An enzyme-based solvent-free green synthesis of lactone sophorolipids method

Abstract

The application belongs to the field of biological chemical industry, and particularly relates to a solvent-free green synthesis method of lactone sophorolipid based on enzymes. Acid-type sophorolipid is used as a substrate, dissolved in an organic monohydric alcohol, and a lipase is used as a catalyst to generate lactone sophorolipid. The application uses a solvent-free system, does not introduce other compounds, and greenly prepares lactone sophorolipid. The application has the advantages of safety, high efficiency, no pollution, no introduction of other chemical reagents, simple operation method, and simple product extraction.

Description

Technical Field

[0001] This invention belongs to the field of biochemical engineering, specifically relating to an enzyme-based solvent-free green synthesis method for ester-type sophorolipids. Background Technology

[0002] Sophorolipids are glycolipid biosurfactants that possess properties such as solubilization, emulsification, wetting, foaming, dispersion, and surface tension reduction. They are also environmentally friendly, non-toxic, and readily biodegradable. These products can be used in petroleum, environmental protection, pharmaceutical, food, cosmetic, detergent, home care, agriculture, and animal feed industries, and can partially or completely replace chemically synthesized surfactants.

[0003] Sophorolipids are produced by microbial fermentation, resulting in high yields and large-scale production. Naturally produced sophorolipids by microbial fermentation are a composite substance containing both acidic and lactone types, with carbon chain lengths ranging from 16C to 22C, mostly 16C or 18C. Different structural types of sophorolipids exhibit different performance characteristics, and sophorolipid derivatives containing different functional groups also show significant performance differences. Natural sophorolipid products are classified into lactone and acidic types, formed by the linkage of long-chain fatty acids and sophorobiose via O-glycosidic bonds. Exoesterified sophorolipids refer to the reaction between the carboxyl group of long-chain fatty acids and alcohols, forming exoesterified sophorolipids. Compared to natural sophorolipids, ester-type sophorolipids have longer carbon chain lengths, resulting in enhanced surface activity, antibacterial properties, and bioactivity (Amanda, Koh, Robert, et al. Effect of Sophorolipid n-Alkyl Ester Chain Length on Its Interfacial Properties at the Almond Oil-Water Interface. [J]. Langmuir the Acs Journal of Surfaces & Colloids, 2016.; Todd, Katherine, Koh, et al. Fundamental Characterization of the Micellar Self-Assembly of Sophorolipid Esters [J]. Langmuir the Acs Journal of Surfaces & Colloids, 2017.; Amanda Koh, et al. Influence of Sophorolipid Structure on Interfacial Properties of Aqueous-Arabian Light Crude and Related Constituent Emulsions [J]. Journal of the American Oil Chemists' Society Volume). 94, pages107–119(2017). TotsinganF, Liu F, Gross R A. Structure–ActivityRelationship Assessment of Sophorolipid Ester Derivatives against ModelBacteria Strains[J]. Molecules, 2021(10).).

[0004] Chemical modification of sophorolipids can increase structural diversity and effectively improve product performance. Currently, literature reports on chemically modified sophorolipids including esterification to produce exoester-type sophorolipids, quaternary ammonium salt modification to produce quaternary ammonium salt-type sophorolipids, and sulfonation modification with sulfonating agents (Gross RA, Schofield M H. Sophorolipid Analog Compositions:, US20120022241A1[P]. 2012.; A new class of antimicrobial biosurfactants: quaternary ammonium sophorolipids, Green Chem., 2015, 17, 3373-3377.; CN112442098A).

[0005] Compared to natural sophorolipids, esterified sophorolipids have increased carbon chain length, resulting in enhanced surface activity, antibacterial properties, and bioactivity. Currently, there are two methods reported in the literature for preparing esterified sophorolipids: one is to react a mixture of sophorolipids with alkoxides in an anhydrous environment to obtain esterified sophorolipids, which requires the use of elemental sodium and is highly dangerous (Bisht K S, Gross RA, Kaplan D L. Enzyme-Mediated Regioselective Acylations of Sophorolipids[J]. Journal of Organic Chemistry, 1999, 64(3):780.); the other is to use long-chain sulfonates as catalysts to catalyze the reaction of lactone-type sophorolipids with monohydric alcohols to obtain esterified sophorolipids, which introduces soluble long-chain sulfonates during the reaction, and requires adjusting the pH of the system to neutral with NaOH after the reaction, resulting in a mixture with low purity (CN114106807A). The former method involves reacting a mixture of sophorolipids with alkoxides in an anhydrous environment. This requires elemental sodium, posing a high risk, and necessitates strict control of the anhydrous environment, placing stringent requirements on reaction conditions. The latter method uses long-chain sulfonates as catalysts to catalyze the reaction of lactone-type sophorolipids with monohydric alcohols. This introduces soluble long-chain sulfonates into the reaction process, requiring the addition of alkali to adjust the pH to neutral. The resulting product has low purity and is difficult to separate and purify. Furthermore, the introduction of other compounds into the reaction violates green manufacturing principles. In summary, current methods for preparing exoester-type sophorolipids are limited, and the yield and purity are insufficient to meet application requirements.

[0006] Therefore, it is very important to develop efficient, simple and green methods for synthesizing ester-type sophorolipids, improve product performance, and meet the needs of multiple applications. Summary of the Invention

[0007] This invention establishes an enzyme-based, solvent-free, green method for the synthesis of exoester-type sophorolipids, solving the problems of harsh reaction conditions and low purity of reaction products in existing exoester-type sophorolipid preparation methods.

[0008] The present invention provides a method for preparing esterified sophorolipids, which uses acidic sophorolipids as a substrate, dissolved in an organic monohydric alcohol, and catalyzed by lipase to generate esterified sophorolipids. This establishes an enzyme-based, solvent-free, and green method for preparing esterified sophorolipids.

[0009] This invention provides a green, enzyme-based method for preparing exoester-type sophorolipids. It employs a solvent-free system, introduces no other compounds, and provides a green preparation method for exoester-type sophorolipids. This method offers advantages such as safety, high efficiency, no pollution, no introduction of other chemical reagents, simple operation, and convenient product extraction. More specifically, its advantages include the following aspects:

[0010] 1) Raw materials for enzyme-catalyzed reaction: The sophorolipid raw material used in this invention is the acidic component of sophorolipid or mixed sophorolipid, obtained by crystallization after microbial fermentation. Existing technologies typically use lactone-type or mixed sophorolipids as raw materials; in this technology, lactone-type sophorolipids do not participate in the reaction. 2) Enzyme-catalyzed reaction system: The reaction system of this invention is a solvent-free system, where sophorolipids are completely soluble in organic monohydric alcohols and participate in the reaction. Enzymatic catalysis generates exoester-type sophorolipids in one step without introducing other soluble compounds. Existing technologies require the use of alkoxides or soluble long-chain sulfonates to produce exoester-type sophorolipids. 3) Product purification method: The product of this invention is purified by rotary evaporation or freeze-drying to remove organic monohydric alcohols, yielding a high-purity exoester-type sophorolipid product in one step. Existing technologies for producing exoester-type sophorolipids lack separation and purification, resulting in a high amount of impurities in the product; furthermore, they require multiple steps of acidification, precipitation, washing, and freeze-drying, which are complex and result in significant losses.

[0011] In summary, this invention provides a one-step enzymatic catalysis for obtaining exoester-type sophorolipids. Compared to chemical catalysis, enzymatic methods offer milder reaction conditions, higher reaction specificity, higher conversion rates, and no byproduct formation. The reaction system is solvent-free, introducing no other soluble compounds, resulting in high-purity, impurity-free products. The organic monohydric alcohols used in the reaction can be removed in one step by rotary evaporation or lyophilization, simplifying product purification, simplifying the operation, and facilitating large-scale industrial processing. Attached Figure Description

[0012] Figure 1HPLC chromatograms of different types of sophorolipids at 0h and 20h. ① Chromatography of acidic sophorolipids at 0h, ② Chromatography of acidic sophorolipids at 20h, ③ Chromatography of lactone-type sophorolipids at 0h, ④ Chromatography of lactone-type sophorolipids at 20h. The HPLC chromatograms of acidic sophorolipids changed before and after the reaction, while the HPLC chromatograms of lactone-type sophorolipids remained unchanged.

[0013] Figure 2 HPLC chromatogram of the acidic sophorolipid substrate. The retention times of the acidic sophorolipid were 5.847 min and 6.870 min.

[0014] Figure 3 LC-MS analysis of the total intensity ratio (M / Z) of acidic sophorolipid substrate from 600 to 900. The major molecular weight is 731.38.

[0015] Figure 4 HPLC chromatogram of the methylated sophorolipid product. The retention times of the methylated sophorolipid were 8.477 min and 9.863 min.

[0016] Figure 5 LC-MS analysis of the methylated sophorolipid product showed an overall intensity ratio (M / Z) of 600 to 900. The major molecular weight was 745.40.

[0017] Figure 6 HPLC chromatogram of the ethyl esterified sophorolipid product. The retention times of the ethyl esterified sophorolipid were 9.370 min and 10.677 min.

[0018] Figure 7 HPLC chromatogram of the propyl-esterified sophorolipid product. The retention times of propyl-esterified sophorolipid were 10.211 min and 11.557 min.

[0019] Figure 8 HPLC chromatogram of the butylated sophorolipid product. The retention times of the butylated sophorolipid were 10.993 min and 12.405 min.

[0020] Figure 9 HPLC chromatogram of hexylesterified sophorolipid. The retention times of hexylesterified sophorolipid were 12.616 min and 14.449 min.

[0021] Figure 10 HPLC chromatogram of octyl esterified sophorolipid. The retention times of octyl esterified sophorolipid were 14.756 min and 17.403 min.

[0022] Figure 11Surface tension of aqueous solutions of sophorolipids of different concentrations. ASLs: acidic sophorolipids; ASLs-ME: methylated sophorolipids; ASLs-EE: ethylated sophorolipids.

[0023] Figure 12 Foaming ability test of different esterified sophorolipids. ASLs: acidic sophorolipids; ASLs-ME: methylated sophorolipids; ASLs-EE: ethylated sophorolipids.

[0024] Figure 13 Emulsifying ability test of different esterified sophorolipids. ASLs: acidic sophorolipids; ASLs-ME: methylated sophorolipids; ASLs-EE: ethylated sophorolipids. Detailed Implementation

[0025] The method of the present invention will be further described below with reference to the embodiments. Experimental methods not specified with particular conditions in the embodiments can generally be performed using conventional experimental methods. Unless otherwise specified, the reagents and instruments used in the following embodiments are commercially available products.

[0026] Natural sophorolipid products are a mixture of acidic and lactone-type sophorolipids, with lactone-type sophorolipids accounting for >60%. The preparation of exoester-type sophorolipids typically uses lactone-type or mixed-type sophorolipids as raw materials, employing alkoxides or long-chain sulfonates as catalysts to catalyze the hydrolysis of lactone-type sophorolipids and participate in the esterification of organic monohydric alcohols. When the inventors used lactone-type sophorolipids as raw materials, the composition remained unchanged before and after the reaction, and the reaction could not proceed normally (see Comparative Example 1). However, when the reactant was replaced with acidic sophorolipids, a new product was surprisingly found to be generated, which, upon testing, was identified as exoester-type sophorolipids.

[0027] Meanwhile, lipase-catalyzed reactions usually need to be carried out at the interface between two phases, and other organic solvents are generally required to ensure the normal progress of the reaction. For example, when Bisht et al. used lipase to catalyze the acetylation of sophorolipids, THF was added to ensure the normal progress of the reaction (Bisht KS, Gross RA, Kaplan DL. Enzyme-Mediated Regioselective Acylations of Sophorolipids[J]. Journal of Organic Chemistry, 1999, 64(3):780.). In the construction of the enzyme-catalyzed reaction system, the present invention unexpectedly found that the reaction can still proceed efficiently without the use of organic solvents. In addition, the inventors found that sophorolipids can be fully dissolved in organic monohydric alcohols (>400 g / L), so the reaction can effectively synthesize ester-type sophorolipids without the need for organic solvents.

[0028] Furthermore, when using fermentation broth containing acidic sophorolipids as the substrate for the catalytic reaction, it was found that only a small portion of the acidic sophorolipid substrate was converted to exoester sophorolipids. This led to the hypothesis that moisture content might affect the reaction. When moisture was removed from the raw materials, the conversion rate significantly increased; when the moisture content in the reaction system was below 5%, acidic sophorolipids could be completely converted to exoester sophorolipids (see Comparative Example 2).

[0029] Through the above research process, the present invention has determined that by replacing the reaction raw materials with acidic sophorolipids and using a solvent-free reaction system, exoester-type sophorolipids can be synthesized efficiently (see Examples 1-6).

[0030] On the other hand, the sophorolipid raw material used in this invention is an acidic sophorolipid product obtained by microbial fermentation. By knocking out key genes in wild-type strains or adjusting the composition of the fermentation medium, a product containing only acidic sophorolipid can be obtained. The acidic sophorolipid raw material used in this invention is obtained by crystallizing the acidic sophorolipid fermentation broth. The crystallized product is washed with water to remove impurities and moisture, and can then be used as the sophorolipid raw material in the catalytic reaction. In this invention, the molecular structure of the acidic sophorolipid raw material is shown in formula (1), which is composed of long-chain fatty acids and sophorobiose linked by O-glycosidic bonds, and the group participating in the catalytic reaction is the carboxyl group of the long-chain fatty acid.

[0031]

[0032] Among them, R 1 =R 2 =H, or R 1 =H,R 2 =Ac, or R 1 =Ac,R 2 =H, or R 1 =R 2 =Ac.

[0033] The organic monohydric alcohol used in this invention is a commercially available industrial product. It can be a C1 to C8 organic monohydric alcohol, such as methanol, ethanol, propanol, butanol, hexanol, octanol, and more preferably a C1 to C4 organic monohydric alcohol.

[0034] The enzyme used in this invention is the commercial lipase novozym435, an immobilized lipase capable of catalyzing esterification or transesterification reactions in various media. In solvent-free systems, using novozym435 to catalyze the esterification of simple acids and alcohols is an effective technique. This invention is the first to utilize novozym435 to catalyze the esterification of sophorolipids and alcohols in a solvent-free system, optimizing the reactants and reaction process to prepare exoester-type sophorolipids.

[0035] The main reaction formulas of the enzyme-catalyzed reaction system described in this invention are shown below. The carboxyl group of acidic sophorolipid undergoes an esterification reaction with an organic monohydric alcohol under the action of lipase to prepare an externally esterified sophorolipid product.

[0036]

[0037] The product of the enzyme-catalyzed reaction of this invention is an externally esterified sophorolipid, which is an esterified sophorolipid with the molecular structure shown in formula (2). The carboxyl groups of the long-chain fatty acids of the sophorolipid are replaced by long-chain esters to generate C1-C8 externally esterified sophorolipid products.

[0038]

[0039] Among them, R 3 = Methanol, ethanol, propanol, butanol, hexanol, octanol.

[0040] The reaction system of this invention is a solvent-free system. Acidic sophorolipids can be completely dissolved in organic monohydric alcohols (>400g / L) and participate in enzyme-catalyzed reactions.

[0041] The reaction temperature in the catalytic system of the present invention can be 30-80℃, preferably 35-45℃, and more preferably 40℃; the reaction time can be 1-20 hours, preferably 3-4 hours.

[0042] In this invention, the substrate has a water content of 0% or less than 30%, preferably less than 5%. When the water content is below 5%, the acidic sophorolipid can be completely converted into the esterified sophorolipid.

[0043] The ester-type sophorolipid product generated by this invention is completely dissolved in an organic monohydric alcohol. The novozym435 lipase is recovered by filtration, and the organic monohydric alcohol is removed by rotary evaporation or freeze-drying, yielding a high-purity ester-type sophorolipid product in one step. C1-C4 organic monohydric alcohols have low boiling points and can be removed by rotary evaporation; C5-C8 organic monohydric alcohols have high melting points and can be removed by freeze-drying.

[0044] Compared to acidic sophorolipid raw materials, the esterified sophorolipid product of this invention exhibits lower surface tension and a lower CMC value (CMC, or critical micelle concentration, refers to the minimum concentration at which surfactant molecules form micelles). When the solution reaches the critical micelle concentration, the surface tension of the solution drops to its minimum value. At this point, further increasing the surfactant concentration does not decrease the surface tension but instead leads to the formation of a large number of micelles. The lower the CMC, the lower the surfactant concentration required to reduce the surface tension of the solution to its minimum value, indicating better surface activity.

[0045] The following provides specific comparative studies and embodiments of the present invention.

[0046] Comparative Example 1: Preparation of Exoester Sophorolipids using Lactone-type Sophorolipids as Substrates

[0047] Weigh 0.5 g of lactone-type sophorolipid into a 50 mL Erlenmeyer flask, add 5 mL of anhydrous ethanol, and after complete dissolution, add 0.05 g of Novozyme 435. React at 40 °C for 20 h. After the reaction is complete, recover Novozyme 435 by centrifugation or membrane filtration. Filter the supernatant from 0 h and 20 h through a 0.22 μm filter membrane and analyze the product by HPLC.

[0048] HPLC spectra of different types of sophorolipids participating in the reaction are as follows: Figure 1 As shown, the HPLC elution time of the acidic sophorolipid changed after the reaction, forming an ethylated exoester sophorolipid; while the lactone sophorolipid could not participate in the reaction, and the spectrum did not change before and after the reaction. The carboxyl group of the lactone sophorolipid is esterified with the hydroxyl group on the sugar group, and Novozyme 435 lipase does not have the ability to catalyze the hydrolysis of lactone sophorolipid, so it cannot participate in the exoesterification reaction.

[0049] Comparative Example 2: Effect of water content on the reaction

[0050] Weigh 0.5 g of acidic sophorolipid into a 50 mL Erlenmeyer flask, and add 5 mL of ethanol with water contents of 0%, 5%, 10%, 15%, 20%, and 30%, respectively. After complete dissolution, add 0.05 g of Novozyme 435 and react at 40 °C for 20 h. After the reaction is complete, recover Novozyme 435 by centrifugation or membrane filtration. Filter the supernatant from 0 h and 20 h through a 0.22 μm filter membrane and analyze the product by HPLC.

[0051] The substrate conversion rates at different water contents in the system are shown in Table 1. Water content in the system inhibits the catalytic reaction; the higher the water content, the lower the conversion rate. When the water content in the system is below 5%, acidic sophorolipids can be completely converted to esterified sophorolipids. When the water content is 10%, only 60% of the acidic sophorolipids can be catalyzed to esterified sophorolipids. And when the water content reaches 30%, only 30% of the acidic sophorolipids can be catalyzed to esterified sophorolipids.

[0052] Table 1 Substrate conversion rates of reaction systems with different water contents

[0053] Moisture content % Peak area before reaction (mAU.s) Peak area after reaction (mAU.s) Substrate conversion rate 0 2401 0 100.0％ 5 2315 0 100.0％ 10 2604 1038 60.1％ 15 2622 1604 38.8％ 20 2651 1562 41.1％ 30 2603 1801 30.8％

[0054] Note: Substrate conversion rate = (Peak area before reaction - Peak area after reaction) / Peak area before reaction * 100%

[0055] Example 1: Preparation of methylated sophorolipid

[0056] Weigh 2g of acidic sophorolipid into a 100mL Erlenmeyer flask, dissolve it completely in 10mL of methanol, add 0.10g of Novozyme 435, and react at 35℃ for 20h. After the reaction is complete, centrifuge or filter membrane to recover Novozyme 435, and remove the solvent from the reaction solution by rotary evaporation to obtain methylated sophorolipid.

[0057] The high-performance liquid chromatography (HPLC) chromatogram of the acidic sophorolipid substrate is shown below. Figure 2 As shown, there are two main peaks with retention times of 5.847 min and 6.870 min, respectively. The acidic sophorolipid substrate was analyzed by LC-MS, and the analytical spectrum is shown below. Figure 3 As shown, the major molecular weight is 731.38, and the composition is C18:O-diacetylated-ASLs(M+Na)+. The acidic sophorolipid substrate was prepared and collected using an HPLC preparative column, and then analyzed by NMR. The NMR analysis results are as follows: 1 H NMR(600MHz,DMSO-d6)δ11.87(s,1H,-COOH),5.56–5.52(m,1H,OH),5.37(s,1H,OH)),5.24(s,1H,OH),5.10(s,2H,O H),4.34(d,J=7.8Hz,1H,H-1”),4.29(d,J=7.7Hz,1H,H-1’),4.15(ddd,J=14.7,11.8,2.1Hz,2H,H-6’b,H-6”b),3.96 (ddd, J = 25.4, 11.8, 6.3 Hz, 2H, H-6'a, H-6”a), 3.59–3.53 (m, 1H, H-17), 3.12–2.92 (8H, H-2'~5', H-2”~5”), 2.11 (t, J = 7.4 Hz, 2H, H-2), 1.93 (s, 6H, 2CH3CO-), 1.41–1.13 (28H, H-3~16), 1.03 (d, J = 6.2 Hz, 3H, H-18), is an acidic sophorolipid substrate containing -COOH.

[0058] The high-performance liquid chromatography (HPLC) chromatogram of the methylated sophorolipid product is shown below. Figure 4 As shown, there are two main peaks with retention times of 8.477 min and 9.863 min, which are the methylated sophorolipid product. The methylated sophorolipid product was analyzed by LC-MS, and the chromatogram is shown below. Figure 5 As shown, the major molecular weight is 745.40, and the molecular weight is C18:0-diacetylated-ASLs-ME(M+Na)+. The methylated sophorolipid product was collected using an HPLC preparative column and analyzed by NMR. The NMR analysis results are as follows: 1H NMR (600MHz, DMSO-d6) δ5.56–5.52(m,1H,OH),5.39–5.35(m,1H,OH),5.23(d,J=5.7Hz,1H,OH),5.10(d,J=5.7Hz,1H,OH),5.02(d,J =4.9Hz,1H,OH),4.34(d,J=7.8Hz,1H,H-1”),4.29(d,J=7.7Hz,1H,H-1’),4.15(ddd,J=15.4,11.8,2.0Hz,2H,H-6’b,H-6”b),3.96(d dd, J = 25.4, 11.8, 6.3 Hz, 2H, H-6'a, H-6”a), 3.55 (p, J = 5.9 Hz, 1H, H-17), 3.51 (s, 3H, -OCH3), 3.27-2.95 (8H, H-2'~5', H-2”~5”), 2.21 (t, J = 7.4 Hz, 2H, H-2), 1.93 (d, J = 1.0 Hz, 6H, 2CH3CO-), 1.47-1.13 (28H, H-3~16), 1.03 (d, J = 6.2 Hz, 3H, H-18), are methylated sophorolipid products containing -OCH3.

[0059] Example 2: Preparation of ethyl esterified sophorolipid

[0060] Weigh 2g of acidic sophorolipid into a 100mL Erlenmeyer flask, dissolve it completely in 10mL of ethanol, add 0.10g of Novozyme 435, and react at 35℃ for 20h. After the reaction is complete, centrifuge or filter membrane to recover Novozyme 435, and remove the solvent from the reaction solution by rotary evaporation to obtain ethyl esterified sophorolipid.

[0061] The high-performance liquid chromatography (HPLC) chromatogram of the ethyl esterified sophorolipid product is shown below. Figure 6 As shown, there are two main peaks with retention times of 9.370 min and 10.677 min, which are ethyl esterified sophorolipid products. LC-MS analysis of the ethyl esterified sophorolipid product revealed a major molecular weight of 759.42, indicating a C18:0-diacetylated-ASLs-EE(M+Na)+.

[0062] Example 3: Preparation of propyl esterified sophorolipid

[0063] Weigh 2g of acidic sophorolipid into a 100mL Erlenmeyer flask, dissolve it completely in 10mL of n-propanol, add 0.10g of Novozyme 435, and react at 40℃ for 20h. After the reaction is complete, centrifuge or filter to recover Novozyme 435, and remove the solvent from the reaction solution by rotary evaporation to obtain the propyl-esterified sophorolipid product.

[0064] The high-performance liquid chromatography (HPLC) chromatogram of the propyl-esterified sophorolipid product is shown below. Figure 7 As shown, there are two main peaks with retention times of 10.211 min and 11.557 min, which are the products of propyl esterified sophorolipid. LC-MS analysis of the propyl esterified sophorolipid product revealed a major molecular weight of 773.43, indicating a C18:0-diacetylated-ASLs-PE(M+Na)+.

[0065] Example 4: Preparation of Butyl-esterified Sophorolipid

[0066] Weigh 2g of acidic sophorolipid into a 100mL Erlenmeyer flask, dissolve it completely in 10mL of n-butanol, add 0.10g of Novozyme 435, and react at 40℃ for 20h. After the reaction is complete, centrifuge or filter to recover Novozyme 435, and remove the solvent from the reaction solution by rotary evaporation to obtain butylated sophorolipid.

[0067] The high-performance liquid chromatography (HPLC) chromatogram of the butylated sophorolipid product is shown below. Figure 8 As shown, there are two main peaks with retention times of 10.993 min and 12.405 min, which are the butyl esterified sophorolipid product. The butyl esterified sophorolipid product was analyzed by LC-MS, and the major molecular weight was 787.45, indicating a C18:0-diacetylated-ASLs-BE(M+Na)+.

[0068] Example 5: Preparation of hexyl esterified sophorolipid:

[0069] Weigh 2g of acidic sophorolipid into a 100mL Erlenmeyer flask, dissolve it completely in 10mL of n-hexanol, add 0.10g of Novozyme 435, and react at 40℃ for 20h. After the reaction is complete, centrifuge or filter to recover Novozyme 435, and remove the solvent from the reaction solution by lyophilization to obtain hexyl esterified sophorolipid.

[0070] The high-performance liquid chromatography (HPLC) chromatogram of the hexyl esterified sophorolipid product is shown below. Figure 9 As shown, there are two main peaks with retention times of 12.616 min and 14.449 min, which are hexylesterified sophorolipid products. LC-MS analysis of the hexylesterified sophorolipid product revealed a major molecular weight of 815.48, indicating a C18:0-diacetylated-ASLs-HE(M+Na)+ composition.

[0071] Example 6: Preparation of Octyl-esterified Sophorolipid

[0072] Weigh 2g of acidic sophorolipid into a 100mL Erlenmeyer flask, dissolve it completely in 10mL of n-octanol, add 0.10g of Novozyme 435, and react at 40℃ for 20h. After the reaction is complete, centrifuge or filter to recover Novozyme 435, and remove the solvent from the reaction solution by lyophilization to obtain octyl esterified sophorolipid.

[0073] The high-performance liquid chromatography (HPLC) chromatogram of the octyl esterified sophorolipid product is shown below. Figure 10 As shown, there are two main peaks with retention times of 14.756 min and 17.403 min, which are octyl esterified sophorolipid products. LC-MS analysis of the octyl esterified sophorolipid product revealed a major molecular weight of 843.51, indicating a C18:O-diacetylated-ASLs-OE(M+Na)+ composition.

[0074] Test Example 1: Surface Tension Measurement

[0075] The ester-type sophorolipid products obtained in Examples 1 and 2 were dissolved in 50% acetonitrile-water, and the products were collected by high-performance liquid chromatography (HPLC). The collected products were then lyophilized to obtain a white powdery solid. Sophorolipid solutions of different concentrations were prepared by dissolving the products in ultrapure water. Surface tension was measured using an SCA20 contact angle meter, and the critical micelle concentration (CMC) was calculated. The surface tension measurement results are as follows: Figure 11 As shown, esterified sophorolipids have a stronger ability to reduce the surface tension of aqueous solutions, and the order is ASLs-EE > ASLs-ME > ASLs. The CMC values ​​of different esterified sophorolipid products are shown in Table 2. Compared to acidic sophorolipids, esterified sophorolipid products have lower CMC values. The CMC value of the acidic sophorolipid substrate is 101.9 mg / L, the CMC value of the methylated sophorolipid product is 33.0 mg / L, and the CMC value of the ethylated sophorolipid product is 16.3 mg / L.

[0076] Table 2 Surface tension and CMC value of exoester-type sophorolipid products

[0077] sample IFT value mN / m CMC value (mg / L) ASLs 45.75 101.9 ASLs-ME 43.92 33.0 ASLs-EE 41.14 16.3

[0078] Test Example 2: Foam Stability Test

[0079] Weigh an appropriate amount of the ester-type sophorolipids from Examples 1 and 2, dissolve them in ultrapure water to prepare a 0.1 mg / mL ester-type sophorolipid solution, and test the foaming ability of the ester-type sophorolipids using a standard foam column.

[0080] The foaming ability of different ester-type sophorolipids, such as Figure 12 As shown, different ester-type sophorolipids all have good foaming ability, and ASLs-EE>ASLs-ME>ASLs, which makes them suitable for use in toiletries.

[0081] Test Example 3: Emulsifying Ability Test

[0082] Weigh appropriate amounts of the esterified sophorolipids from Examples 1 and 2, and dissolve them in ultrapure water to prepare a 10 mg / mL esterified sophorolipid solution. Pipette 2.7 mL of the sophorolipid solution into a sample vial, add 5 μL of 1.0% wt methylene blue indicator and 0.3 mL of caprylic / capric triglyceride containing 0.1% v / v dimethyl yellow indicator, and emulsify using a homogenizer at 20,000 rpm for 3 min. Allow the emulsion to stand at room temperature for 1 h and observe its stability.

[0083] The emulsifying abilities of different ester-type sophorolipids, such as Figure 13 As shown, the external ester type sophorolipid has good emulsifying ability, and the emulsion does not separate after standing at room temperature for 1 hour, exhibiting good emulsion stability.

Claims

1. A method for preparing an exolacciferous sophorolipid, characterized by, The application adopts acid sophorolipid with water content less than 30% as a substrate, dissolves in organic monohydric alcohol, and uses lipase as a catalyst to generate exolipid sophorolipid. The organic monohydric alcohol is C1-C8 organic monohydric alcohol, and the acid sophorolipid has a molecular structural formula shown in formula (1): Formula (1); wherein R 1 =H, or R 2 =Ac, or R 1 =H, R 2 =Ac, or R 1 =H, R 2 =Ac, or R 1 =H, or R 2 =Ac.

2. The production method according to claim 1, wherein The temperature of the catalytic reaction is 30-80 ℃; and the reaction time is 1-20 hours.

3. The production method according to claim 1, wherein The temperature of the catalytic reaction is 35-45 ℃; and the reaction time is 3-4 hours.

4. The production method according to claim 1, wherein In the catalytic reaction system, the water content of the substrate is less than 5%.

5. The production method according to claim 4, wherein In the catalytic reaction system, the water content of the substrate is 0%.

6. The production method according to claim 1, wherein The organic monohydric alcohol is C1-C4 organic monohydric alcohol.

7. The production method according to claim 6, wherein The organic monohydric alcohol is methanol, ethanol, propanol, butanol, hexanol or octanol.

8. The production method according to claim 1, wherein The lipase is novozym435 lipase.

9. The production method according to claim 1, wherein After the reaction is completed, a step of removing the organic monohydric alcohol is further included.

10. The production method according to claim 9, wherein After the reaction is completed, the organic monohydric alcohol is removed by rotary evaporation or freeze drying.

Images