Tokyo Rikakikai, Japan) to remove ethyl acetate. Residual oil was extracted and removed with 30 parts by weight (hexanes) of hexane (v / v), most of the residual oil was a fatty acid consisting of the partial decomposition products of SDO and SDO ( Fig. 1 ).

As a result, vesicle lipid production started in the late exponential phase, and exhaustion of the secondary substrate was observed in the stationary phase ( FIG. 2 ). At this time, biomass, vesicle lipid production and residual glucose amount were compared to clarify the effect of the secondary substrate ( Table 1 ).

Volume (g / ℓ) Sopolori lipid production Biomass Residual glucose SDO 90 12.3 4 Corn oil 100 7.6 2 Soybean oil 24 7.9 6

For corn oil, 100 g / l of phospholipids were produced, and for soybean oil, 24 g / l phospholipids were produced. When SDO was used, 90 g / l of phospholipids were produced, 90-110 g / l of phospholipids from lactose and canola oil (Zhou, et al., J. Am Oil. Soc., 69: 89-91, 1992).

Example 2 Measurement of Characteristics of Sopolori Lipid Produced from SDO

<2-1> Types of Sopolori Lipids

At room temperature and atmospheric pressure, SDO was used to produce two types of vesicle lipids, lactone-type and acid-type. Two types of ratios were measured by Fourier Transform Infrared Spectrophotometer (JASCO co., JAPAN) and TLC / FID (IATROSCAN MK-5, IATRON Co., JAPAN). The types of phospholipids produced from corn oil and soybean oil were predominantly acidic, including 85.49% and 96.06%, respectively. In contrast, the type of phospholipide produced from SDO was 40.26% in lactone form, which was higher in lactone production than other substrates.

<2-2> Fatty Acid Composition of Sopolori Lipid

To analyze the fatty acids and oils of Sopolori lipid, 1 g of each sample was dissolved in 15 ml of 0.5 N KOH / methanol, followed by a sand bath at 100 ° C. for 3 hours to break the ester bonds. Reacted. Then 20 ml of BF ₃ / methanol were added and reacted at 90 ° C. for 15 minutes to form fatty acid methyl ester. The methyl ester formed was extracted with petroleum containing ether and hexane, and GC / FID (GC-14B, SHIMADZU co., Equipped with Supelco Wax 10 column) JAPAN). Thereafter, the fatty acid composition between the secondary substrate and the phospholipids produced therefrom was compared ( Table 2 ).

Raw material oil and phospholipids produced using the same fatty acid(%) C 16: 0 C 18: 0 C 18: 1 C 18: 2 C 18: 3 corn Raw material oil 9.75 1.65 24.65 61.95 0.59 Produced Sopolori Lipid 7.83 0.67 27.41 56.50 2.23 bean Raw material oil 10.3 3.6 21.91 56.13 6.22 Produced Sopolori Lipid 8.27 2.19 26.45 60.88 0.51 SDO Raw material oil 6.50 2.63 16.10 65.41 5.78 Produced Sopolori Lipid 9.20 4.26 22.13 51.10 4.87

week; Left is carbon number of fatty acids

    The right side shows the number of double bonds in fatty acids

As a result, the fatty acid composition of the raw material oil and the oleic acid and linoleic acid of the phospholipids produced therefrom showed generally similar composition ratios. In addition, the fatty acid composition of the phospholipids produced from other raw materials showed a similar tendency. As a result, it was confirmed that SDO can be replaced by secondary substrates such as vegetable oils such as corn and soybeans.

<2-3> Measurement of surface tension, critical micelle concentration (CMC) and molar solubility ratio (MSR)

Measure the surface tension of the vesicle lipids with a du Nuoy tensiometer (Surface Tensiomat 21, Fisher Scientific, Pittsburgh, PA) using a platinum ring and ring tenometer at 25 ° C. CMC was measured. In addition, for the solubility experiment of aqueous solution containing sorbodilipide, 10 ml of 0.1 g / l, 0.5 g / l and 1.0 g in the presence of excess 4-methylnaphthalene as a hydrocarbon It was carried out in 20 ml vials with a phophorolipid concentration of / l. After the aqueous vial was equilibrated for 48 hours, 2 ml of the sample was collected and extracted with 4 ml of hexane. 4-methylnaphthalene was analyzed from the extract using gas chromatography equipped with FID (GC-14B, Shimadzu, Japan). Molar solubility ratio (MSR) represents the molar concentration of hydrocarbons dissolved in aqueous solution containing phospholipid ( Table 3 ).

Raw material Surface tension (mN / m) CMC (mg / L) MSR SDO 48.0 150 0.2 Corn oil 41.0 82 0.2 Soybean oil 40.5 88 0.5

week; CMC-Critical Micelle Concentration,

    MSR-Molar Solubility Ratio.

As a result, it was confirmed that the phospholipids produced from SDO exhibited the same properties as the surfactants. Specifically, the CMC and minimum surface tension of the vesicle lipids produced from SDO in aqueous solution were 150 mg / l and 48.0 mN / m, respectively. On the other hand, CMC and minimum surface tensions produced from corn oil were 82 mg / L and 41.0 mN / m, respectively, and CMC and minimum surface tensions produced from soybean oil were 88 mg / L and 40.5 mN / m, respectively. In addition, for phospholipids produced from SDO, the MSR, which represents the solubility of hydrocarbons in aqueous surfactant solution, was 0.201, which was similar to that of phospholipids produced from other substrates.

<2-4> Measurement of Dispersion Force

0.01 g of ferric oxide (Fe ₂ O ₃ ) was added to 0.02 parts by weight (w / v) of phospholipid and an aqueous solution of a surfactant in a 10 cm test tube, followed by vigorous mixing for 30 seconds, followed by standing at room temperature. After 2 hours and 24 hours, samples were taken at a depth of 5 cm from the surface of the test tube solution and the absorbance was measured at 640 nm using a spectrophotometer (UV-160A, SHIMADZU co., JAPAN) ( Table 4 ).

Dispersion force (OD 640 nm) 2 hours 24 hours water 0.12 0.02 Corn Oil Sopolori Feed 0.64 0.13 SDO Sopolori Feed 0.39 0.09 Soybean oil Sopolori lipid 0.92 0.25 Chemical surfactants Tween 80 1.93 0.71 SDS 0.20 0.07 Brij 30 0.66 0.10

As a result, the dispersing power of the phospholipids produced from vegetable oils was higher than the chemical synthetic surfactants SDS (sodium dodecyl sulfate) and Brij 30 (Polyoxyethylene (4) lauryl ether). Although, after 2 hours, the dispersibility of phospholipids produced from SDO was weaker than that of phospholipids and chemical surfactants produced from vegetable oils other than SDS, after 24 hours, they were similar to those of SDS and Brij 30. The figures are shown.

Example 3 Determination of Antimicrobial Activity of Sopholori Lipids Produced from SDO

In order to test the antimicrobial activity of vesicle lipids produced from SDO, several bacterial strains were obtained from Korea Culture Type Collection (KCTC). Propionibacterium acne cultures were used with Reinforced Clostrial Medium (Topley House, ENGLAND) (Gribbon, et al., J. Gen. Microbiol. 139: 1745-1751, 1993). For other bacterial strains, LB medium was used. Inhibitory concentration (IC) refers to the concentration at which the colony count of propionibacterium acne cultured at 37 ° C. for 6-7 days under the conditions of an anaerobic jar begins to decrease, whereas The mean concentration of starting to decrease the number of colonies cultured for 1-2 days ( Table 5 ).

microbe IC (mg / L) Bacillus subtilis 4 Propionibacterium Acne 0.5 Escherichia coli No inhibitory activity

As a result, the ICs for Bacillus subtilis and Propionibacterium acne were 4.0 and 0.5 mg / l, respectively. Interestingly, no inhibition of E. coli cell growth was observed. The results were similar to the ICs of phospholipids produced in canola oil (Kim, et al., J. Microbiol. Biotechnol. , 12: 235-241, 2002). Sophorolipid exhibited antimicrobial activity against the propionibacterium acne, an acne-induced bacterium.

In addition, the IC of Sophorolipid against Propionibacterium acne was compared with commercial antimicrobial agents ( Table 6 ).

Corn oil parcel lipid SDO parcel feed Soybean oil phospholipid Canola oil capsule lipid Irgasan DP-300 IC (mg / L) <1.5 <1.5 <16 <0.5 <3

week; Irgasan DP-300 is a commercial sanction.

As a result, the IC of Sophorolipid against Propionibacterium acne was <1.5 mg / l, which was lower than Irgasan DP-300. The results disclose that vesicle lipids can be used in health supplement products as antimicrobial agents. Since phospholipid has both surface activity and antimicrobial activity as a surfactant, it can be used for cleaning agents having antimicrobial activity such as body shampoo and the like.

As described above, after incubating the yeast bacteria of the genus Candida in a medium containing soybean dark oil (SDO), the culture suspension was inoculated in a fermentor, and then eluted by treating with hexane and ethyl acetate. Sopolori lipids are environmentally friendly because they can recycle SDOs. In addition, the use of inexpensive SDO can not only mass-produce vesicle lipids as a microbial surfactant at low cost, but also can be applied to antibacterial cleaning agents due to antimicrobial activity.

Figure 1 is a schematic diagram showing the separation process of the sophorolipid (sophorolipid). Candida bombicola is grown in a medium containing soybean dark oil (SDO), and then the culture broth is extracted with the same volume of ethyl acetate. The ethyl acetate layer is then evaporated with a rotary vacuum evaporator to remove ethyl acetate, and residual oil is removed with hexane to separate pure phospholipids.

Figure 2 is a graph showing the production (g / ℓ) of vesicle lipids by Candida Bombi Cola over the course of the culture day. Dry cell weight (△), phospholipid (■), and feeding oil when fed-batch with 100 g / l initial glucose and 100 g / l SDO ), And residual oil (?).

Claims (6)

(1) culturing candida yeast in medium containing soybean dark oil (SDO); And (2) after treating the yeast culture medium of step 1 with ethyl acetate to extract phophorolipid, hexane treatment to remove residual oil. The method of claim 1, wherein the SDO of step 1 is characterized in that the medium is contained in 0.5 ~ 20.0 parts by weight (w / v) of vesicle lipids. The method of claim 1 wherein the Candida genus yeast of step 1 is Candida spring rain coke (Candida bombicola), Candida Bahia coke (Candida apicola), and Candida petroleum parcel, characterized in that one selected from the group consisting of (Candida petrollium) Laurie Method of production of feed. The method of claim 1, wherein the ethyl acetate of step 2 is treated with 100 to 200% by volume (v / v) with respect to the yeast culture of step 1, and hexane is treated by 10 to 50% by volume (v / v). Method of producing phospholipids. Antimicrobial vesicle lipids produced by the method of claim 1. Surfactant comprising the phospholipid of claim 5.