CN108619996B

CN108619996B - Separation and purification method of mannosylerythritol lipids

Info

Publication number: CN108619996B
Application number: CN201810382133.8A
Authority: CN
Inventors: 龙旭伟; 沈亮; 金明杰; 马科锋; 朱孟婕
Original assignee: Nanjing University of Science and Technology
Current assignee: Nanjing University of Science and Technology
Priority date: 2018-04-26
Filing date: 2018-04-26
Publication date: 2023-01-03
Anticipated expiration: 2038-04-26
Also published as: CN108619996A

Abstract

The invention discloses a separation and purification method of mannose trichophytol ester, which is used for separating and purifying MELs by utilizing a methanol-water-n-hexane ternary system based on the polarity difference of each component in a crude MELS product generated by fermentation. The MELs are green biosurfactants and are secondary metabolites generated by fermentation of aphid mimicry yeast or ustilago. The methanol-water-n-hexane ternary extraction system provided by the invention can effectively remove impurities such as grease and free fatty acid in crude MELS products, realizes efficient and rapid separation and purification of MELS, and is simple and efficient in operation process.

Description

Separation and purification method of mannosylerythritol lipids

Technical Field

The invention relates to a novel separation and purification method, in particular to a novel separation and purification method of mannose trichophytol lipids (MELs).

Background

The surfactant is called 'industrial monosodium glutamate' and has wide application in industry and daily life. However, petroleum-based chemical surfactants are mostly used at present, and a large amount of chemical surfactants can bring adverse effects on soil, water and organisms and cause great harm to the environment and human health. The biosurfactant is produced by microbial fermentation, has the advantages of low toxicity, degradability, ecological safety, high surface activity and the like, and can replace a chemical surfactant in multiple fields. Mannose Erythritol Lipids (MELs) are a class of biosurfactants produced by fermentation of pseudomyces aphidicola (Pseudozyma aphidis) or Ustilago rhodotorula (Ustilago aphidis) using fats and sugars as carbon sources, and are also one of the biosurfactants currently considered to have the most potential for use. MELs have high-efficiency surface/interface activity, and also have the functions of moisture retention, oxidation resistance and the like, and in addition, the MELs also have special physiological activity, such as inhibiting the growth of microorganisms, inducing cell differentiation, improving gene transfection efficiency and the like, so the MELs have wide application prospects in various fields and can be used for replacing chemical surfactants. However, due to the high production cost, MELs have not been widely used in industry. At the very least, the low-level and low-efficiency separation process is the main reason for the high cost.

At present, the MELs are mainly separated and purified by resin adsorption, foam fractionation, solvent extraction and the like. Wherein the resin adsorption recovery rate and the product purity are low and the operation is complicated; foam fractionation is only used to separate low concentration products and the equipment investment and operating costs are high. The solvent extraction is a separation and purification means developed based on different affinities of MELs in an organic solvent-water phase system, and is also a separation means widely researched at present. Generally, the extractant used for separating and recovering MELs includes: chloroform, ethyl acetate, methyl tert-butyl ether, cyclohexane and the like, but most solvents cannot remove free fatty acid and unconsumed grease in the fermentation liquor, so that the problems of low product purity, serious low recovery rate and the like are caused.

Disclosure of Invention

Aiming at the defects of the prior MELs separation and purification method, the invention provides the MELs separation and purification method.

The purpose of the invention is realized by the following technical scheme: a method for separating and purifying mannitol trichophytol lipid comprises the following steps:

(1) Dissolving the crude product of the mannose trichophytol lipid in methanol, adding water and n-hexane, and fully mixing, wherein the volume ratio of the methanol to the water to the n-hexane is (2-5);

(2) Separating the two phases by standing or centrifuging, and recovering the lower methanol phase rich in mannitol and erythritol;

(3) Adding methanol and water into the upper-layer normal hexane phase separated in the step 2, uniformly mixing, wherein the volume ratio of the methanol to the water to the normal hexane is 2-5;

(4) Combining the methanol phases in the step 2 and the step 3, adding n-hexane, wherein the volume of the n-hexane is 0.2-1 time of that of the methanol phase, and uniformly mixing; separating the two phases by standing or centrifuging to obtain a lower high-purity MELS methanol phase;

(5) And 4, removing the methanol from the MELS methanol phase in the step 4 through rotary evaporation and drying to obtain a pure product of the mannose trichophytol.

Further, in the step 1, the crude product of the mannose trichophytol lipid can be a fermentation liquid in a fermentation process; or solid or semi-solid paste precipitated from the fermentation liquid; or the extract liquid of the fermentation liquor extracted by the organic solvent.

Further, in the step 1, the pH of the added water is preferably 2 to 5.

Further, in the step 3, the pH of the added water is preferably 2 to 5.

Further, in the step 5, the technological parameters of the rotary evaporation drying for removing the methanol are as follows: the vacuum degree is 100-500Pa, the rotating speed is 60-150rpm, and the drying temperature is 70-80 ℃.

The beneficial effect of the invention is that,

1. the invention adopts a solvent extraction method, changes the polarity of an extraction system by adjusting the proportion of methanol, water and normal hexane according to the polarity difference of each component in crude products of MELs, removes impurities such as fatty acid, grease and the like from the crude products, and realizes the purpose of efficiently separating and purifying the MELs.

2. The method has the advantages of simple operation, low toxicity of the used solvent, and high product recovery rate and purity.

Drawings

FIG. 1 is a schematic diagram of a separation and purification scheme for MELs;

FIG. 2 shows upper and lower phase TLC patterns of each step in the extraction process.

Detailed Description

MELs are a class of biosurfactants produced by fermentation of aphidicolor (Pseudozyma aphidis) or Ustilago (Ustilago aphidis) using fats and sugars as carbon sources. Generally speaking, MELs in the fermentation broth mainly exist in four structures (MEL a, MEL B, MEL C, MEL D), each having the following structural formula:

in the invention, the crude MELS can be fermentation liquor; or semisolid paste precipitated from fermentation liquor, or extract extracted from the fermentation liquor by an organic solvent. Based on the polarity difference of components such as MELs, grease, free fatty acid and the like in crude MELs products, the invention develops a novel methanol-water-n-hexane ternary extraction system for efficiently separating and purifying the MELs. The MELS separation and purification process provided by the invention can meet the requirements of different MELS recovery rates and purities by adjusting the proportion of the ternary extraction system, and the operation process is simple and efficient.

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

As shown in fig. 1 and fig. 2, the method for separating and purifying MELs of the present invention comprises the following steps:

(1) Dissolving crude MELS in methanol, adding water and n-hexane, and mixing thoroughly;

(2) Separating the two phases by standing or centrifuging, and recovering the lower methanol phase rich in MELs;

(3) Adding methanol and water into the upper n-hexane phase separated in the step 2, uniformly mixing, separating the two phases by standing or centrifuging, and recovering the lower methanol phase;

(4) Combining the methanol phases in the step 2 and the step 3, adding n-hexane, and uniformly mixing; separating the two phases by standing or centrifuging to obtain a lower high-purity MELS methanol phase;

(5) And (4) carrying out rotary evaporation or vacuum drying on the methanol phase in the step 4 to obtain pure MELS.

The present invention will be described in detail below based on examples, and the objects and effects of the present invention will become more apparent.

Example 1

And (3) filtering the MELs-rich fermentation liquor by using filter paper, and dissolving the semi-solid filter residue by using enough methanol to obtain a methanol solution of the MELs. Then according to the methanol: water: n-hexane =4, 1, adding water and n-hexane in corresponding volumes, controlling the pH of the aqueous phase at 2,5, 7, 9, 11, mixing well on a suspension mixer for 2min, then centrifuging at 4000rpm for 2min, and taking out the lower methanol phase. As shown in Table 1 below, the separation and purification of MELs was achieved by this method, and the aqueous phase was preferably at an acidic pH.

TABLE 1

pH of the aqueous phase	Recovery of MELS
		2	72±3％
5	60±4％
		7	44±2％
9	22±5％
		11	15±5％

Example 2

And (3) filtering the MELs-rich fermentation liquor by using filter paper, and dissolving semisolid filter residues by using enough methanol to obtain a methanol solution of the MELs. According to the following methanol: water: n-hexane = 2. The results are shown in Table 2 below, and the separation of MELs can be achieved by this method.

TABLE 2

Example 3

And (3) filtering the MELs-rich fermentation liquor by using filter paper, and dissolving the semi-solid filter residue by using enough methanol to obtain a methanol solution of the MELs. According to the following methanol: water: n-hexane =2, 1. As shown in Table 3 below, the separation of MELs was achieved by this method.

TABLE 3

Example 4

And (3) filtering the MELs-rich fermentation liquor by using filter paper, and dissolving the semi-solid filter residue by using enough methanol to obtain a methanol solution of the MELs. According to the following methanol: water: n-hexane =3, 1. As shown in Table 4 below, the separation of MELs was achieved by this method.

TABLE 4

Example 5

And (3) filtering the MELs-rich fermentation liquor by using filter paper, and dissolving the semi-solid filter residue by using enough methanol to obtain a methanol solution of the MELs. According to the following methanol: water: n-hexane =2, 1, to which water and n-hexane are added, and the aqueous phase is adjusted to pH 2. Mix well on the suspension mixer for 2min and then centrifuge at 4000rpm for 2min.

And (3) taking out the extracted methanol phase, adding n-hexane which is 0.2,0.4,0.6,0.8,1 times of the total volume, uniformly mixing for 2min by a suspension mixer, centrifuging for 2min at 4000rpm, and taking out the lower methanol phase, wherein the results are shown in the following table 5, and the purpose of removing impurities can be achieved.

TABLE 5

Adding the n-hexane into the mixture according to the volume ratio	Content of impurities
		0.2	6±2％
0.4	5±1％
		0.6	5±1％
0.8	7±2％
		1	6±1％

Example 6

And (3) filtering the MELs-rich fermentation liquor by using filter paper, and dissolving the semi-solid filter residue by using enough methanol to obtain a methanol solution of the MELs. According to the following methanol: water: n-hexane =2, 1, to which water and n-hexane are added, and the aqueous phase is adjusted to pH 2. Mixing on a suspension mixer for 2min, centrifuging at 4000rpm for 2min, and taking out methanol phase.

The n-hexane phase after extraction was taken off, then methanol: water: adding methanol and water into n-hexane in proportion, mixing on a suspension mixer for 2min, centrifuging at 4000rpm for 2min, and recovering the lower methanol phase.

And (3) combining the methanol phases in the two separation steps, adding 0.5-time volume of n-hexane, shaking up, centrifuging again for layering, and recovering the bottom methanol phase. The methanol phase is dried and concentrated by a rotary evaporator under the following conditions: the pure MELs was obtained by removing methanol by rotary evaporation at 70 ℃ for 30min under a vacuum of 100Pa and 120rpm. Through the separation steps, the recovery rate of MELs can reach more than 92%, and the purity is higher than 90%.

The above description is only a preferred example of the present invention, and is not intended to limit the scope of the present invention. In addition to the above embodiments, the present invention may have other embodiments. All technical solutions formed by adopting equivalent substitutions or equivalent changes fall into the protection scope of the claims of the present invention.

Claims

1. A separation and purification method of mannosylerythritol lipids is characterized by comprising the following steps:

(1) Dissolving the crude product of the mannitol erythritol ester in methanol, adding water and n-hexane, and fully mixing, wherein the volume ratio of methanol to water to n-hexane is (2-5);

(2) Separating the two phases by standing or centrifuging, and recovering the lower methanol phase rich in mannosylerythritol lipids;

(3) Adding methanol and water into the upper n-hexane phase separated in the step (2), uniformly mixing, wherein the volume ratio of methanol to water to n-hexane is (2-5);

(4) Combining the methanol phases in the step (2) and the step (3), adding n-hexane, wherein the volume of the n-hexane is 0.2-1 time of that of the methanol phase, and uniformly mixing; separating the two phases by standing or centrifuging to obtain a lower high-purity MELS methanol phase;

(5) Removing methanol from the MELS methanol phase in the step (4) through rotary evaporation and drying to obtain a pure product of the mannitol erythritol ester; in the step (1), the pH value of the added water is 2;

in the step (3), the pH value of the added water is 2-5.

2. The method for separating and purifying mannosylerythritol lipids according to claim 1, wherein in the step (1), the crude mannosylerythritol lipids are fermentation broth in a fermentation process; or solid or semi-solid paste precipitated from the fermentation liquor; or the extract liquid of the fermentation liquor extracted by organic solvent.

3. The method for separating and purifying mannosylerythritol lipids according to claim 1, wherein in the step (5), the technological parameters for removing methanol by rotary evaporation and drying are as follows: the vacuum degree is 100-500Pa, the rotating speed is 60-150rpm, and the drying temperature is 70-80 ℃.