KR20150034360A - Method for producing sophorolipid using coconut oil - Google Patents

Abstract

The present invention relates to a method for producing Sophorolipid using coconut oil wherein the method can culture a Candida bombicola strain in a culture medium with coconut oil and glucose and can accordingly produce Sophorolipid, thereby being applicable in mass-producing Sophorolipid which has an outstanding washing effect for fabric fabrics contaminated by oil and an antimicrobial effect for Staphylococcus aureus and Escherichia coli.

Description

The present invention relates to a method for producing sophorolipid using coconut oil,

The present invention relates to a method for producing a sophorolipid using palm oil, and more particularly, to a method for producing sophorolipid using palm oil, which comprises culturing yeast in a medium containing coconut oil and glucose, A method for producing sophorolipid containing the sophorolipid, and a method for producing the sophorolipid, which comprises the sophorolipid produced by the above method, as an active ingredient, and a composition for cleaning.

Parcel Lori feed (Sophorolipid) Candida is a spring rain Cola (Candida bombicola , C. apicola , C. batistae , Wickerhamiella is a glycolipid-type surface-active material synthesized by some yeast species, such as domericqiae and Rhodotorula bogoriensis (Konishi et al. , J. Oleo. Sci. 57 , 359-369, 2008). The annual consumption of sophorolipid is estimated at about 10 million tonnes and is mainly used as a household / laundry detergent, but has some potential applicability in the chemical, textile, food, paper and cosmetic industries (Van Bogaert et al ., Appl. Microbiol. Biotechnol. 76 (1), 23-34, 2007).

Commercialization of Sophorolipid-based products is rapidly taking up the market share of herbal cosmetics. The company Soliance (http://www.groupesoliance.com) and MG Intobio Co. of Korea Ltd has been producing Sophorolipid-based cosmetics and skin care products. Since soporolipids have antibacterial properties against Propionibacterium acnes and Corynebacterium xerosis as well as surfactant properties and exhibit low cytotoxicity, they can be used in cosmetic formulations, It becomes a better emulsifier than surfactant. In addition, various advantages such as anticarcinol of soporolipid, promotion of dermal fibroblast metabolism, free radical elimination, epidermal layer exfoliation and whitening, hair protection and sanitization have been reported to be caused by pharmacodermatological (Shao et al. , J. Surg. Res. 173 (2), 286-291, 2012).

Of the individuals producing sporroripizide, candida bombicola is the highest in terms of production. In fact, Candida spring rain coke (C. bombicola) feeds the parcels Lowry synthesized by a mixture of molecules with diversity in the structural aspects, such as some types, lactonization (lactonization) and acetylation pattern of fatty acids. This diversity has also been reported to affect the hydrophobicity of the sophorolipid molecules (Van Bogaert et al. , Process Biochem. 46 (4), 821-833, 2011). In nature, the sophorolipid molecules are synthesized as a mixture of acidic and lactonic forms and show very large structural diversity (Kim et al. , Bioresour. Technol. 100 (23), 6028-6032, 2009). It has been reported that the biological activity of macromolecular compounds such as sophorolipid is class-dependent and the size, structural anomalies and purity of the compounds also contribute significantly to their role (Morya et < RTI ID = 0.0 > al . , Appl . Microbiol . Biotechnol . 93 (1), 71-82, 2012).

The integrated, de-novo, and bioconversion production of the sophorolipid by candida bovis cola is an additional benefit for choosing it as a producer. The products of de-novo synthesis are mainly derivatives of palmitic acid, stearic acid and oleic acid (Van Bogaert et al. , Appl. Microbiol. Biotechnol. 76 (1), 23-34, 2007). Biological conversion of the oil for the production of the sophorolipid is carried out by the individual as a co-substrate of glycerol in the oil after the synthesis of the de novo, and the fatty acid is used as a raw material (Kim et al. Bioresour, Technol., 100 (23), 6028-6032, 2009). Thus, the oil can be used as nutrients as well as raw materials during the production of the sophorolipid.

Over the years, a variety of vegetable oils have been screened for the production of soporolipids with a variety of biological activities (Kim et al. , Bioresour. Technol. 100 (23), 6028-6032, 2009). The production variability of many substrates used has been a major constraint on the industrial application of sophorolipid as a surfactant. In previous studies, a variety of substrates have been used for intermediate chain sophorolipid production, but many restrictions have not been applied to industrial processes. In 1998, Brakemeier and colleagues used secondary alcohols (C12 and C14) as substrates with glucose (carbon source) as an alternative method to produce intermediate chain sophorolipids (Brakemeier et al. , Appl. Microbiol. Biotechnol., 50, 161-166, 1998). However, the method has not been adopted in the industry due to the high cost of secondary alcohols (Van Bogaert et al. , Process Biochem. 46 (4), 821-833, 2011).

A number of substrates and culture conditions have been attempted to acquire new properties of the soporolipid and to extend the application (Kim et al. , Bioresour. Technol. 100 (23), 6028-6032, 2009) Or a higher fatty acid. It has been reported that the surfactant ability of the sophorolipid is mainly determined by the fatty acid chain length and the respective strains, i.e., the unsaturated bonds and the side groups, and the long-chain length of the fatty acid domain limits the bioavailability and also the biological properties (Van Bogaert et al. , Process Biochem. 46 (4), 821-833, 2011). On the other hand, medium or small sized fatty acids are easily metabolized through β-oxidation, but the yield of sophorolipid is low during fermentation. Therefore, optimization of fermentation conditions to solve these problems remains a major challenge for researchers (Van Bogaert et al. , Process Biochem. 46 (4), 821-833, 2011).

Palm oil, on the other hand, contains mainly lauric acid (CH ₃ (CH ₂ ) ₁₀ COOH) as a major fatty acid component. More than 90% of palm oil fatty acids have been reported to have saturated fatty acids (iodine values 7 to 12) with the lowest risk of oxidative damage. Palm oil is one of the most abundant sources of medium chain fatty acids (MCFA). In addition, palm oil has been reported to contain 47.5% of lauric acid (C12) and 18.1% of myristic acid (C14) in total fatty acids (Gordon and Rahman, J AOCS 68 (8), 574-576, 1991) .

The present invention uses palm oil as a substrate for the production of sporeolipids in the medium chain to solve the biological activity and yield problems in the production of the sophorolipid. The fermentation conditions (pH, temperature, The experiments were carried out to design and establish. In addition to these fermentation conditions, the surface properties, antibacterial properties and molecular weight of the sophorolipid have been further studied.

Korean Patent No. 0569238 discloses a method for producing a soporolipid using soybean oil by-product, and Korean Patent No. 1125189 discloses a method for producing an anti-inflammatory sophorolipid. However, as described in the present invention, the production method of the sophorolipid using palm oil has not been disclosed.

The present invention has been made in view of the above-mentioned needs, and the present inventors have found that when a Candida bombicola strain is cultured in a culture medium containing coconut oil and glucose, it contains C12 and C14 fatty acids (Sophorolipid) is produced efficiently, the present invention has been completed.

In order to solve the above problems, the present invention provides a method for producing sophorolipid comprising culturing yeast in a culture medium containing coconut oil and glucose and extracting sophorolipid from the culture medium, And a method for producing the same.

The present invention also provides an antimicrobial composition and a cleaning composition containing the sophorolipid produced by the above method as an active ingredient.

Using the method of producing a sophorolipid by culturing a Candida bombicola strain in a medium containing coconut oil and glucose according to the present invention, , Which can produce a large amount of a microporous lysozyme exhibiting antimicrobial activity against Staphylococcus aureus and Escherichia coli .

Figure 1 shows the fermentation conditions for producing sophorolipid from palm oil in fed-batch cultivation using Candida bombicola (ATCC 22214) strain.
Fig. 2 shows the result of measuring the foaming power and stability. The foam height was measured immediately after and 5 minutes after the foaming of the linear alkylbenzenesulfonate (LAS), sugar ester, palm oil derived sophorolipid and corn oil based sophorolipid.
Fig. 3 shows the results of measuring the surfactant properties and emulsifying power of the soporolipid from palm oil and corn oil. LAS and sugar ester were used as control. (A) Minimum surface tension and CMC curve, (B) Dispersion force, (C) Cleaning force, and (D) Emulsion force measurement results. LAS: Linear alkylbenene sulphonate, Sugar ester: Sugar ester, SL: Sophorolipid, JIS: Composite contaminated cloth, AS-10: Stained contaminated cloth (CFT AS-10) and WFK 10D:
Fig. 4 shows antimicrobial activity of sapororipid derived from palm oil and corn oil. (A) Antibacterial activity against E. coli , (B) Antimicrobial activity against S. aureus . SL: Sophorolipid, DCF-100: Grape seed extract.

In order to accomplish the object of the present invention, the present invention relates to a method for producing a microorganism which comprises culturing yeast in a culture medium containing coconut oil and glucose, and extracting the microorganism from the culture broth sophorolipid. < / RTI >

In a method according to an embodiment of the present invention, the yeast may be Candida bombicola strain, but is not limited thereto.

In the culture method according to an embodiment of the present invention, the culture may be a two-step batch cultivation, more preferably 14 to 16 g / L of coconut oil and 8 to 12 g / L of glucose at a pH of 4.8 to 5.2 and culturing the yeast at a pH of 3.4 to 3.6 containing 14 to 16 g / L of palm oil and 95 to 105 g / L of glucose Producing a sophorolipid in a medium, more preferably a medium of pH 5.0 containing 15 g / L of coconut oil and 10 g / L of glucose, And culturing the yeast in a medium at a pH of 3.5 containing 15 g / L of palm oil and 100 g / L of glucose. It is not.

The culture conditions of the 2-step batch culture were inoculated with 4 to 6% (v / v) inoculum of the total medium volume for growth of the strain, and the agitation speed was 150 to 250 rpm, the culture temperature was 24 to 32 ° C, Culturing the cells at a rate of 0.4 to 1.2 vvm, and culturing the cells at a stirring speed of 150 to 250 rpm, a culture temperature of 24 to 32 ° C, and a vigorous growth rate of 0.4 to 1.2 vvm for the production of the sophorolipid, Culturing the cells at a stirring speed of 200 rpm, a culture temperature of 30 캜 and a vigorous growth rate of 1.0 vvm, inoculating a 5% (v / v) volume of the seed medium, and culturing the cells at a stirring speed of 200 rpm, Lt; / RTI > vvm, but not limited thereto.

In addition, in the culture method according to an embodiment of the present invention, the culture may be fed-batch cultivation, more preferably 8-12 g / L of coconut oil, After culturing the yeast in a medium with a pH of 4.5 to 5.0 containing 12 g / L of glucose, palm oil and a glucose of 8 to 12 g / day / L ratio were maintained at a ratio of 13 to 16 g / day / L and more preferably 10 g / L of coconut oil and 10 g / L of glucose, in a medium in which the pH is adjusted to 3.4 to 3.6. After the yeast was cultured in a medium of pH 4.8, the cells were cultured in a fermentation culture to produce a soporolipid in a medium of 15 g / day / L of palm oil and 10 g / day / L of glucose maintained at a pH of 3.5 But is not limited thereto.

The culture conditions of the above-mentioned fed-batch culture were inoculated with 1.0-6.0% (v / v) of the total medium volume for growth of the strain, and the agitation speed was 150-250 rpm, the culture temperature was 24-32 ° C, vvm for 2 to 4 days and then cultured for 12 to 14 days at a stirring speed of 150 to 250 rpm, a culture temperature of 24 to 32 ° C and an expansion rate of 0.4 to 1.2 vvm for the production of sophorolipid , Preferably 5% (v / v) of the total medium volume is inoculated and cultured for 3 days at a stirring speed of 150 rpm, a culture temperature of 30 ° C and a vigorous 0.4 vvm, At a rate of 250 rpm, at a culture temperature of 27 ° C, and at an expiratory flow rate of 1.0 vvm for 13 days, but the present invention is not limited thereto.

The culture medium for the production of the above-described soporolipidem and for the production of the sophorolipid is, for example, YM medium, LB medium, Trypticase Soy Agar medium, BHI (Brain heart infusion) medium, TBS ) Medium, Sabouraud Dextrose (SD) medium and MRS (de Man, Rogosa and Sharpe) medium can be used. If the medium induces normal soporolipid production without adversely affecting the growth of the strain, Do not. In addition, the culturing of the microorganism producing microorganism can be carried out by any of the conventional methods used for culturing microorganisms.

In a method according to one embodiment of the present invention, the sophorolipid can be a sophorolipid including C12 fatty acids and C14 fatty acids, preferably 52-55% C12 fatty acids and 20-23% C14 fatty acids , More preferably about 53% C12 fatty acid and about 21% C14 fatty acid, but is not limited thereto.

The present invention also provides an antimicrobial composition containing the sophorolipid produced by the above method as an active ingredient.

In one embodiment of the invention, the soporolipid may have, but is not limited to, antimicrobial activity against Staphylococcus aureus and Escherichia coli .

The term " antimicrobial " means the ability to reduce, prevent, inhibit, or eliminate the growth or survival of a microorganism at a particular concentration. The antimicrobial composition may contain, as an active ingredient, a cultured Candida albicans strain culture broth according to the present invention or a sophorolipid extracted from the culture broth, and may be added to the composition in a solution or other form. In addition, suitable excipients or carriers may be further included within the scope of not adversely affecting the antimicrobial properties of the soporolipid.

In addition, the present invention provides a cleaning composition containing the sophorolipid produced by the above method as an active ingredient. The cleaning composition may contain, as an active ingredient, a culture medium of Candida viridis cola strain according to the present invention or a sophorolipid extracted from the culture solution, characterized in that it is free of bubbles, and may be added to a composition in a solution or other form . In addition, a suitable carrier or carrier may be further included within the scope of not adversely affecting the cleaning ability of the sophorolipid.

Excipient means a compound, component or structure that is combined with the < RTI ID = 0.0 > sophorolipid < / RTI > of the present invention and refers to a compound, component or structure that assists in or facilitates the manufacture of the composition and is suitable for storage, administration, delivery, Lt; / RTI > That is, excipients may be selected to minimize degradation of the active ingredient and minimize additional side effects. Examples of suitable aqueous and non-aqueous vehicles, diluents and solvents include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, etc.), vegetable oils and suitable mixtures thereof.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Materials and methods

1. Strain, medium and culture conditions

Candida bombicola (ATCC 22214) strain was used to produce sporolipid . Candida bovis cola cryopreserved at -70 ° C was recovered in YM broth medium (3.0 g / L yeast extract, 3.0 g / L malt extract, 5.0 g / L peptone and 1.0 g / L glucose) At 250 rpm for 24 hours. A portion of this culture broth was transferred to the production medium and the remainder was preserved for further cell storage. The composition of the production medium is as follows: 100 g of glucose, 5.0 g of yeast extract, 1.0 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 peptone and 10% (w / v) vegetable oil. All chemicals were purchased from Sigma-Aldrich. The culture medium and components were purchased from Difco and Merck respectively, and palm oil and corn oil were purchased from Shindongbang Co. (Republic of Korea).

In the present invention, the inoculum size of the soporolipid producing strains was 5% (v / v) of the total production medium volume. The fermentation was carried out in a 30 L fermenter (Kobiotech, Korea) with a working volume of 15 L. The conditions for the batch culture used in the present invention were a temperature of 28 DEG C, a pH of 3.5, a stirring speed of 150 rpm, a wake rate of 0.5 vvm and a culturing period of 3 to 8 days. The degree of oxygen saturation was adjusted to 20% by changing the stirring speed. If necessary, the culture pH was titrated with 6 N NaOH. Samples were taken from the culture at 1 day intervals for further analysis.

2. Fatty acid composition

The fatty acid composition of the oil and each of the sophorolipids was determined by gas chromatography equipped with a flame ionization detector (GC-FID, HP 6890, HP, USA) using standard methods reported by Kim et al . Technol., 100 (23), 6028-6032, 2009).

3. Optimization of culture conditions and hydrophilic and lipophilic carbon source

Optimization of the glucose consumption rate for maximum production of the sophorolipid has been achieved through two-step batch fermentation. During the growing period (0-72 hours), 10 g / L of glucose was supplemented to the standard medium for proper cell growth. After optimal cell growth, varying concentrations of glucose (10-200 g / L) were supplemented in different media with 15 g / L palm oil each during the production (72-96 hours). The culture was carried out at a stirring speed of 250 rpm, an initial pH of 5.0, a culture temperature of 25 ° C, a culture volume of 50 mL in a 250 mL flask, and a culture period of 4 days (3 days of growth and 1 day of production). After incubation, the culture was collected and analyzed for residual oil, glucose, sophorolipid content and dry biomass.

4. Extraction of residual oil and extraction of the soporolipid

The residual oil was separated from the culture filtrate by extraction with n-hexane (Shin et al. , Bioresour. Technol. 101 (9), 3170-3174, 2010). The upper non-aqueous layer containing vegetable oil was separated using a separatory funnel. The separated oil was recovered after evaporating n-hexane using a rotary vacuum evaporator (Eyela, Japan) and weighed. In the aqueous layer, the same volume of ethyl acetate was used to extract the soporolipid three times. The crude sophorolipid was collected by evaporation of ethyl acetate using rotary vacuum evaporator (Eyela, Japan) and weighed.

5. Measurement of Residual Glucose and Dry Cell Weight

After extraction of the sophorolipid and residual oil, the remaining culture was evaporated in a rotary vacuum evaporator (Eyela, Japan) to remove traces of solvent. The aqueous culture broth was centrifuged at 7000 xg for 15 minutes. The residual glucose was estimated from the supernatant, and the pellet was dried for estimation of biomass and then weighed (Kim et al. , Bioresour Technol. 100 (23), 6028-6032, 2009). Glucose levels were investigated using Accu-Check (Loche) to immediately supply deficient glucose during incubation and reaffirmed via HPLC (Aminex HPX-87H column; mobile phase, 0.01 NH ₂ SO ₄ , flow rate 1.0 mL / min) Respectively.

6. Feed rate control

Fed-batch cultivation was carried out in a 30 L industrial fermenter (Kobiotech, Korea) with a culture volume of 15 liters. The bacterial strains were inoculated in a steam sterilized, suitably stable medium at a ratio of 1: 100 (inoculum: medium). Initial fermentation conditions were carried out at a stirring rate of 130 rpm for 3 days at a culture temperature of 30 ° C and a vigorous growth rate of 0.3 vvm (growth period). The degree of oxygen saturation was adjusted to 30% by changing the stirring speed. After 3 days of growth, the final culture conditions were set at a stirring speed of 250 rpm, a culture temperature of 27 ° C, and an expansion rate of 1.0 vvm for 13 days. Samples of the culture medium were periodically taken from the fermentation broth and analyzed for changes. The fed medium contained 10% glucose and 15% palm oil in final concentrations during the production of the sophorolipid. To control foaming, 10% palm oil was added at the beginning of the culture and the pH was maintained at 3.5 using 6N NaOH.

7. Measurement of surfactant properties and emulsifying power

The surface tension of the extracted sophorolipid was measured by the du Nuoy method (model 21, Fisher Scientific, Pittsburgh, Pa., USA) using a platinum iridium ring (circumference 5.935 cm) (Ma et al. , J. Colloid. Interface. Sci. 376 (1), 165-172, 2012). The surface tension of the sophorolipid at various concentrations (0-300 ppm) contained in distilled water was measured and the solution was allowed to react for 30 minutes before being used for surface tension measurements. The critical micelle concentration (CMC), which corresponds to the threshold of minimum surface tension, was measured at 25 ° C using the same tensionmeter as above, with the concentration at which the sophorolipid begins to form micelles. CMC and minimum surface tension values were obtained from the relationship curve between the sophorolipid concentration and the surface tension of the corresponding concentration (Ma et al. , J. Colloid. Interface. Sci. 376 (1), 165-172, 2012).

Dispersion power was measured according to a standardized protocol (Ma et al. , J. Colloid. Interface. Sci. 376 (1), 165-172, 2012).

The foaming power was measured at 50 DEG C by Ross and Miles method according to the international standard ISO 696 (Urum and Pekdemir, Chemosphere. 2004 Dec; 57 (9), 1139-1150, 2004).

The cleansing power was measured by standard procedures (Shin et al. , Bioresour. Technol. 101 (9), 3170-3174, 2010), and briefly, oleic acid, triolein, cholesterol oleate, paraffin wax, squalene, cholesterol, Composite contaminated cloth (JIS) contaminated with gelatin, clay and carbon black; Dirt stained with pigment, oil, milk (CFT AS-10); And oil, contaminated with pigment and sebum (WFK 10D). The reflectance of the fabric (contaminated cloth) was measured using a standard procedure, and the rinsing power was calculated by the following formula (Shin et al. , Bioresource Technol. 101 (9), 3170-3174, 2010).

Cleaning power P (%) = {(RR ₀ ) / (65-R ₀ )} 100

R _o represents the reflectivity before cleaning, R represents the reflectance after cleaning, and P represents the relative cleaning power.

The emulsifying power of the sophorolipid to soybean oil was measured using standard methods. Briefly, 2.0 mL of 0.1% (w / v) soporolipid solution was mixed with 2.0 mL of phosphate buffer (200 mM, pH 7.0) along with 2.0 mL of soybean oil and vortexed for 1 minute. The emulsification power was measured at 5 minutes intervals by measuring the absorbance at 540 nm (Shin et al. , Bioresource Technol. 101 (9), 3170-3174, 2010).

8. Antimicrobial activity

The antimicrobial activity of sophorolipids against Staphylococcus aureus and Escherichia coli was determined according to the method described by Shin et al. (Shin et al. , Bioresour. Technol. 101 (9), 3170-3174 , 2010). DF-100 (grape seed extract) was used as a standard (Defer et al. , Aquaculture. 293 (1-2), 1-7, 2009).

9. HPLC and LC-MS analysis of the sophorolipid

The extracted soporolipid from palm oil was dissolved in ethanol and analyzed by analytical HPLC using a 4.6 mm x 150 mm Spherisorb ODS2 column (Waters Inc). The acetonitrile / water mixture was used as a mobile phase at a flow rate of 0.5 mL / minute with an acetonitrile gradient from 60% to 80% for 35 minutes followed by an acetonitrile gradient from 40% to 60% for 15 minutes. The injection volume was 25 ㎕, and the eluent was measured with a UV detector at 195 nm.

The LC-MS system (HP 1100 series with UV-Visible diode-array detector-Bruker's Esquire electrospray MSn analyzer) was used to isolate and characterize the structure of each of the sophorolipid components. The mobile phase used is acetonitrile / H ₂ O (8/2, v / v). Sophorolipids separated by HPLC were analyzed via a mass spectrometer (Tran et al. , Bioresor. Technol. 115, 84-87, 2012). Molecular ions were collected in the ion trap and mass / charge (m / z) values were detected. Ions in the ion trap can be separated and can be further divided by collision with helium molecules. The molecular structure is shown from LC-MS data as described in the prior art (Tran et al. , Bioresor. Technol. 115, 84-87, 2012).

10. Statistical Analysis

The experiment was repeated three times and the results were averaged together with the standard deviation (SD) indicated by the error bars in the graph.

Example 1. Analysis of fatty acid residues

The GC-FID results showed that the palm oil used in the present invention contained 49.83% C12 and 17.55% C14. In addition, the sophorolipid produced from palm oil contained 53.27% C12 and 21.54% C14. On the other hand, C12 and C14 fatty acids were not present in the soporolipids from corn oil and corn oil used as a control (Table 1). These results suggest that the sophorolipids from palm oil are mainly derived from lauric acid (C12) and myristic acid (C14) derivatives. In addition, the yield of C12 sophorolipid derived from palm oil produced in the present invention was high enough to be purified. However, previous methods using n-alkanes and glucose as substrates for certain types of sophorolipid production have been low yields and very expensive to produce (Van Bogaert et al. , Appl . Microbiol . Biotechnol . 76 23-34, 2007).

Fatty acid content analysis (%) fatty acid C8 C10 C12 C14 C16 C18 C18: 1 C18: 2 C18: 3 Palm oil 8.66 6.95 49.83 17.55 8.81 0.94 5.76 1.51 0.00 Sophorolipid (palm oil) 3.01 4.47 53.27 21.54 9.51 0.00 8.20 0.00 0.00 Corn oil 0.00 0.00 0.00 0.00 13.64 4.41 25.58 51.96 4.41 Sophorolipid (corn oil) 0.00 0.00 0.00 0.00 7.41 0.79 27.00 64.80 0.00

Example 2. Optimization of culture conditions and hydrophilic and lipophilic carbon sources

During two-step batch fermentation, the production of the soporolipid with the Candida bombicola (ATCC 22214) strain was observed to be optimal at a glucose concentration of 100 ± 5 g / L, g / L of the glucose concentration. Regular monitoring and supplementation of glucose was carried out for optimal production of the sophorolipid, and the neutralization of palm oil-derived fatty acids was controlled with 6 N NaOH. The pH during the strain growth was maintained at about pH 5 ± 0.1 for optimal growth. As the pH of the medium was found to be greatly reduced during the sophorolipid producers and pH 3.5 was confirmed to be an optimum pH suitable for the production of the sophorolipid, 6 N NaOH was used during the incubation for the production of the sophorolipid The pH of the medium was maintained at 3.5.

The cell growth of Candida bovis cola reached a stopper after 48 hours of incubation, and the dry cell weight was about 16 g / L. However, full-fledged production of the sophorolipid was carried out from the middle of the exponential phase (after 4 days of culture) and was continuously produced during the production period.

Example 3. Feed-rate control Fed-batch cultivation < RTI ID = 0.0 >

The present invention is designed to produce the largest possible sophorolipid using palm oil and glucose under fed-batch culture conditions. As shown in Fig. 1, the fed-batch cultivation was performed using an initial glucose concentration of 10 g / L and palm oil of 10 g / L, and the feeding rate control fed-batch culture was almost doubled to the stationary phase. As a result, 54 g / L of sophorolipid was obtained after 10 days of fermentation. Since the viscosity of the culture broth after fermentation became very high, it was impossible to carry out fermentation for more than 10 days.

The supply of palm oil has been limited for the protection of metabolism and for the production of sophorolipids. Consumption of palm oil was associated with the pH control of the medium, which was controlled with 6 N NaOH.

Example 4. Surface activity and emulsifying power

The results of the measurement of the surfactant properties and the emulsifying force of the palm oil-derived sophorolipid are shown in Fig. In both cases, LAS (Linear Alkylbenzen Sulfonate) and sugar ester were used as controls.

As a result of measuring the foaming power and stability using the bubble generation force measuring device, the sophorolipid derived from palm oil and corn oil showed a relatively lower foamability as compared with LAS and sugar ester. The bubble stability was also found to be very low as measured by the foam height after 5 minutes. The foaming power of the sophorolipid is known to decrease as the carbon chain length of the fatty acid chain increases. The soporolipid from corn oil has a slightly higher foaming power than the cocoyl-derived soporolipid (Fig. 2).

In addition, it is well known that the foaming power of the surfactant solution increases at low surfactant concentrations until the critical micelle concentration (CMC) is reached (Montufar et al. , Mater. Sci. Eng: , 1498-1504, 2011). Therefore, calculation of CMC and minimum surface tension is important. The surface tension of the palm oil and corn oil-derived sophorolipid was increased from about 70 dyn / cm to 25-35 dyn (75 ppm for palm oil-derived sophorolipids and 100 ppm for corn oil-derived sophorolipids) / cm < / RTI > (Figure 3A). The minimum surface tension of palm oil derived sophorolipid was slightly lower than that from corn oil. This indicates that the palm oil-derived sophorolipid is smaller in size but higher in hydrophobicity than the corn oil-derived soporolipid.

The CMC values of the sophorolipid varied from 20-35 mg / L and the two sophorolipids exhibited a significantly lower CMC than the standard surfactants (LAS and sugar ester) (data not shown).

The dispersing ability of palm oil and corn oil based sophorolipid showed better than LAS. The palm oil derived sophorolipid showed a higher dispersing power than the corn oil-derived soporolipid (Fig. 3B).

The contaminated foam washing test showed that the soporolipid from palm oil and corn oil had lower detergency than LAS and sugar ester. Palm oil and corn oil-derived soporolipid showed almost similar cleansing power, especially clearing power against oily contaminants (WFK 10D) (FIG. 3C).

Along with the surfactant properties, good emulsifying power is also an important consideration for bio-surfactants applied in the industry. The sophorolipid obtained from palm oil has lower emulsifying power than the standard materials (LAS and sugar ester) (Figure 3D). The emulsifying activity of the surfactant molecule was measured not only by surface tension, but also by direct interaction of the hydrophobic substrate with the hydrophobic part of the molecule (Shin et al. , Bioresour. Technol. 101 (9), 3170-3174, 2010).

Example 5. Antibacterial activity

The antimicrobial efficacy data of palm oil and corn oil based sophorolipid against E. coli and S. aureus are shown in FIG. The antimicrobial activity of sophorolipid against Escherichia coli was found to be higher than that of DF-100 (grape seed extract), while the sophorolipid synthesized from palm oil showed similar activities to those of corn oil-derived sophorolipid. However, antimicrobial activity against Staphylococcus aureus was higher than that of corn oil - derived soporolipid.

The antimicrobial activity of the soporolipids against the bacteria may encourage the use of sophorolipid for screening for other pathogenic bacteria and may also be used as a bactericide suitable for the washing of fruits and vegetables.

Example  6. Sophorolipid HPLC  analysis

Detailed analysis of the form of the sophorolipid was performed by HPLC analysis, and the HPLC results showed several peaks (data not shown). As shown in Table 1, the sophorolipid produced from palm oil is a compound containing lactone and acid sophorolipid. The HPLC data showed peaks between 4-7 min with acid sophorolipid binding and 10-20 min with lactone sophorolipid binding, with a larger peak area of lactone sophorolipid. The palm oil derived sophorolipid has a short chain length but a high hydrophobicity due to the high proportion of lactone form in the partially purified sophorolipid. Acetylation as well as lactonization also contribute to the high hydrophobicity of the molecule.

Claims (10)

Culturing the yeast in a culture medium containing coconut oil and glucose; And
And extracting the sophorolipid from the culture liquid. The method according to claim 1, wherein the yeast is a Candida bombicola strain. 2. The method of claim 1, wherein the culture is a two-step batch cultivation or fed-batch cultivation. 4. The method according to claim 3, wherein the two-stage batch culture is carried out at a stirring speed of 150-150 ° C in a medium containing 14-16 g / L of coconut oil and 8-12 g / L of glucose at a pH of 4.8-5.2, Culturing the yeast at 250 rpm, at a culturing temperature of 24 to 32 ° C and an aeration rate of 0.4 to 1.2 vvm and culturing the yeast at pH 3.4 to 3.6 containing 14 to 16 g / L of palm oil and 95 to 105 g / L of glucose In a culture medium comprising a medium of agar medium at a stirring speed of 150 to 250 rpm, a culture temperature of 24 to 32 ° C, and an expansion rate of 0.4 to 1.2 vvm. 4. The method according to claim 3, wherein the fed-batch culture is performed at a stirring speed of 150-250 m in a medium of pH 4.5-5.0 containing 8-12 g / L of coconut oil and 8-12 g / L of glucose , Yeast was cultured for 2 to 4 days at a culture temperature of 24 to 32 ° C and an expansion rate of 0.4 to 1.2 vvm. Then, coconut oil at a ratio of 13 to 16 g / day / L and glucose at a rate of 8 to 12 g / day / In a medium having a pH adjusted to 3.4 to 3.6, with a stirring speed of 150 to 250 rpm, a culturing temperature of 24 to 32 ° C and an exposing rate of 0.4 to 1.2 vvm for 12 to 14 days to obtain a sophorolipid . The process of claim 1, wherein the sophorolipid comprises 52-55% C12 fatty acids and 20-23% C14 fatty acids. 7. An antimicrobial composition comprising a sophorolipid produced by the method of any one of claims 1 to 6 as an active ingredient. The antimicrobial composition according to claim 7, wherein the sophorolipid has antimicrobial activity against Staphylococcus aureus and Escherichia coli . A cleaning composition comprising as an active ingredient a sophorolipid produced by the method of any one of claims 1 to 6. A cleaning composition comprising, as an active ingredient, a bubble-free sophorolipid produced by the method of any one of claims 1 to 6.

Images