CN113980070B - A kind of purification method of hydrophilic mannose erythritol lipid - Google Patents

Abstract

The invention discloses a method for purifying hydrophilic Mannose Erythritol Lipids (MELs). The hydrophilic MELs are prepared by acid hydrolysis of a MELs mixture obtained by fermentation in a methanol solution, and the main impurities in the MELs mixture are free fatty acids to form fatty acid methyl esters, which can be removed by n-hexane extraction, and finally the hydrophilic MELs with high purity are obtained. The MELs are glycolipid biosurfactants produced primarily by yeast fermentation. The method provided by the invention can realize the rapid preparation of hydrophilic MELs, can also effectively remove free fatty acid in crude MELs, realizes the efficient and rapid separation and purification of products, provides a new scheme for purifying the conventional structure of MELs, and is beneficial to promoting the industrial production and application of MELs.

Description

A kind of purification method of hydrophilic mannose erythritol lipid

Technical field

The present invention relates to a new method for the separation and purification of glycolipid biosurfactants, and in particular to a method for the purification of hydrophilic mannoserythritol lipids (MELs).

Background technique

Surfactant is a compound with an amphiphilic structure that can significantly reduce the surface tension of liquids at low concentrations. It has been widely used in many fields such as the petroleum industry, environmental engineering, and food industry, and is known as "industrial monosodium glutamate." However, most of the current surfactants are synthesized through chemical methods using petroleum as raw materials, which will bring about a series of environmental problems during their production and use. Biosurfactants are a class of surface-active amphiphilic compounds mainly produced by microbial fermentation. Compared with chemical surfactants, biosurfactants have higher structural diversity, foaming properties, environmental friendliness and unique biological activities. . Mannosylerythritol lipids (MELs) are one of the most widely studied glycolipid biosurfactants. They have good surface/interface activity, emulsifying properties and other special biological activities. They are widely used in cosmetics, pharmaceuticals, It has good application prospects in many aspects such as daily chemicals. The structures of common MELs are divided into MEL-A, B, C, and D according to the number and position of acetyl groups. Most of the natural MELs produced by fermentation are MEL-A, but MEL-A has poor water solubility, which limits its wide application. As the application market of MELs continues to expand, the preparation of more hydrophilic MELs becomes particularly important.

The main purification method for MELs is column chromatography, but column chromatography separation and purification technology is often costly, resulting in a large amount of waste of products and organic solvents, and cannot meet the needs of industrial production of MELs.

Contents of the invention

In view of the shortcomings of the existing technology, the present invention provides an efficient purification method for hydrophilic mannose erythritol lipids MELs.

The purpose of the present invention is achieved through the following technical solutions:

A method for purifying hydrophilic mannose erythritol lipids, including the following steps:

(1) Dissolve the mannose erythritol lipid obtained from the fermentation broth of Pseudozyma aphidis in methanol. The content of the solute mannose erythritol lipid should be within the solubility range of methanol. Add concentrated sulfuric acid to The volume fraction of sulfuric acid is 0.167-1.33%, react at 40-60°C for 30-120 minutes, and obtain an acidic methanol solution of hydrophilic mannose erythritol lipid.

(2) Adjust the pH value of the acidic methanol solution obtained in step (1) to 6-8, centrifuge, discard the upper liquid, and obtain a concentrated solution of hydrophilic mannose erythritol lipid.

(3) Add methanol to the concentrated solution of hydrophilic mannose erythritol lipid obtained in step (2), adjust the concentration of mannose erythritol lipid to 40-200g/L, and then add n-hexane, as described The volume ratio of the methanol solution of mannose erythritol lipid to n-hexane is 1:1-3, mix thoroughly and then centrifuge, collect the methanol phase (lower phase), use n-hexane to repeatedly wash the methanol phase, and finally obtain purification After the mannose erythritol lipid methanol solution.

(4) After drying the purified mannose erythritol lipid methanol solution obtained in step (3) under reduced pressure at 40-50°C, a pure hydrophilic mannose erythritol lipid is obtained.

Further, the mannose erythritol lipid in step (1) is the precipitate produced by the fermentation broth during the fermentation process, or is the crude product obtained after simple separation of the fermentation broth.

Further, the pH adjustment in step (2) can be done by washing with deionized water. The volume ratio of the acidic methanol solution to deionized water is preferably 1:1-3; solids such as CaCO ₃ can also be added for adjustment.

Further, in the step (3), the concentration of hydrophilic mannose erythritol lipid in the methanol phase is preferably 160g/L.

Further, in the step (3), the volume ratio of the methanol solution of the mannose erythritol lipid to n-hexane is preferably 1:3.

The beneficial effects of the present invention are:

1. The present invention is based on the fact that the acetyl group (-Ac) in MELs is easily lost under acidic conditions, thereby generating more hydrophilic MELs. In addition, under the catalysis of concentrated sulfuric acid, free fatty acids, the main impurities in MELs, easily react with methanol to generate fatty acid methyl esters. ; Then based on the polarity difference between the product and impurities, a series of new extraction systems were developed to remove impurities such as fatty acids and oils, and achieve the preparation and efficient purification of hydrophilic MELs.

2. The present invention starts from the crude MELs obtained during the fermentation process, uses chemical reactions to achieve the preparation of hydrophilic MELs, and modifies the most important impurities (free fatty acids) in the crude product, and then achieves the purification of MELs through simple extraction operations. It avoids the use of traditional column chromatography separation and solves the problem of low product concentration in existing separation processes. It is suitable for the large-scale preparation of hydrophilic MELs. Achieving efficient and rapid separation and purification of products also provides a new solution for the purification of conventional structures of MELs, which helps to promote the industrial production and application of MELs.

Description of the drawings

Figure 1 is a schematic flow chart of the preparation and purification method of hydrophilic MELs.

Detailed ways

Mannosylerythritol lipids (MELs) are a type of glycolipids mainly produced by yeasts (such as Pseudozyma aphidis) using hydrophilic (glucose) and hydrophobic carbon sources (soybean oil, olive oil) as raw materials Biosurfactants. Most of the products obtained during microbial fermentation are a mixture of MEL-A as the main component and supplemented by other structures. The impurities mainly include unconsumed vegetable oil and intermediate free fatty acids. The main structural formula of MELs obtained from fermentation is as follows:

(MEL-A: R1＝R2＝Ac; MEL-B: R1＝Ac, R2＝H; MEL-C: R1＝H; MEL-D: R1＝R2＝H, R2＝Ac)

The crude MELs in the present invention can be the fermentation broth; it can also be the semi-solid paste precipitated during the fermentation process; it can also be the extract or its concentrate after simple extraction and separation. The present invention achieves the preparation of hydrophilic MELs through acid-promoted chemical reactions, and at the same time reacts free fatty acids into more hydrophobic fatty acid methyl esters in methanol solution, significantly increasing the polarity difference between hydrophilic MELs and impurities. A corresponding new extraction system was developed to effectively remove impurities and obtain high-concentration products, which is conducive to the large-scale preparation of hydrophilic MELs.

The present invention will be further described in detail below in conjunction with the accompanying drawings and examples.

Example 1

Take 2.5g of the crude MELs paste, dissolve it in 30 ml of methanol, add 50-400 μL of concentrated sulfuric acid until the volume fraction of sulfuric acid is 0.167-1.33%, react the above mixture at 40-60°C for 30-120 minutes, and pass TLC Observe the structural changes of the product and impurities. The concentrated sulfuric acid functions as a catalyst in methyl esterification and as an H ⁺ provider in the structural change of MELs. The addition amount of acid will mainly affect the reaction time.

The rate of this reaction depends on the temperature and the amount of acid added. As shown in Table 1 below, the methyl esterification reaction of free fatty acids is relatively fast. With concentrated sulfuric acid as a catalyst, the reaction can be completed within 10-30 minutes, and the conversion rate is close to 100%. However, it takes a long time to obtain hydrophilic MELs through this reaction. As the amount of acid added increases, the time required gradually decreases, ranging from 120 to 500 minutes. But at the same time, but as the reaction time increases, MELs will degrade, resulting in product loss (>20%).

Table 1: Relationship between concentrated sulfuric acid concentration and preparation time of methyl esterification and hydrophilic MELs

Example 2

Take 2.5g of the crude MELs paste, dissolve it in 30 ml of methanol, add 50 μL (0.167%) of concentrated sulfuric acid until the volume fraction of sulfuric acid is 0.167%, and react the above mixture at 50°C for 30 minutes. After the reaction, 2 g of CaCO ₃ was added to methanol, the pH was adjusted to 6-7, and the solid was removed by filtration. Add methanol to the resulting neutral solution or concentrate by evaporation to adjust the concentration of MELs to 40-300g/L.

Divide the methanol solution of mannose erythritol lipid into three parts and add different proportions of n-hexane respectively. After the concentration adjustment, the volume ratios of the MELs methanol solution and n-hexane are 1:1, 1:2, and 1:3 respectively. .

Shake and mix thoroughly on a spin mixer, then centrifuge at 12,000 rpm for 5 minutes, and then discard the upper n-hexane phase. Then repeat the above extraction process three times with n-hexane, collect the lower layer (methanol phase), and wash repeatedly to obtain a high-concentration MELs methanol solution. Collect the methanol phase of the purified MELs and perform vacuum distillation and drying at 40-50°C to obtain pure MELs.

The results show that when the volume ratio of n-hexane to the methanol solution of mannose-erythritol lipid is greater than 2:1, the two organic solvents can be separated into layers better and the recovery of MELs can be achieved. In addition, the concentration of MELs in methanol should be <200g/L, otherwise it will affect the stratification effect and lead to a decrease in the purity and recovery rate of MELs. Therefore, based on the separation and purification effect and the amount of organic solvent used, the optimal concentration of MELs in methanol during the separation process is 160g/L, and the volume ratio of n-hexane to methanol phase is 3:1. Under this condition, the purity of the MELs obtained was close to 100%, and the recovery rate of MELs was 76.8%.

Example 3

Take 2.5g of the crude MELs paste, dissolve it in 30 ml of methanol, add 400 μL of concentrated sulfuric acid until the volume fraction of sulfuric acid is 1.33%, and react the mixture at 50°C for 120 min. After the reaction, add three volumes of pure water to the methanol phase, shake and mix evenly, centrifuge at 8000 rpm for 5 minutes, remove the upper liquid phase, and collect the neutral precipitate of hydrophilic MELs in the lower layer. The pH adjustment method in the method of the present invention is to adjust the solute of MELs so that the solubility of MELs decreases and H ⁺ can be transferred to the liquid and removed together.

Methanol was added to the resulting precipitate to dilute it to different concentrations (40-300g/L). After the concentration is adjusted, n-hexane is added to the methanol solution of MELs for extraction. The volume ratio of the methanol solution of the mannose erythritol lipid to n-hexane is 1:1-3. Shake and mix thoroughly on a spin mixer, and then Centrifuge at 12,000 rpm for 5 min, and discard the upper n-hexane phase. The above n-hexane washing process was repeated three times to almost completely remove impurities. Collect the purified high-concentration methanol phase of MELs and perform vacuum distillation and drying at 40-50°C to obtain pure MELs.

The results show that adjusting the pH by washing with water has no effect on subsequent separation and purification. The better conditions are also: the concentration of MELs in methanol is 160g/L, and the volume ratio of n-hexane to methanol phase is 3:1. At this time, the purity of MELs obtained was close to 100%, and the recovery rate was 46.63%.

Example 4

Take 2.5g of the crude MELs paste product, dissolve it in 30 ml of methanol, add 50 μL of concentrated sulfuric acid until the volume fraction of sulfuric acid is 0.167%, and react the mixture at 50°C for 30 minutes. After the reaction, add three volumes of pure water to the methanol phase, and centrifuge at 8000 rpm for 5 min. Discard the upper liquid phase and collect the MELs precipitate in the lower layer. Add methanol to the precipitate to dissolve the MELs so that the concentration of MELs is 160g/L. Then add 3 times the volume of n-hexane to the methanol phase, shake and mix thoroughly on a spin mixer, then centrifuge at 12,000 rpm for 5 min, and then discard the upper n-hexane phase. The above-mentioned n-hexane washing operation was carried out three times in total, and the impurities were almost completely removed. Collect the purified high-concentration methanol phase of MELs and perform vacuum distillation and drying at 40-50°C to obtain pure MELs. The results show that during the entire separation process, the total recovery rate of MELs is 66.37%, and the purity of MELs products can reach 100%, which is far better than the traditional separation process.

The above are only preferred examples of the patent of the present invention and are not intended to limit the scope of protection of the patent of the present invention. In addition to the above embodiments, the present invention may also have other embodiments. Any technical solution formed by adopting equivalent substitutions or equivalent changes shall fall within the protection scope required by the present invention.

Claims (7)

1. A method for purifying hydrophilic mannose erythritol lipid, which is characterized in that it includes the following steps: (1) Dissolve mannose erythritol lipid in methanol, add concentrated sulfuric acid until the volume fraction of sulfuric acid is 0.167-1.33%, and react at 40-60 ^o C for 30-120 minutes to obtain hydrophilic mannose erythritol. Acidic methanol solution of sugar alcohol lipid; the mannose erythritol lipid is obtained from the fermentation broth of Pseudozyma aphidis ; (2) Adjust the pH value of the acidic methanol solution obtained in step (1) to 6-8, centrifuge, discard the upper liquid, and obtain a concentrated solution of hydrophilic mannose erythritol lipid; (3) Add methanol to the concentrated solution of hydrophilic mannose erythritol lipid obtained in step (2) to obtain a methanol solution of mannose erythritol lipid, in which the concentration of mannose erythritol lipid is 40-200g/L, then add n-hexane, the volume ratio of the methanol solution of the mannose erythritol lipid to n-hexane is 1:1-3, mix thoroughly and then centrifuge, collect the methanol phase, and use n-hexane to The methanol phase is washed repeatedly to finally obtain the purified mannose erythritol lipid methanol solution; (4) Dry the purified mannose erythritol lipid methanol solution obtained in step (3) under reduced pressure at 40-50°C to obtain pure hydrophilic mannose erythritol lipid. 2. The purification method of hydrophilic mannose erythritol lipid according to claim 1, characterized in that the mannose erythritol lipid in the step (1) is produced by Pseudozyma aphidis during the fermentation process. The product precipitates, or is the crude product obtained after simple separation of the fermentation broth. 3. The purification method of hydrophilic mannose erythritol lipid according to claim 1, characterized in that the pH is adjusted in step (2) by washing with deionized water or adding alkaline solids. . 4. The method for purifying hydrophilic mannose erythritol lipid according to claim 3, characterized in that the alkaline solid is CaCO ₃ . 5. The purification method of hydrophilic mannose erythritol lipid according to claim 3, characterized in that the volume ratio of the acidic methanol solution and deionized water in the deionized water washing is 1:1- 3. 6. The purification method of hydrophilic mannose erythritol lipid according to claim 1, characterized in that, in the step (3), the concentration of hydrophilic mannose erythritol lipid in the methanol phase is 160g/L. 7. The purification method of hydrophilic mannose erythritol lipid according to claim 1, characterized in that in the step (3), the methanol solution of the mannose erythritol lipid and n-hexane The volume ratio is 1:3.