CN114874272B

CN114874272B - Preparation and purification method of glucose ester

Info

Publication number: CN114874272B
Application number: CN202210472471.7A
Authority: CN
Inventors: 龙旭伟; 崔天佑; 赵梦倩
Original assignee: Nanjing University of Science and Technology
Current assignee: Nanjing University of Science and Technology
Priority date: 2022-04-29
Filing date: 2022-04-29
Publication date: 2023-08-01
Anticipated expiration: 2042-04-29
Also published as: CN114874272A

Abstract

The invention discloses a preparation and purification method of glucose grease, which is prepared by acid hydrolysis and esterification of a crude sophorolipid mixture obtained by fermentation in methanol solution and then de-esterification reaction in alkaline environment. And carrying out multistage extraction on the reacted product to finally obtain the high-purity glucose fat. The glucose ester is a green biosurfactant, is a product with a structure similar to that of sophorolipid, and only one beta-D-glucose in a hydrophilic head group. The preparation and separation and purification process provided by the invention can realize the rapid preparation and purification of the glucose ester, can be used for preparing a large amount of glucose ester, and promotes the practical application of the glucose ester.

Description

Preparation and purification method of glucose ester

Technical Field

The invention relates to a novel method for preparing, separating and purifying glycolipid biosurfactant, in particular to a novel method for preparing, separating and purifying glycolipid.

Background

The surfactant is an amphiphilic compound with hydrophilic groups and hydrophobic groups, and directional adsorption is easy to occur on an interface which is mutually insoluble, so that the surface/interface tension is obviously reduced. Because the surfactant has the characteristics of dissolution, decontamination, wetting, foaming and the like, the surfactant is widely applied to various industries which are closely related to our lives, such as daily chemicals, petroleum, textiles, environmental protection, agriculture, medicines, foods and the like. Most of the surfactants used at present come from petrochemical industry, are hard to degrade and are harmful to human body and environment. Furthermore, with the continued consumption of energy, the production and use of chemical surfactants will be greatly limited. The biosurfactant is a substance which is derived from natural plants or has excellent surface activity through microbial fermentation, is low in toxicity and biodegradable, is environment-friendly, and is considered to be an ideal substitute for chemical surfactants. Sophorolipids (SLs) are a class of glycolipid biosurfactants, which are the most studied at present and are the biosurfactants for realizing industrial production and application at first. SLs fall into two main categories as to whether fatty acid chains are cyclized with glycogen or not: lactone type sophorolipids and acid type sophorolipids. Wherein the fatty acid chain end of the acid type sophorolipid contains a free carboxyl group, so that the acid type sophorolipid has pH sensitivity; in addition, the open-loop structure of acid type sophorolipids and the presence of free carboxyl groups on the fatty acid chains can provide more convenience and possibility for the synthesis of high added value derivatives of sophorolipids. The glycolipid G-C18:1 (glucolipids G-C18:1, GL) structure is similar to that of sophorolipids, except that the number of glycogen in the hydrophilic head is different: the sophorolipid consists of sophorose (two beta-D-glucoses are combined by beta-1, 2 glycosidic bonds), and the hydrophilic group of the glucolipid only contains one beta-D-glucose, so that the sophorolipid has better fat solubility, and lays a good foundation for the application of the sophorolipid in the fields of daily chemicals and the like. Therefore, how to quickly and efficiently obtain GLs is particularly important for enriching the types of the biosurfactants and promoting the high added value application of the biosurfactants.

Currently, there are bio-enzymatic methods and methods for constructing genetic strains for the preparation of GL. The enzyme preparation has strict requirements on reaction conditions, low conversion rate and difficult acquisition of the enzyme; the method for constructing the gene strain has the advantages of high cost, complex operation and multi-component mixture of products, complicated column chromatography separation, low recovery rate (less than 20 percent) and difficult mass preparation.

Disclosure of Invention

The invention provides a preparation, separation and purification method of glucose ester aiming at the defects of the prior art.

The aim of the invention is realized by the following technical scheme: the invention provides a preparation and purification method of glucose ester, which comprises the following steps:

(1) Dissolving sophorolipid in alcohol solution, adding concentrated hydrochloric acid until the volume fraction of the concentrated hydrochloric acid is 0.5-6%, and reacting at 40-60 ℃ for 8-24h to obtain acidic alcohol solution of glucose lipid esterification product;

(2) Adjusting the pH value of the acidic alcohol solution obtained in the step (1) to be more than 10.0, and reacting for 12 hours at room temperature to obtain an alkaline alcohol solution of glucose fat;

(3) Adding pure water with the volume ratio of 1:1.5-3 into the glucose fat alkaline alcohol solution obtained in the step (2), fully and uniformly mixing, and regulating the pH value to 2.0-6.0 to obtain a weakly acidic alcohol-water mixed reaction solution;

(4) Adding dichloromethane into the weakly acidic alcohol-water mixed reaction solution obtained in the step (3), fully and uniformly mixing the alcohol-water mixed reaction solution of the glucose fat and the dichloromethane according to the volume ratio of 1:1, and then standing or centrifuging to separate two phases and collecting a dichloromethane phase rich in the glucose fat;

(5) Removing the solvent from the methylene dichloride solution of the glucose ester obtained in the step (4) by rotary evaporation and vacuum drying to obtain a paste-like substance of a crude product of the glucose ester;

(6) Adding n-hexane into the solid of the crude glucose ester product obtained in the step (5) for fully mixing, washing for 3-4 times, standing or centrifuging to separate two phases, and collecting a glucose ester phase, wherein the volume ratio of the crude glucose ester product to the n-hexane is 1:5-10;

(7) And (3) drying the product liquid in the step (6) at 40-50 ℃ under reduced pressure to obtain a pure glucose fat product.

Further, the sophorolipid in the step (1) is sediment of fermentation liquor in the fermentation process, or crude products of the fermentation liquor after treatment, or acid sophorolipid, or esterification products of the acid sophorolipid, or a mixture of the acid sophorolipid and esterification products of the acid sophorolipid.

Further, the alcohol solvent in the step (1) is preferably methanol, ethanol or n-propanol.

Further, the amount of the acid added in the step (1) is preferably 3 to 6% by volume.

Further, the heating temperature in the step (1) is preferably 60 ℃.

Further, the volume ratio of the reaction solution to the pure water in the step (3) is preferably 1:1.5-2.

Further, the preferred value of the pH adjustment in the step (3) is 3.0-5.0.

Compared with the prior art, the invention has the beneficial effects that:

1. the invention is based on a two-step reaction strategy, and uses crude sophorolipid as a raw material to efficiently prepare the glucose lipid. First, in an acidic environment, crude sophorolipids are susceptible to chain scission of acetyl groups, lactone bonds and β -1,2 glycosidic bonds in the alcohol phase, thereby producing esterified products of glucose, and then, under alkaline conditions, to cleavage of lipid bonds, thereby producing glycolipids. The reaction strategy effectively avoids the formation of large amounts of homologs and products of similar structure. Subsequently, the invention develops a multistage solvent extraction method, and realizes efficient preparation and purification of the glucose ester according to the difference of the affinity and hydrophobicity between the glucose ester and impurities contained in the glucose ester.

2. The invention starts from natural sophorolipid, prepares the structural analogue glucose lipid of sophorolipid by means of chemical reaction, has simpler reaction conditions, improves the conversion rate and the recovery rate, lays a foundation for industrially preparing the glucose lipid in a large scale, avoids using the traditional biological enzyme method or constructing the gene strain fermentation method, can economically, simply and efficiently prepare, separate and purify the glucose lipid, and lays a good foundation for high added value application of the glucose lipid.

Drawings

FIG. 1 is a schematic flow chart of a method for producing and purifying a glucose ester GL.

Detailed description of the preferred embodiments

Reference will now be made in detail to exemplary embodiments, examples of which are illustrated in the accompanying drawings. When the following description refers to the accompanying drawings, the same numbers in different drawings refer to the same or similar elements, unless otherwise indicated. The implementations described in the following exemplary examples do not represent all implementations consistent with the invention. Rather, they are merely examples of apparatus and methods consistent with aspects of the invention as detailed in the accompanying claims.

The following describes in detail a method for producing and purifying a glycolipid according to the present invention with reference to the accompanying drawings. The features of the examples and embodiments described below may be combined with each other without conflict.

The glycolipid surfactant is a surfactant with a hydrophilic group as a glycosyl group and a hydrophobic group as saturated or unsaturated fatty alcohol and fatty acid chains. Sophorolipids are a class of biosurfactants produced by fermentation of candida bumblebee (Candida bombicola) with oil and sugar as carbon sources. In general, sophorolipids in fermentation broths exist mainly in two structures (acid-type sophorolipids and lactone-type sophorolipids), and the respective structural formulas are shown below:

the product of the invention, namely the glycolipid G-C18:1 (glucolipids G-C18:1, GL), is a structural analogue of sophorolipids, except that the number of sugars in the hydrophilic head group is different: sophorolipids consist of sophorose (two beta-D-glucoses are bound by a beta-1, 2 glycosidic bond), whereas the hydrophilic head of the glycolipid is a beta-D-glucosyl element. The structure is as follows:

the sophorolipid crude product of the reaction substrate can be semisolid paste separated out in the fermentation process, can be the crude product of fermentation liquor after acid precipitation sterilization treatment, and can be acid type sophorolipid or the esterification product of acid type sophorolipid. According to the invention, the preparation of GL is realized by a chemical modification method, and hydrophilic impurities such as glucose, alkyl glycoside and the like and hydrophobic impurities such as grease, free fatty acid and the like in the GL crude product are effectively removed by a solvent extraction method based on the hydrophilic-hydrophobic difference between GL and impurities contained in GL, so that GL separation and purification are realized economically, rapidly and efficiently.

The invention will be described in further detail with reference to the drawings and examples.

As shown in FIG. 1, the present invention provides a method for preparing and purifying a glucose ester, which is prepared by hydrolyzing and esterifying a crude sophorolipid mixture obtained by fermentation in methanol solution by acid, and then performing de-esterification reaction in alkaline environment. And carrying out multistage extraction on the reacted product to finally obtain the high-purity glucose fat. The glucose ester is a green biosurfactant, is a product with a structure similar to that of sophorolipid, and only one beta-D-glucose in a hydrophilic head group. The method specifically comprises the following steps:

(1) Dissolving sophorolipid in alcohol solution, adding concentrated hydrochloric acid to volume fraction of 0.5-6%, preferably 3% -6%. The method comprises the steps of carrying out a first treatment on the surface of the Reacting at 40-60 ℃ for 8-24h, preferably heating at 60 ℃, to obtain an acidic alcohol solution of the esterified product of the glucose fat; the alcohol solvent is preferably methanol, ethanol or n-propanol.

(3) Adding pure water, preferably 1:1.5-2, in a volume ratio of 1:1.5-3 into the glucose fat alkaline alcohol solution obtained in the step (2), and fully and uniformly mixing, and then adjusting the pH value to 2.0-6.0, preferably 3.0-5.0, so as to obtain a weakly acidic alcohol-water mixed reaction solution;

Example 1

Taking 0.1g of sophorolipid crude product, dissolving in 5ml of methanol, adding concentrated hydrochloric acid until the volume fraction of the concentrated hydrochloric acid is 0.5-6%, reacting at 60 ℃, observing structural changes of the product and impurities by TLC, and recording the time for the product to be normalized to one glycolipid point. Then adjusting the pH of the acidic methanol solution to be more than 10.0, and reacting for 12 hours at room temperature to obtain an alkaline methanol solution of the glucose ester, detecting the structural change of the product by TLC, wherein the conversion rate of the product is 100%.

The rate of the reaction depends on the amount of acid added. As shown in the following Table 1, sophorolipids of different structures can be completely converted into glucose methyl ester products and alkyl glycosides under the condition of using concentrated hydrochloric acid as a catalyst. When the acid addition amount is 0.5-6%, the time required for completely converting sophorolipid into the mixture of glucose methyl ester and alkyl glycoside gradually decreases along with the increase of the acid addition amount, and the conversion rate is close to 100 percent, wherein the time is 6.5-23 hours. As shown in the following Table 1, the conversion time is shorter and the conversion efficiency is higher when the volume fraction of the concentrated hydrochloric acid is preferably 3% -6%. And as the reaction time is prolonged, the methyl glucose ester is gradually converted into alkyl glycoside, resulting in loss of product.

Table 1: time table for preparing concentrated hydrochloric acid concentration and glucose methyl ester product

Concentrated hydrochloric acid addition (v/v)	Time for complete conversion to methyl glucose ester (h)
		0.5％	23
1％	10.5
		3％	7.5
6％	6.5

Example 2

Taking 0.1g of sophorolipid crude product, dissolving in 5ml of methanol, adding concentrated hydrochloric acid with volume fraction of 6%, reacting at different temperatures, respectively at room temperature and 40 ℃ and 60 ℃, observing structural changes of the product and impurities by TLC, and recording the time for the product to be normalized to one glycolipid point. Then adjusting the pH of the acidic methanol solution to be more than 10.0, and reacting for 12 hours at room temperature to obtain an alkaline methanol solution of the glucose ester, detecting the structural change of the product by TLC, wherein the conversion rate of the product is 100%.

The rate of the reaction also depends on the heating temperature at the time of the reaction. The sophorolipids of different structures were normalized for structure under acid catalysis as shown in table 2 below. This reaction requires energy intake and the reaction speed increases with increasing temperature. At room temperature-60 ℃, the time required for converting the sophorolipid into the mixture of the glucose methyl ester and the alkyl glycoside is gradually reduced, and the end point can be reached only for 6.5 hours at 60 ℃, and the conversion rate is close to 100%. But at room temperature, the time at 48h was not yet fully converted to methyl glucose. Therefore, the heating temperature is preferably 60 ℃.

Table 2: reaction temperature and preparation time of glucose methyl ester product

Reaction temperature (. Degree. C.)	Time for complete conversion to methyl glucose ester (h)
		Room temperature	>48
40	30
		60	6.5

Example 3

Taking 0.1g of sophorolipid crude product, dissolving in 5ml of branched alcohol solutions of different types, namely methanol, ethanol, n-hexanol and n-octanol respectively, adding concentrated hydrochloric acid with the volume fraction of 6%, reacting at 60 ℃, observing structural changes of the product and impurities by TLC, and recording the time for the product to be normalized to one glycolipid point. Then, the pH of the acidic methanol solution is adjusted to be more than 10.0, the reaction is carried out for 12 hours at room temperature, and an alkaline methanol solution of the glucose fat is obtained, and the structural change of the product is detected by TLC.

The results show that: similar reactions can occur with different types of linear alcohols, and there is no significant difference in reaction time. Namely, the sophorolipid can be converted into the glucose ester derivative and the alkyl glycoside with different chain lengths by heating under the condition of taking concentrated hydrochloric acid as a catalyst, the specific shift value of the glucose ester derivative and the impurity alkyl glycoside on TLC gradually becomes larger along with the increase of the length of the straight-chain alcohol, and the hydrophobicity of the substance is enhanced. The glycolipid conversion rate of different alcohols can reach 100 percent.

Example 4

0.1g of the crude sophorolipid product is taken and dissolved in 5ml of methanol solution, concentrated hydrochloric acid with the volume fraction of 3% is added, and the reaction is carried out for 8 hours at 60 ℃. Then adjusting the pH value of the acidic methanol solution to be more than 10.0, and reacting for 12 hours at room temperature to obtain the alkaline methanol solution of the glucose ester, wherein the conversion rate of the product is 100%. Adding pure water with the volume ratio of 1:0-3 into glucose fat alkaline methanol solution, fully and uniformly mixing, regulating the pH value to 1.0-10.0, adding dichloromethane solvent with the volume ratio of 1:1 into the methanol-water mixed reaction solution of glucose fat, fully and uniformly mixing, standing overnight to separate two phases, discarding the upper methanol-water mixed solution, and collecting dichloromethane phase rich in glucose fat. In the invention, the reason for adding pure water and adjusting the pH is that the delamination of the alcohol solution and the methylene dichloride is facilitated, and the separation of hydrophilic impurities and the crude product of the glucose ester is realized. The methylene chloride was removed by rotary evaporation and vacuum drying, and then n-hexane was added to the crude glucose ester product in an oily liquid in a volume ratio of 1:5-10, and thoroughly mixed, and after washing 3-4 times, the n-hexane phase was discarded. And then the glucose fat is dried under reduced pressure at the temperature of 40-50 ℃ to obtain the pure glucose fat product.

The results show that: pure water is added into the methanol reaction liquid, and the pH value of the methanol-water mixed solution is reduced, so that the layering of dichloromethane and the mixed solution and the removal of hydrophilic alkyl glycoside impurities can be realized. When the volume ratio of the added water is 1:0-1.5, effective layering cannot be achieved, and in addition, effective extraction of the glucose ester cannot be achieved when the pH of the mixed solution of methanol and water is adjusted to 6.0-10.0. The preferable condition of the volume ratio of the added water is 1:1.5-2.0, and the preferable condition of adjusting the pH of the methanol-water mixed solution is 3.0-5.0. At this time, the total recovery rate of the glycolipid was 50% -60%, and the purity of the glycolipid was 95%.

Example 5

5.23g of the crude sophorolipid product after the preliminary acid precipitation treatment is taken and dissolved in 50ml of methanol solution, and is added with 3% concentrated hydrochloric acid to react for 13.5 hours at 60 ℃. Then adjusting the pH value of the acidic methanol solution to be more than 10.0, and reacting for 12 hours at room temperature to obtain the alkaline methanol solution of the glucose ester, wherein the conversion rate of the product is 100%. Adding pure water with the volume ratio of 1:1.5-2 into glucose fat alkaline methanol solution, fully and uniformly mixing, regulating the pH value to 3.0-5.0, adding dichloromethane solvent with the volume ratio of 1:1 into the methanol-water mixed reaction solution of glucose fat, fully and uniformly mixing, standing overnight to separate two phases, discarding the upper methanol-water mixed solution, and collecting dichloromethane phase rich in glucose fat. The methylene chloride was removed by rotary evaporation and vacuum drying, and then n-hexane was added to the crude glucose ester product in an oily liquid in a volume ratio of 1:5-10, and thoroughly mixed, and after washing 3-4 times, the n-hexane phase was discarded. And then the glucose fat is dried under reduced pressure at the temperature of 40-50 ℃ to obtain the pure glucose fat product. The results show that: in the whole separation process, the total recovery rate of the glucose ester is 50% -60%, and the purity of the glucose ester is more than 95%.

In summary, the invention develops a multistage solvent extraction method, and realizes efficient preparation and purification of the glucose ester according to the difference of the affinity and hydrophobicity between the glucose ester and impurities contained in the glucose ester. The preparation and separation and purification process provided by the invention can realize the rapid preparation and purification of the glucose ester, can be used for preparing a large amount of glucose ester, and promotes the practical application of the glucose ester. The invention starts from natural sophorolipid, prepares the structural analogue glucose lipid of sophorolipid by means of chemical reaction, has simpler reaction conditions, improves the conversion rate and the recovery rate, lays a foundation for industrially preparing the glucose lipid in a large scale, avoids using the traditional biological enzyme method or constructing the gene strain fermentation method, can economically, simply and efficiently prepare, separate and purify the glucose lipid, and lays a good foundation for high added value application of the glucose lipid.

The foregoing is merely a preferred example of the present invention and is not intended to limit the scope of the present invention. In addition to the embodiments described above, other embodiments of the invention are possible. All technical schemes formed by adopting equivalent replacement or equivalent variation fall within the protection scope of the invention.

Claims

1. A method for preparing and purifying a glucose ester, comprising the steps of:

2. The method for producing and purifying a glucose ester according to claim 1, wherein the sophorolipid in the step (1) is a precipitate of a fermentation liquid during fermentation, or a crude product of a fermentation liquid after treatment, or an esterified product of acid sophorolipid, or a mixture of acid sophorolipid and an esterified product of acid sophorolipid.

3. The method for producing and purifying a glucose ester according to claim 1, wherein the alcoholic solvent in the step (1) is methanol, ethanol or n-propanol.

4. The method for producing and purifying a glucose ester according to claim 1, wherein the amount of the acid added in the step (1) is 3% to 6% by volume.

5. The method for producing and purifying a glycolipid according to claim 1, wherein the heating temperature in the step (1) is 60 ℃.

6. The method for producing and purifying a glucose ester according to claim 1, wherein the volume ratio of the reaction solution to the pure water in the step (3) is 1:1.5-2.

7. The method for producing and purifying a glycolipid according to claim 1, wherein the pH is adjusted to 3.0 to 5.0 in the step (3).