CN114716488A

CN114716488A - Sophorolipid deacetylation modification method and sophorolipid

Info

Publication number: CN114716488A
Application number: CN202210282851.4A
Authority: CN
Inventors: 张鹏; 强耀锋; 樊冰; 李庆廷
Original assignee: Shanghai Longyin Biotechnology Co ltd
Current assignee: Shanghai Longyin Biotechnology Co ltd
Priority date: 2022-03-22
Filing date: 2022-03-22
Publication date: 2022-07-08

Abstract

The invention provides a sophorolipid deacetylation modification method and sophorolipid. The color, performance and bond breaking effects generated in the deacetylation modification process can be minimized.

Description

Sophorolipid deacetylation modification method and sophorolipid

Technical Field

The invention belongs to the technical field of sophorose ester modification, and particularly relates to a preparation method for deacetylation modification of sophorose ester.

Background

Sophorolipids (SLs), also known as Sophorolipids, are glycolipid biosurfactants produced by fermentation of Candida buminus (Candida bombicola) using fats and sugars as carbon sources, and are one of the most potential biosurfactants currently. The differences in groups within the SLs molecule give it 20 major structures and more than 100 minor structures. Referring to fig. 1 and 2, the main configuration thereof includes lactone-type sophorolipids, which have free carboxyl groups (-COOH) at the ends of the fatty acid chains, compared to lactone-type sophorolipids, thereby imparting pH sensitivity thereto, and acid-type sophorolipids. Secondly, the differences in configuration also include the length of the carbon chain of the fatty acid side chain of sophorolipid (12 to 24 carbonic acid), and the number of unsaturated bonds (0-3) and position of the fatty acid side chain. In addition, the two acetylation sites on the side chain of sophorose ester sugar can be three types of non-acetylation, 2 types of single-site acetylation and double-acetylation. Deacetylation of this site can significantly increase the hydrophilic character of sophorose esters.

Currently, most (90-99%) sophorolipids obtained by natural fermentation exist in an acetylated form, so that deacetylation modification is required to obtain deacetylated sophorolipids. In the prior art, it has been reported that modification of deacetylation function of sophorolipid can be carried out from a source-producing cell, and that acetylation function of sophorolipid in Candida bombesi is modified by Acetyltransferase (AT), and only nonacetylated sophorolipid is produced in Candida bombesi by knocking out or inactivating Acetyltransferase gene (Transformation of S.bombicola endo a plant organization, DOI: 10.1007/978-1-4939-7795-6-5). However, inactivation of the acetyltransferase gene affects the exocrine capacity of cells to sophorolipid, thereby greatly reducing the yield of sophorolipid. The heating esterification and the hydrolysis of sophorolipid in a strong alkaline aqueous system by KOH and NaOH also belong to the deacetylation derivatization modification of sophorolipid in the prior art. For example, PCT patent Application PCT advanced lipophilic Production (International Application Number: PCT/EP20ll/059306) used three times the molar equivalent of KOH in aqueous solution and alkaline hydrolysis at 80 ℃ for 4h to completely dissociate the acetyl groups of the side chains of the sugar chains. However, under alkaline heating lipolysis conditions, brown reaction occurs to the sophorose, the color becomes dark brown, and the appearance, color and use performance of the product are affected. Secondly, under the esterification condition, the chain breaking condition of part of the fatty acid chains of the sophorose ester can occur, and the service performance of the product is further influenced.

Disclosure of Invention

The technical problem to be solved by the present invention is to overcome the above disadvantages of the prior art, and to provide a method for deacetylation modification of sophorolipid, which can minimize the influence of color, performance and bond cleavage during the deacetylation modification process.

The technical problem to be solved can be implemented by the following technical scheme.

A sophorolipid deacetylation modification method is characterized by comprising the step of catalyzing the esterolysis reaction of sophorolipid under the anhydrous condition by using sodium alkyl alcoholate as a catalyst.

As a further improvement of the technical proposal, the adding proportion of the catalyst is 0.2 times higher than the equivalent of the sophorolipid substance. In a preferred embodiment of the present invention, the sodium alkyl alkoxide is any one of sodium ethoxide and sodium methoxide.

Also as a further improvement of the technical proposal, the anhydrous catalytic reaction condition of the sodium alkyl alcoholate is 25-30 ℃.

Further, the esterification reaction is carried out for more than 5 hours under the condition of magnetic stirring at the temperature of 25-30 ℃.

Also as a further improvement of the technical scheme, the method also comprises the step of further adding water after the completion of the esterification reaction and recovering the organic solvent in the water.

Further, the organic solvent is methanol or ethanol.

As a preferred embodiment of the present invention, the sophorolipid is a natural sophorolipid.

Another technical problem to be solved by the present invention is to provide a sophorolipid, which is a non-acetylated sophorolipid prepared by the method for deacetylation modification of sophorolipid.

Further, the non-acetylated sophorolipid is prepared by deacetylation modification of natural sophorolipid.

Compared with the prior art, the method for deacetylating and modifying sophorolipid and sophorolipid thereof adopting the technical scheme have the following advantages and beneficial effects: firstly, the technical scheme provided by the application does not cause the deacetylation of sophorolipid and the breaking of fatty acid side chains in the ring opening process of lactone, so as to form degradation products such as octenyl sophorose ester (figure 9), and reduce the biological activity and functional efficacy of the product. Compared with the prior art, the scheme for deacetylating and esterifying sophorolipid provided by the invention has the advantages that the color generated in the process is lighter, and the sophorolipid product with better chroma quality can be achieved only by a simpler decoloring means and lower decoloring cost.

Drawings

FIG. 1 is an acid type sophorolipid structure of sophorolipid;

FIG. 2 shows the lactone-type sophorolipid structure of sophorolipid;

FIG. 3 shows the structure of deacetylated sophorose ester; FIG. 4 is a total proton flow diagram of the deacetylated sophorolipid prepared in example 1;

FIG. 5 is a secondary mass spectrum of the peak retention time of the deacetylated sophorolipid prepared in example 1, 4.457 min;

FIG. 6 is a secondary mass spectrum of the 4.625min peak retention time of the deacetylated sophorolipid peak prepared in example 1;

FIG. 7 is a total proton flow diagram of the deacetylated sophorolipid prepared in example 3;

FIG. 8 is a secondary mass spectrum of the peak retention time of 3.53min for the deacetylated sophorolipid prepared in example 3;

FIG. 9 shows the molecular structure of octenyl sophorose ester corresponding to the peak at retention time 3.53min in FIG. 8.

Detailed Description

The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.

Example 1: deacetylation reaction of sophorolipid

2g of sophorolipid dry powder is placed in a 100mL round-bottom flask which is connected with a condensing reflux pipe (the temperature of a refrigerant is 10 ℃), and is also connected with a protection pipe filled with anhydrous calcium chloride to prevent moisture in the atmosphere from influencing the reaction. During the reaction, 25mL of a 0.025mol/L sodium methoxide methanol solution was added, and the mixture was magnetically stirred at 25 ℃ for 5 hours to effect an esterification reaction. Then, 25mL of pure water was added, and the reaction was magnetically stirred for 1 hour. And then taking out the reaction solution, placing the reaction solution in a rotary evaporator for vacuum concentration at 35 ℃, removing and recovering methanol in the reaction system, thereby obtaining the deacetylated acid sophorose ester solution. The sophorose ester powder samples were obtained from deacetylated acid sophorose ester solutions, air dried in an air-blown drying oven and ground to a white powder.

Example 2: deacetylation of sophorose esters

2g of sophorolipid dry powder is placed in a 100mL round-bottom flask which is connected with a condensing reflux pipe (the temperature of a refrigerant is 10 ℃), and is also connected with a protection pipe filled with anhydrous calcium chloride to prevent moisture in the atmosphere from influencing the reaction. During the reaction, 25mL of 0.02mol/L ethanol solution of sodium ethoxide is added, and the reaction is carried out for 5 hours under the magnetic stirring at the temperature of 30 ℃ to carry out the esterification reaction. Then, 25mL of pure water was added, and the reaction was magnetically stirred for further transesterification for 1 hour. And then taking out the reaction solution, placing the reaction solution in a rotary evaporator for vacuum concentration at 40 ℃, and removing and recovering ethanol in the reaction system to obtain the deacetylated acid sophorose ester solution. The sophorose ester powder samples were obtained from deacetylated acid sophorose ester solutions, air dried in an air-blown drying oven and ground to a white powder.

Example 3: alkaline hydrolysis control reaction

The alkali-hydrolytically prepared deacetyl sophorolipid was prepared as described in example 19 with reference to PCT patent Application PCT/EP20 ll/059306. The sophorolipid aqueous solution having a solid content of 50% was reacted for 4 hours by adding 3 times molar equivalent of KOH and 2 times water, after which the reaction mixture was heated to 50 ℃, stirred for 10 minutes, and then passed through a glass fiber filter twice, and then the mixture was heated to 80 ℃ to complete the total hydrolysis and obtain deacetylated sophorolipid. The pH of the reacted mixture was adjusted to pH 1.5 using 37% HCl and left to precipitate overnight. The acidic sophorolipid was separated from the supernatant salt solution (KCl and potassium acetate) by centrifugation. The sophorolipid collected by centrifugation was washed twice with 1% diluted HCl, and the deacetylated acid sophorose ester solution was collected by centrifugation. The sophorose ester powder samples were obtained from deacetylated acid sophorose ester solutions, air dried in an air-blown drying oven and ground to a white powder.

Example 4: compositional analysis of sophorose ester samples

The composition of the sophorose ester samples was analyzed by LC-MS/MS (Ultimate 3000UHPLC-Q active LC MS). The test sample is diluted by 100 times by pure water, filtered by 0.22 mu m microporous filter membranes respectively and directly subjected to LC-MS/MS analysis. The chromatographic conditions are as follows: a chromatographic column: eclipse Plus C18100mm X4.6 mm, 5 μm; column temperature: 30 ℃; sample introduction amount: 5.0 mu L; mobile phase: a: 0.1% formic acid water; b: acetonitrile; gradient elution conditions are as in table 1:

table 1: gradient elution conditions

Sample analysis mass spectrometry conditions were as follows: mass spectrometry: thermo Scientific Q exact; an ion source: HESI; the qi rising rate: 40 mL/min; auxiliary gas rate: 10 mL/min; spray voltage: negative ions of 3.2 kV; capillary temperature: 320 ℃; temperature of the auxiliary gas: 300 ℃; s-lens: 50 percent; scanning mode: fullms/dd-ms2top 10; scanning range: primary scanning: the resolution is 70000, and the range is 100-1500 m/z; secondary scanning: resolution 17500, starting ion 50 m/z; collision voltage: NCE 30.

Example 5: comparison of deacetylation efficiency

The untreated sophorolipid, the deacetylated sophorolipid samples obtained by the methods of examples 1 to 3, were subjected to LC-MS/MS detection according to the methods listed in example 4, respectively. The detection results are summarized in Table 2, and the sophorose ester deacetylated by the sodium alkoxide method has higher deacetylation efficiency.

Table 2: peak area ratio of sophorolipid with different degrees of acetylation

Example 6: performance comparison

(1) Degree of integrity of the fatty acid side chain of the deacetylated sophorolipid

FIG. 4 is a total proton flow diagram of the deacetylated sophorolipid prepared in example 1, wherein a secondary mass spectrum of a peak with a retention time of 4.457min is shown in FIG. 5, and a secondary mass spectrum of a characteristic peak with a retention time of 4.625min is shown in FIG. 6, and a secondary mass spectrum of a characteristic peak with a fatty acid side chain of linoleic acid is shown in FIG. 5. No sophorose molecular fragments with side chain cleavage were found in the sophorose esters prepared in example 1.

FIG. 7 is a total proton flow diagram of the deacetylated sophorolipid prepared in example 3, wherein the second-order mass spectrum of the peak with retention time of 3.53min is shown in FIG. 8, and the peak is a non-acetylated sophorolipid with octenyl as a side chain (the molecular structure is shown in FIG. 9, and the molecular structure is shown in FIG. 3 for a normal non-acetylated sophorolipid molecule), which is derived from the bond breaking at the C6 position of fatty acid in the non-acetylated acid type sophorolipid of fatty acid side chains of oleic acid and linoleic acid. It is shown that the ester bond of the sophorose ester molecule is not only affected during the alkaline cleavage process of example 3, but also the fatty acid side chain of sophorose ester is partially destroyed.

(2) Height of frothing

The underivatized sophorolipid and the nonacetylated sophorolipid obtained in examples 1 to 3 were each prepared as a 0.5% aqueous solution, 200mL of each was placed in a 1000mL measuring cylinder, and stirred rapidly with a glass rod to observe the position of the highest graduation surface reached by the formation of foam on the liquid surface. The results are as follows: raw sophorose ester (reached 425mL scale), non-acetylated sophorose ester prepared in example 1 (reached 950mL scale), non-acetylated sophorose ester prepared in example 2 (over 1000mL scale), non-acetylated sophorose ester prepared in example 3 (reached 825mL scale).

(3) Color and appearance

Example 1 the sophorolipid solution prepared was transparent and pale yellow. Example 3 the sophorose ester prepared was a transparent dark brown color. Because the daily cosmetic field has higher requirements on appearance, color and texture, the light-colored transparent solution has more competitive advantages, and the decoloring cost of the product is greatly reduced in the subsequent treatment process.

Example 7: optimization of molar ratio of sodium alkoxide

2g (2.9mmoL) sophorolipid dry powder is put into a 100mL round-bottom flask which is connected with a condensing reflux pipe (the temperature of a refrigerant is 10 ℃), and is also connected with a protection pipe filled with anhydrous calcium chloride to prevent moisture in the atmosphere from influencing the reaction. During the reaction, 25mL of ethanol solutions of sodium ethoxide (the concentrations are shown in Table 3) with different concentrations were added, and the reaction was carried out at 25 ℃ for 5 hours with magnetic stirring to carry out the esterification reaction. Then, 25mL of pure water was added. The sample after the reaction was analyzed for the composition of sophorose ester according to the analysis method described in example 4, and the results are shown in Table 3. When the ratio of the amounts of the substances exceeds 1:0.2 (sophorolipid: sodium ethoxide), the sophorolipid has a higher degree of deacetylation.

Table 3: peak area ratio of sophorolipid of sodium alkoxide concentration

Claims

1. A method for deacetylation modification of sophorolipid is characterized by comprising the step of catalyzing the esterification reaction of sophorolipid by using sodium alkyl alcoholate as a catalyst under anhydrous condition.

2. The method for deacetylation modification of sophorolipid as claimed in claim 1, wherein the catalyst is added in a proportion of 0.2 times higher than the equivalent amount of sophorolipid.

3. The method for deacetylation modification of sophorolipid as claimed in claim 1, wherein the sodium alkyl alkoxide is any one of sodium ethoxide or sodium methoxide.

4. The method for deacetylation modification of sophorolipid as claimed in claim 1, wherein the anhydrous catalytic reaction conditions of sodium alkyl alcoholate are 25-30 ℃.

5. The method for deacetylation modification of sophorolipid according to claim 1, wherein the esterification reaction is performed at 25 to 30 ℃ for 5 hours or longer under magnetic stirring.

6. The method for deacetylation modification of sophorolipid as claimed in claim 1, further comprising a step of adding water after completion of the esterification reaction and recovering the organic solvent therein.

7. The method for deacetylation modification of sophorolipid as claimed in claim 6, wherein the organic solvent is methanol or ethanol.

8. The method for deacetylation modification of sophorolipid according to claim 1, wherein the sophorolipid is a natural sophorolipid.

9. A sophorolipid, characterized in that a non-acetylated sophorolipid obtained by the deacetylation modification method of a sophorolipid according to any one of claims 1 to 7 is used.

10. Sophorolipid as claimed in claim 9, wherein the non-acetylated sophorolipid is prepared from a natural sophorolipid by deacetylation modification.