Method for purifying modified sophorolipid

Abstract

The invention discloses a method for purifying modified sophorolipid, which comprises the following steps: adding a salt solution into lactone type crude sophorolipid obtained by fermenting candida bumblebee, stirring, standing, and collecting a lower layer sophorolipid phase; according to the method, the produced crude sophorolipid with low or high emulsification degree is purified, impurities are removed, good appearance conditions are achieved, and the appearance requirement of the commodity sophorolipid is met. And meanwhile, the sophorolipid is modified in the purification process by utilizing the chemical properties of different salts, so that the decoloration of the sophorolipid is realized, the concentration of the lactone type sophorolipid is improved, and the stability of the lactone type sophorolipid is improved at the same time.

Description

Technical Field

[0001] The invention belongs to the technical field of sophorolipid preparation, and particularly relates to a method for purifying modified sophorolipid. Background Art

[0002] Sophorolipids, as glycolipid biosurfactants, are usually produced by the metabolism of microorganisms such as bumblebee Candida albicans. They have the advantages of low toxicity, multiple biological activities, excellent surface properties, and biodegradability. They are in line with the general direction of green and sustainable development. Their good anti-inflammatory and antibacterial activities, antiviral and anticancer activities, and excellent surface activity make them have huge application potential and broad application prospects in many fields such as personal care and cosmetics, medicine, food, oil extraction, and agriculture.

[0003] The sophorolipids produced by fermentation of Candida albicans are mainly lactone-type, and can be found as the third phase below the oil phase and the water phase during the fermentation process. After the bacteria are inactivated, the lactone-type sophorolipids produced can be obtained by liquid separation, which provides great convenience for separating the produced sophorolipids, but their appearance is turbid and the color is darker. It does not meet the appearance requirements of commercial sophorolipids. And the lactone-type sophorolipids are not stable enough. Summary of the invention

[0004] The purpose of this section is to summarize some aspects of embodiments of the present invention and to briefly introduce some preferred embodiments.

[0005] As one aspect of the present invention, the present invention provides a method for purifying modified sophorolipids, comprising adding a salt solution to a lactone-type crude sophorolipid obtained by fermentation of Candida bombus, stirring, and collecting a lower sophorolipid phase after standing;

[0006] Wherein, the salt solution includes sodium chloride and / or sodium sulfite solution, and the mass concentration of the salt solution is 10% to 30%.

[0007] As a preferred embodiment of the method for purifying modified sophorolipids according to the present invention: it also includes collecting the lower sophorolipid phase and further extracting and decolorizing: adding acetate and acid solution to the lower sophorolipid phase, stirring and then standing, and taking out the sophorolipid phase; wherein the mass concentration of acetate is 8-16%, and the mass concentration of acid is 8-16%.

[0008] As a preferred embodiment of the method for purifying modified sophorolipids of the present invention: the method for preparing the lactone type crude sophorolipids comprises fermenting and culturing Candida bombus in a culture medium containing vegetable oil, allowing to stand for stratification after sterilization, and collecting the crude sophorolipids in the lower layer.

[0009] As a preferred embodiment of the method for purifying modified sophorolipids of the present invention, it further comprises adding hot water to the crude sophorolipids in the lower layer, stirring, standing for stratification, and collecting the crude lactone sophorolipids in the lower layer.

[0010] As a preferred embodiment of the method for purifying modified sophorolipids according to the present invention: the salt solution consists of an aqueous solution of sodium chloride and sodium sulfite.

[0011] As a preferred embodiment of the method for purifying modified sophorolipids of the present invention: the mass concentration of sodium chloride is 10-15%; the mass concentration of sodium sulfite is 10-15%.

[0012] As a preferred embodiment of the method for purifying modified sophorolipids according to the present invention: the stirring temperature is 60-90°C.

[0013] As a preferred embodiment of the method for purifying modified sophorolipids according to the present invention: the acetate includes sodium acetate, and the acid includes citric acid.

[0014] As a preferred embodiment of the method for purifying modified sophorolipids of the present invention: the stirring is followed by standing, the stirring temperature is 60 to 90° C., and the standing time is 4 to 14 hours.

[0015] The beneficial effects of the present invention are as follows: the present invention purifies crude sophorolipids with a low or high degree of emulsification, removes impurities and achieves good appearance conditions, meeting the appearance requirements of commercial sophorolipids. At the same time, the chemical properties of different salts are utilized to modify sophorolipids during the purification process: decolorization of sophorolipids is achieved, the concentration of lactone sophorolipids is increased, and the stability of lactone sophorolipids is improved at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solution of the embodiment of the present invention, the following briefly introduces the drawings required for describing the embodiment, wherein:

[0017] Figure 1 It is a crude sophorolipid after static separation.

[0018] Figure 2 The sophorolipid prepared in Example 1.

[0019] Figure 3 The sophorolipids prepared in Example 2 and Research Example 1.

[0020] Figure 4 This is the sophorolipid after static separation in Control Example 1.

[0021] Figure 5 This is the sophorolipid of control example 2 after static separation. DETAILED DESCRIPTION

[0022] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with specific embodiments.

[0023] The crude sophorolipids (INC1 name is glycolipid or glycolipid or Candida albicans / glucose / rapeseed oleic acid methyl ester fermentation product) used in the present invention are obtained by fermentation with Candida albicans bumblebee (ATCC 22214) and only the crude sophorolipids product in which the bacteria are inactivated is completed.

[0024] The method for preparing crude sophorolipids is a conventional method, and an example of the method for preparing crude sophorolipids is as follows:

[0025] (1) Bacterial culture:

[0026] Activate the strain, pick the strain and streak it on the YPD solid medium plate / test tube slope, culture it at 30℃ for 48h, and obtain a single colony.

[0027] (2) Seed liquid shake flask culture:

[0028] Pick a single colony from the plate and inoculate it into 50mL YPD liquid medium, and culture it in a shaking incubator at 30℃ and 220rpm for 48h. Inoculate the above strain culture solution into 100mL liquid medium at a 5% (v / v) inoculation amount, and culture it in a shaking incubator at 30℃ and 220rpm for 48h. Add 3.75g rapeseed oil as fermentation raw material. Ferment in a shake flask for 9d, and adjust the pH to about 3.5 with 1mol / L NaOH every 24h.

[0029] (3) Fermentation tank culture:

[0030] The liquid volume of the 5L fermentation tank is 4L, the speed is controlled at 350-450rpm, the temperature is maintained at 30℃, and the ventilation volume is maintained at 3-6L / min. The pH is controlled to be maintained at 5.5-6.0 for 16 hours before fermentation, and the pH is maintained between 3.5 and 3.8 after 16 hours. When the residual sugar concentration is lower than 30g / L, solid glucose is added to more than 30g / L; when the residual oil concentration is lower than 20g / L, rapeseed oil is added to more than 20g / L. No more feeding is performed at the end of fermentation to allow the hydrophilic and hydrophobic carbon sources to be naturally consumed.

[0031] (4) Bacterial inactivation:

[0032] After the fermentation is completed, the temperature of the fermenter is raised to 60°C, sterilized for 30 minutes, the mechanical stirring is turned off and the ventilation is stopped. All the fermentation liquid is collected in a separatory funnel, and after standing overnight for stratification, the sophorolipid at the bottom is separated.

[0033] (5) Hot water extraction purification: Take 100 g of the above-mentioned sophorolipid, add 100 g of boiling water, mix, place in an 80°C water bath and stir at 100 rpm for 30 min. After standing overnight, the system is separated into three phases (see Figure 1 ), take out the lower layer of sophorolipid phase, weigh and obtain 110.5g of crude sophorolipid in this phase. The crude sophorolipid solution is opaque and turbid. Figure 1 .

[0034] Embodiment 1:

[0035] S1) Extraction and purification with aqueous NaCl solution: 100 g of the crude sophorolipid obtained in step (5) above was taken, 100 g of boiling water was added thereto, and then 30 g of NaCl was added, the mixture was placed in an 80°C water bath and stirred at 100 rpm for 30 min. After standing overnight, the system was separated into two phases, and the lower sophorolipid phase was taken out (see Figure 2 ), weighing to obtain 89.5 g of this phase.

[0036] The solid content SC (Solid content) of the sophorolipid product was determined by the following method: the solid content of the sophorolipid was determined by using an Ohaus rapid moisture analyzer MB90, the temperature was 105°C, the program was automatic, and the moisture content Ww (unit %) was obtained, then the solid content SC = 100% - Ww, and the solid content of the sophorolipid purified in step S1 was measured to be 82.82%. The purification steps of the crude sophorolipid in the control group were: repeating the operation of the above step (5), that is, after two hot water purifications, the solid content of the sophorolipid purified twice by hot water in the control group was measured to be 62.00%. Figure 2 The left side shows the sophorolipid obtained by two hot water purifications in the control group, and the right side shows the sophorolipid obtained in step S1.

[0037] Impurity protein test method: Coomassie Brilliant Blue G-250 protein measurement method. Coomassie Brilliant Blue G250 was used to determine the protein content in the purified sophorolipids. Within a certain protein concentration range (1-1000 μg / g), the absorbance of the solution at a wavelength of 595 nm is linearly related to the protein concentration. A standard curve was drawn based on the standard protein solution, and the protein concentration of the sample was calculated based on the absorbance at a wavelength of 595 nm; then the soluble protein content of the biological sample was calculated according to the formula.

[0038] The standard protein solution is used as the standard, the protein solution concentration is the abscissa (X), and the absorbance is the ordinate (Y) to draw a standard curve, the relationship is Y = 36293.3917X + 0.04188 (mg / g), the correlation coefficient R2 = 0.995, and there is a good linear relationship. The results show that within the concentration range of 0-0.510 mg / g, the Coomassie Brilliant Blue method has a good color development effect, which can be used as an impurity protein quality evaluation curve of the sophorolipid. The sophorolipid impurity protein content obtained by purifying the sophorolipid with NaCl aqueous solution in step S1 is 2.99%, and the sophorolipid impurity protein content of the control group twice purified by hot water is 3.25%.

[0039] Impurity oil test method: Take 10g of sophorolipids, use an equal mass of n-hexane to extract the impurity oil in the sophorolipids, then use a rotary evaporator to evaporate the n-hexane to obtain the impurity oil, and weigh the impurity oil mass m o , then the impurity oil content W o =(m o / 10)*100%, the impure oil content of the sophorolipids obtained by purification with NaCl aqueous solution in step S1 was measured to be 6.50%, and the impure oil content of the sophorolipids obtained by two hot water purifications in the control group was 7.50%.

[0040] The present invention found that using NaCl can effectively demulsify the sophorolipids, thereby making the obtained sophorolipids clear and transparent, and reducing the impurity content of the obtained product. Simply purifying the same batch of sophorolipids twice with hot water cannot obtain clear and transparent sophorolipids (see Figure 2 , left side), while the solid content of the sophorolipid prepared by the purification method of the present invention is significantly increased, and the product is clearer and more transparent, and the impurity content is significantly reduced (see Figure 2 , right side).

[0041] Embodiment 2:

[0042] S1) Extraction and purification with NaCl and Na2SO3 aqueous solution: Take 100 g of the crude sophorolipid obtained in step (5) above, add 100 g of boiling water, 15 g of NaCl and 15 g of Na2SO3, mix and place in an 80°C water bath and stir at 100 rpm for 30 min. After standing, the system is separated into two phases, and the lower sophorolipid is taken out.

[0043] S2) Extraction and decolorization with sodium acetate and citric acid: 100 g of boiling water was added to the sophorolipid obtained in step S1, and then 15 g of sodium acetate and 15 g of citric acid were added, mixed and placed in an 80 ° C water bath and stirred at 100 rpm for 30 min. After standing, the system was separated into two phases, and the lower sophorolipid phase was taken out and weighed to obtain 91.6 g of sophorolipid.

[0044] The mixed salt solution of NaCl and Na2SO3 used in step S1 of Example 2 can not only obtain a product with a transparent appearance, but also the contents of impurity oil and impurity protein are 6.42% and 2.96%, respectively, which are similar to those in Example 1. The product obtained in step S2 of Example 2 was dissolved in ethanol, and after characterization by high performance liquid chromatography, the peak area ratio of acid-type sophorolipid (peak before 10 min) decreased, and the peak area ratio of lactone-type sophorolipid (peak after 10 min) increased, indicating that Example 2 obtained a higher purity lactone-type sophorolipid and increased the proportion of lactone-type sophorolipid. The peak areas of acid-type sophorolipid and lactone-type sophorolipid obtained after treating the crude sophorolipid once according to Example 1 and Example 2 are shown in Table 1.

[0045] Chromatographic conditions are as follows: mobile phase is acetonitrile-water; detection wavelength is 207 nm; injection volume is 20 μL; flow rate is 1 mL / min. Gradient elution (volume fraction): acetonitrile content increases from 40% to 60% from 0 to 15 min, acetonitrile content increases from 60% to 70% from 15 to 30 min, acetonitrile content increases from 70% to 90% from 35 to 40 min, and acetonitrile content is maintained at 90% from 40 to 55 min.

[0046] Table 1

[0047] Treatment Solid content (%) Acid type peak area (%) Lactone type peak area (%) Crude Sophorolipids 68.23 23.25 76.75 Example 1 82.82 20.58 79.42 Example 2 78.58 9.65 90.35

[0048] Compared with crude sophorolipids, Example 2 significantly increased the proportion of lactone sophorolipids in the prepared sophorolipids, and the mass fraction of lactone sophorolipids in the sophorolipids reached more than 90%. In addition, due to the introduction of SO3 in Example 2 2- In an acidic environment, H2SO3 generated in the aqueous phase and the sophorolipid phase has a reductive bleaching effect. The method for determining the decolorization rate is based on the principle of complementary colors. The absorbance at 470nm is selected to represent the depth of color. The absorbance before decolorization is A0, and the absorbance after decolorization is A n , then the decolorization rate %R = (A0-A1) / A0*100%. Compared with the simple NaCl extraction treatment in Example 1, the decolorization rate of Example 2 is 68.15%, which is Figure 3 (Left side). However, Example 1 has no obvious decolorization effect.

[0049] Study Example 1:

[0050] S1) Extraction and purification with NaCl and Na2SO3 aqueous solution: Take 100 g of the crude sophorolipid obtained in step (5) above, add 100 g of boiling water, 22.5 g of NaCl and 7.5 g of Na2SO3, mix and place in an 80°C water bath and stir at 100 rpm for 30 min. After standing, the system is separated into two phases, and the lower sophorolipid phase is taken out.

[0051] S2) Extraction and decolorization with sodium acetate and citric acid: 100 g of boiling water was added to the sophorolipid obtained in step S1, and then 15 g of sodium acetate and 15 g of citric acid were added. The mixture was placed in an 80° C. water bath and stirred for 30 min. After standing, the system was separated into two phases, and the sophorolipid phase was taken out.

[0052] The decolorization rate was measured to be 54.12%. Figure 3 (right side).

[0053] Study Example 2:

[0054] S1) Extraction and purification with NaCl and Na2SO3 aqueous solution: Take 100 g of the crude sophorolipid obtained in step (5) above, add 100 g of boiling water, then add 15 g of NaCl and 15 g of Na2SO3, mix and place in an 80 ° C water bath and stir at 100 rpm for 30 min. After standing, the system is separated into two phases, and the lower sophorolipid is taken out.

[0055] S2) Extraction and decolorization with sodium acetate and citric acid: 100 g of boiling water was added to the sophorolipid obtained in step S1, and then 15 g of sodium acetate was added. After mixing, the mixture was placed in an 80° C. water bath and stirred for 30 min. After standing, the system was separated into two phases, and the sophorolipid phase was taken out.

[0056] In order to study the effect of citric acid on the decolorization of purified sophorolipids, no citric acid was added because SO3 2- It is not converted into H2SO3 and no significant decolorization effect is found.

[0057] Study Example 3:

[0058] S1) Extraction and purification with NaCl and Na2SO3 aqueous solution: Take 100 g of the crude sophorolipid obtained in step (5) above, add 100 g of boiling water, then add 15 g of NaCl and 15 g of Na2SO3, mix and place in an 80 ° C water bath and stir at 100 rpm for 30 min. After standing, the system is separated into two phases, and the lower sophorolipid is taken out.

[0059] S2) Extraction and decolorization with sodium acetate and citric acid: 100 g of boiling water and 15 g of citric acid were added to the sophorolipid obtained in step S1, and the mixture was placed in a water bath at 80° C. and stirred for 30 min. After standing, the system was separated into two phases, and the sophorolipid phase was taken out.

[0060] The sophorolipids obtained in Example 2 and Research Example 3 were placed in an oven at 50°C for stability study, and HPLC analysis was performed after 7, 15 and 30 days. Since the more acetyl groups a sophorolipid contains, the stronger its hydrophobicity, the later the peak in the chromatogram, the peak area after 40 minutes was calculated (see Table 2), and the sophorolipid acetyl content was analyzed.

[0061] Table 2

[0062] Treatment Day 0 7 days 15 days 30 days Example 2 46.05％ 44.59％ 43.04％ 42.68％ Study 3 45.69％ 42.28％ 38.76％ 36.35％

[0063] It can be seen from Table 2 that the technical solution of Example 2 improves the stability of the lactone-type sophorolipid compared to that of Research Example 3.

[0064] Comparative Example 1:

[0065] S1) Extraction and purification with CaCl2 aqueous solution: Take 100 g of the crude sophorolipid obtained in step (5) above, add 100 g of boiling water thereto, then add 30 g of CaCl2, mix, place in an 80°C water bath and stir at 100 rpm for 30 min, let stand overnight, the system is separated into two phases, and the lower sophorolipid phase is taken out.

[0066] like Figure 4 As shown, the obtained sophorolipids were not clear and transparent.

[0067] Comparative Example 2:

[0068] S1) Extraction and purification with Na2SO4 aqueous solution: Take 100 g of the crude sophorolipid obtained in step (5) above, add 100 g of boiling water thereto, then add 30 g of Na2SO4, mix and place in an 80°C water bath and stir at 100 rpm for 30 min. After standing overnight, the system is separated into two phases, and the lower sophorolipid phase is taken out.

[0069] like Figure 5 As shown, the sophorolipids are not clear and transparent and appear crystalline.

[0070] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for purifying modified sophorolipids, characterized in that: The method comprises adding a salt solution to the lactone-type crude sophorolipid obtained by fermenting Candida bombus, stirring, and collecting the lower sophorolipid phase after standing; Wherein, the salt solution includes sodium chloride and / or sodium sulfite solution, and the mass concentration of the salt solution is 10% to 30%.

2. The method for purifying modified sophorolipid according to claim 1, characterized in that: It also includes collecting the lower sophorolipid phase and further extracting and decolorizing: adding acetate and acid solution to the lower sophorolipid phase, stirring and then standing, and taking out the sophorolipid phase; wherein the mass concentration of acetate is 8-16%, and the mass concentration of acid is 8-16%.

3. The method for purifying modified sophorolipid according to claim 1 or 2, characterized in that: The preparation method of the lactone type crude sophorolipid comprises the steps of fermenting and culturing Candida bombus in a culture medium containing vegetable oil, sterilizing, standing and stratifying, and collecting the crude sophorolipid in the lower layer.

4. The method for purifying modified sophorolipid according to claim 3, characterized in that: The method also includes adding hot water to the lower layer of crude sophorolipid, stirring, standing and stratifying, and collecting the lower layer of lactone-type crude sophorolipid.

5. The method for purifying modified sophorolipid according to claim 1 or 2, characterized in that: The salt solution consists of sodium chloride and sodium sulfite aqueous solutions.

6. The method for purifying modified sophorolipid according to claim 5, characterized in that: The mass concentration of the sodium chloride is 10-15%; the mass concentration of the sodium sulfite is 10-15%.

7. The method for purifying modified sophorolipid according to claim 1, characterized in that: The stirring temperature is 60-90°C.

8. The method for purifying modified sophorolipid according to claim 2, characterized in that: The acetate includes sodium acetate and the acid includes citric acid.

9. The method for purifying modified sophorolipid according to claim 2, characterized in that: The stirring is followed by standing still, the stirring temperature is 60-90° C., and the standing time is 4-14 hours.

Images