KR20210068195A - Purification method of lactonic sophorolipid - Google Patents

Abstract

The present invention relates to a method for purifying lactone-type sophorolipids, and more particularly, to a method for purifying lactone-type sophorolipids with high purity by recovering oxidized and lactone-type sophorolipid precipitates produced by culturing a yeast of the genus Candida having a sophorolipid-producing ability, and performing ultrafiltration using the principle of formation of a micelle structure among the surfactant properties of sophorolipids.

Description

Purification method of lactonic sophorolipid

The present invention relates to a method for purifying a lactone-type sophorolipid, and more particularly, recovering the oxidized and lactone-type sophorolipid precipitates produced by culturing a yeast of the genus Candida having a sophorolipid-producing ability, and therefrom It relates to a method of purifying lactone-type sophorolipids with high purity through ultrafiltration using the principle of forming a micelle structure among the surfactant properties of sophorolipids.

A surfactant is a compound having a hydrophilic group and a hydrophobic group, and has a function of reducing the interfacial tension by adsorbing to the interface formed by two or more substances. Such surfactants can be broadly classified into ionic surfactants that exhibit ions in aqueous solution and nonionic surfactants that do not ionize, and as another classification criterion, chemical surfactants produced by chemical synthesis, bacteria, and yeast and biosurfactants produced by microorganisms such as fungi. Among them, biosurfactants show low toxicity to the human body and are easily biodegradable due to their simple structure and constituent materials.

In addition, when the surfactant exceeds a certain concentration in the aqueous solution state, the interfacial tension is not reduced any more and is maintained while simultaneously forming micelles. The concentration that forms micelles at this time is called Critical Micelle Concentration (CMC), and biological surfactants have a lower CMC compared to surfactants made by chemical synthesis, so they show better cleaning power than chemical surfactants. A lot of research is being done as an alternative to

Biosurfactants refer to all kinds of surfactants produced through the biosynthetic metabolic pathway of microorganisms such as bacteria, yeast and mold, and depending on the structure, glycolipid, lipopeptide and lipoprotein, neutral It is classified into Neutral lipid and Phospholipid, Polymeric surfactant and Particulate surfactant.

Sophorolipid is a surfactant with a glycolipid structure. Sophorose, in which two glucose molecules are β-1,2 bonded, and hydroxyl groups present in one molecule of unsaturated fatty acids, mainly palmitic acid (C16) and stearic acid (C18). It has a simple structure combined with , and has high cleaning power and high antibacterial activity against bacteria and fungi, so it is used in various fields such as cosmetics and food. The structure of the sophorolipid produced from yeast is largely divided into two types: a closed type of lactonic sophorolipid and an open type of acidic sophorolipid. This appears differently. The current study is that Candida bombicola, a strain of the genus Candida, is bombicola ) strain exhibits high productivity, and many studies are being conducted on the strain, and it is being used for production research.

Oxidized Sophorolipid Lactone Sophorolipid

[Formula 1] [Formula 2]

The yeast, Candida bombicola, is known to show a sophorolipid production concentration of up to 300 g/L through fed-batch culture, and in general, in the case of sophorolipid produced by culturing Candida bombicola, , the oxidized type and the lactone type are composed of 40% and 60%, respectively. The lactone-type sophorolipid exhibited a lower CMC than the oxidized sophorolipid, and had better cleaning power. Oxidized sophorolipids show little antibacterial activity against bacteria, while lactone-type sophorolipids show antibacterial activity against acne bacteria such as Propionibacterium acnes. The oxidized type has a higher HLB (Hydrophilic Lipophilic Balance) value than the lactone type, which means that the oxidized type has high solubility in water and can act as a solubilizer as a hydrophilic surfactant.

Methods for separation and purification of surfactants having a glycolipid structure including sophorolipid include purification using an organic solvent, purification using a column, etc., which include purification of a mixture of lactone and oxidized sophorolipids or single purification of oxidized sophorolipids or It may be purified differently depending on the content and purpose of the target substance, such as a single tablet of lactone-type sophorolipid. In the case of the purification method using the organic solvents hexane and ethyl acetic acid, the purity of sophorolipid is low, and if the organic solvents remain toxic to the human body, there may be restrictions on use, and purification using a column may be Recovery is difficult, the amount is small, and since an organic solvent is used, it may be a problem for the use of products such as cosmetics as in the solvent extraction method (Saraya Co., Ltd. (2015). EP Patent No. 2821495A1)

On the other hand, purification of a material using membrane filtration separates the material based on a specific pore size according to the molecular weight of the material to be purified. Microfiltration (MF), ultrafiltration (UF), nano Nanofiltration (NF) and reverse osmosis (RO) are representative examples of membrane filtration. In general, microfiltration is 0.1 to 10 μm, ultrafiltration is 10 to 100 nm, nano filtration is 1 to 10 nm, and reverse osmosis membrane filtration is commonly used as having a pore size of 1 nm or less. Separation of substances that either pass through or do not pass through the membrane.

European Patent Publication EP 2821495A: High-purity acid-form sophorolipid (sl) containing composition and process for preparing same Korean Patent Laid-Open No. 2019-0125878: Method for producing lactone-type sophorolipids

The present inventors provide a culture solution composition containing sugars and oils from a sophorolipid precipitate produced by culturing a yeast of the genus Candida capable of producing sophorolipids, which are biosurfactants, proteins produced during culture, impurities such as pigments as metabolites, and Separation and purification of oxidized sophorolipids using membrane filtration, more specifically ultrafiltration, was conducted, and as a result, the production of highly purified lactone-type sophorolipids was confirmed, thereby completing the present invention.

Accordingly, an object of the present invention is to provide a method for producing a biosurfactant, sophorolipid, particularly a lactone-type sophorolipid, from a culture solution precipitate of a yeast of the genus Candida by using ultrafiltration and purification to separate and purify the sophorolipid.

In order to achieve the above object, the present invention,

A first step of obtaining a sophorolipid mixture containing lactone-type and oxidized sophorolipid from a yeast culture medium of the genus Candida having a sophorolipid-producing ability;

a second step of dissolving the oxidized sophorolipid by mixing a buffer with the mixed solution;

a third step of filtering the mixed solution mixed with the buffer through an ultrafiltration membrane having a molecular weight cutoff of 1,000 to 3,000 Da to separate a concentrate (retantate) containing lactone-type sophorolipids in which micelles are formed;

A fourth step of recovering a filtrate (permeate) containing lactone-type sophorolipids in which micelles are decomposed by heating the concentrate (retantate) and filtering it through an ultrafiltration membrane having a molecular weight cutoff of 1,000 to 3,000 Da

It provides a method for purifying a lactone-type sophorolipid comprising a.

According to the purification method of the present invention, the sophorolipid precipitate produced through the culturing of yeast bacteria of the genus Candida having sophorolipid-producing ability in a culture solution composition including sugars and oils is recovered, and sugars and oils, which are largely the contents of the culture solution in the sediment High-purity lactone-type sophorolipids by separating and purifying impurities such as proteins produced during culturing, pigments produced through metabolic processes, and oxidized sophorolipids through micelle structure formation and molecular weight according to solubility can get

1 is a schematic diagram illustrating a series of processes for isolating and purifying lactone-type sophorolipids through ultrafiltration from sophorolipid precipitates produced through the cultivation of yeast in the genus Candida.
2 is a pre-treatment of the sophorolipid precipitate using an ultrafiltration membrane having a pore size corresponding to a molecular weight cutoff of 10,000, followed by HPLC (High-Performance Liquid Chromatography) to confirm the intermediate purity of the filtrate. will be.
3 shows the results of HPLC analysis of the purity of the finally obtained powdered lactone-type sophorolipid.

Hereinafter, the present invention will be described in detail.

The purification method of lactone-type sophorolipid of the present invention is

A first step of obtaining a sophorolipid mixture containing lactone-type and oxidized sophorolipid from a yeast culture medium of the genus Candida having a sophorolipid-producing ability;

a second step of dissolving the oxidized sophorolipid by mixing a buffer with the mixed solution;

a third step of filtering the mixed solution mixed with the buffer through an ultrafiltration membrane having a molecular weight cutoff of 1,000 to 3,000 Da to separate a concentrate (retantate) containing lactone-type sophorolipids in which micelles are formed;

A fourth step of recovering a filtrate (permeate) containing lactone-type sophorolipids in which micelles are decomposed by heating the concentrate (retantate) and filtering it through an ultrafiltration membrane having a molecular weight cutoff of 1,000 to 3,000 Da is included.

A detailed look at each step is as follows.

Step 1: Sophorolipid productivity from the culture medium of yeast bacteria of the genus Candida with rock tone and oxidized Sophorolipid containing Sophorolipid get a mixture

The yeast of the genus Candida having the sophorolipid-producing ability is a bacterium capable of producing sophorolipids endogenously or by transformation, specifically, Candida bombicola can be used.

The yeast culture medium is obtained by culturing the yeast in a culture medium, and contains the metabolites of microorganisms, and may contain microorganisms, or the microorganisms may be isolated cultures.

A sophorolipid mixed solution containing lactone-type and oxidized sophorolipids is obtained from the yeast culture solution. In this case, the lactone-type and oxidized sophorolipid precipitates can be recovered and used by using the density difference.

At this time, a pretreatment process of removing cells and cell by-products in the culture medium by filtering the sophorolipid mixture containing the lactone-type and oxidized sophorolipids with an ultrafiltration membrane having a molecular weight cutoff of 5,000 to 10,000 Da can be performed, and the filtrate ( permeate) is used in the subsequent process.

Step 2: Mixing the buffer with the mixed solution oxidized Sophorolipid dissolve

The purification method of the lactone-type sophorolipid of the present invention uses the structural characteristics of a surfactant including sophorolipid to separate and purify the lactone-type sophorolipid from the sophorolipid precipitate through ultrafiltration.

Surfactants have an amphiphilic structure having an aqueous group and a hydrophobic group at the same time, thereby forming a micelle structure under specific conditions according to temperature, pH, and the concentration of the surfactant in a solution or organic solvent. The surfactant is adsorbed to the interface to lower the interfacial tension. When the concentration is higher than a certain concentration, the interfacial tension is not reduced and the level is maintained, and the concentration is set as the critical micelle concentration (CMC). this is formed A micelle is an aggregate in which surfactant monomers are gathered and integrated.

In aqueous solution, the hydrophobic functional group of the surfactant forms a central nucleus and the hydrophilic functional group faces outward. This type of structure is called a micelle. The number of surfactant monomers in the aggregate varies depending on the structure, temperature, concentration, etc. of the material, which _{can be represented by the Aggregation Number (N Agg} ), and based on this principle, the lactone-type sophorolipid can be separated and purified.

The sophorolipid precipitate has a high viscosity and is dissolved in a buffer to provide a liquid flow in the membrane filtration unit. A buffer solution was used in order to increase the solubility of sophorolipids, particularly oxidized sophorolipids, with the composition of the aqueous phase culture medium, proteins, metabolites, and the like. To check solubility according to pH, citrate buffer pH 5.0 and phosphate buffer pH 5.5, pH 6.0, pH 6.5, pH 7.0, pH 7.5, pH 8.0 were prepared, and defoamed in each buffer Dissolution proceeded so that the lolipid precipitate was 1% concentration. As a result, the solubility was the highest in the phosphate buffer at pH 6.5, and loss such as the degradation of sophorolipids at pH 7.0 or higher was confirmed.

Step 3: The mixed solution mixed with the buffer has a molecular weight cutoff of 1,000 to 3,000 Da's with ultrafiltration membrane by filtration micelles formed lactone Sophorolipid Separate the concentrate (retantate) containing it.

Membrane filtration was performed with a phosphate buffer solution in which the sophorolipid precipitate was dissolved. Sophorolipids showed CMC of 0.1 wt% and 0.01 wt%, respectively, in the standard state of the oxidized and lactone forms, and the average number of _{sophorolipids} (Aggregation number, N Agg) above the concentration was 6, that is, Six sophorolipid monomers form one micelle. Since the molecular weight of sophorolipid is 706 g/mol for the oxidized type and 688 g/mol for the lactone type, more specifically, sophorolipid has an average micelle structure in 1 wt% phosphate buffer solution per 1 mol (mol). The oxidized form has a molecular weight of 4240 g, and the lactone form has a molecular weight of 4130 g.

Sophorolipids, especially lactone-type sophorolipids, were compared with the molecular weight of sophorolipid micelles with major constituents, proteins, and metabolites in the culture medium to select the appropriate pore size of the filtration membrane when separating lactone-type sophorolipids according to molecular weight. Among the composition of the culture medium, sugar represented by glucose as the main material is 180 g/mol, oil represented by canola oil with a high content of oleic acid is 280 g/mol, and pigment is 300 to 400 g /mol.

Based on the above information, an ultrafiltration membrane having a pore size corresponding to a molecular weight cut-off of 1,000 to 3,000 Da among membrane filtration was selected. Although the oxidized sophorolipid has a molecular weight of 4240 g/mol in the micellar structure, most of the oxidized sophorolipid dissolved in a pH 6.5 phosphate buffer and present as a monomer having a molecular weight of 706 g/mol is filtered. The lactone-type sophorolipid is hardly dissolved even in the phosphate buffer and forms a micellar structure, so it is not filtered and is concentrated in the filtration membrane.

The phosphate buffer solution of the sophorolipid precipitate was subjected to ultrafiltration using a filtration system device. When a filtration membrane having a pore size of 1,000 to 3,000 Da, more preferably 3,000 Da, is used, most substances in the culture medium, such as sugars and oils, pass through the membrane and exit as a filtrate (permeate), and do not pass through the membrane. Proteins, lactone-type sophorolipids of micellar structure, undissolved oxidized sophorolipids, and impurities with a molecular weight of 3,000 Da or more are concentrated (retantate) in the membrane. Among the ultrafiltration membranes with a molecular weight cutoff of 1,000 to 3000 Da, the ultrafiltration membrane corresponding to 3,000 Da was selected when the lactone-type sophorolipid micellar structure was used. Clogging can be minimized.

Step 4: Concentrate ( retantate ) after heating, molecular weight fraction of 1,000 to 3,000 Da's Filtration with an ultrafiltration membrane Michelle decomposed lactone Sophorolipid The filtrate (permeate) containing it is recovered.

Proteins with a molecular weight of 3000 Da or more, lactone-type sophorolipids and oxidized sophorolipids having a micellar structure that were not permeated by ultrafiltration, and a concentrate (retantate) containing impurities were recovered and the solution was heated.

This is to decompose the micellar structure formed by the sophorolipids in the solution by increasing the temperature of the solution through heating. Since sophorolipids are decomposed when heated to 120 ℃ or higher, the temperature is controlled by dividing the temperature sections of 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃, and after adjusting the temperature, the same molecular weight of 3000 Filtration was performed using a filtration membrane. As a result, there was no significant difference in the amount of lactone-type sophorolipid recovered at 50° C. or higher, and after filtration, the recovered filtrate (permeate) was dried to obtain a high-purity lactone-type sophorolipid.

Hereinafter, preferred examples and experimental examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the content of the present invention is not limited by the examples.

Example One

1-1 molecular weight cut off 10,000 Da out of bounds filter membrane Removal of cells and cell by-products in the culture medium using

In order to remove yeast cells and cell by-products remaining in the culture medium, filtration was performed using an ultrafiltration membrane corresponding to a molecular weight fraction of 10000 Da. In addition to cells and cell by-products, proteins with a molecular weight of 10,000 Daltons or more account for a significant amount of the total protein, so the molecular weight is primarily molecular weight before filtration of lactone-type sophorolipids using an ultrafiltration membrane with a pore size corresponding to a molecular weight cutoff of 1000 to 3000 Da. It serves as a pretreatment to filter out these large substances.

This is also to prevent the ultrafiltration membrane having a pore size corresponding to a cut molecular weight of 1000 to 3000 Da before filtering the lactone-type sophorolipid from clogging from proteins and impurities with a molecular weight of 10000 Daltons or more.

Substances subject to separation and purification are metabolites such as proteins and pigments, composition substances in culture including sugars and oils, and since the main purpose of filtration using an ultrafiltration membrane with a molecular weight cutoff of 10,000 Da is to remove proteins, analysis of proteins Whether or not it was removed was confirmed as follows.

Protein analysis was performed using a protein analysis kit, and a calibration curve was prepared using a UV-Vis spectroscopy using a standard protein material provided in the kit, and then the filtered sample was prepared at a specific concentration and analyzed.

In order to confirm the sophorolipid separated by filtration, the analysis of the oxidized sophorolipid and the lactone-type sophorolipid was performed using HPLC (High-Performance Liquid Chromatography). Finally, an analysis sample was prepared by dissolving sophorolipid in 20% MeOH. The column used for sophorolipid HPLC analysis is Optimapak C18, and the analysis conditions are column temperature 25 ℃ UV detector 207 nm, Acetonitrile (A) and Concentration gradient for each time period using water as the mobile phase (0-15 min 20% A, 15-40 min 80% A, 40-50 min 100% A, 50-70 min 100% A, 70-80 min 20% A ) and proceeded with a flow rate of 0.5 ml/min. The results are shown in FIG. 2 .

Referring to FIG. 2 , the peak present between 30 min and 42 min is oxidized sophorolipid, and the peak present in 51 min and 55 min is lactone-type sophorolipid, and the rest are considered impurities. did. In addition, the main purpose of the pretreatment is for the primary removal of protein, and the result of protein content analysis confirmed that it decreased from 20.5% before filtration to 2.1% after filtration.

1-2. Molecular weight cutoff using phosphate buffer 3,000 Da out of bounds filtration purification

From the sophorolipid filtrate from which cells and cell by-products, proteins and impurities with a molecular weight of 10,000 Da or more have been removed, composition materials such as sugars and oils in the culture medium, proteins with a molecular weight of 10,000 Da or less, pigments as metabolites and oxidized sophorolipids, etc. Filtration was performed using an ultrafiltration membrane having a pore size corresponding to a molecular weight cutoff of 3,000 Da to remove them.

After the pretreatment in 1-1, the filtrate (permeate) was dissolved in a pH 6.5 phosphate buffer at a concentration of 1 wt%. The phosphate buffer solution was filtered through an ultrafiltration membrane with a pore size corresponding to a molecular weight cutoff of 3,000 Da. In this process, sugars represented by glucose, oils represented by canola oil, proteins and pigments, and oxidized sophorolipids and impurities were removed. At the same time, it was confirmed that the color gradually became pale as the filtration proceeded. Most of the lactone-type sophorolipids form micellar structures and have a molecular weight of 3,000 daltons or more, so they are not filtered and are concentrated in the filtration membrane.

1-3. Molecular weight fraction after heating 3,000 Da out of bounds through filtration lactone Sophorolipid refine

A concentrate (retantate) from which most major impurities except for lactone-type sophorolipid and a small amount of oxidized sophorolipid and protein in the sophorolipid precipitate were removed, and heating was performed so that the temperature was set to 50 °C. In a phosphate buffer solution of 50 ° C or higher, the solubility of sophorolipids increases and the micellar structure is broken. Therefore, when the solution is filtered through an ultrafiltration membrane with a pore size corresponding to a molecular weight cutoff of 3000 Da, lactone-type sophorolipids and a small amount of oxidation The type sophorolipid is permeated, and proteins and impurities remaining in the solution are filtered out. Finally, the filtrate (permeate) was collected and freeze-dried, and the purity of the powdered lactone-type sophorolipid was analyzed, and the results are shown in FIG. 3 .

Referring to FIG. 3 , the purity of the sophorolipid in the dry sample was 85.2%, and the oxidized type and the lactone type were 21.3% and 63.9%, respectively. Glucose was not detected when analyzed and measured using HPLC and RI (Refractive Index) detector, and the protein content was found to be 0.1% after filtration from 20.5% before filtration.

Claims (4)

A first step of obtaining a sophorolipid mixture containing lactone-type and oxidized sophorolipid from a yeast culture medium of the genus Candida having a sophorolipid-producing ability;
a second step of dissolving the oxidized sophorolipid by mixing a buffer with the mixed solution;
a third step of filtering the mixed solution mixed with the buffer through an ultrafiltration membrane having a molecular weight cutoff of 1,000 to 3,000 Da to separate a concentrate (retantate) containing lactone-type sophorolipids in which micelles are formed;
A fourth step of recovering a filtrate (permeate) containing lactone-type sophorolipids in which micelles are decomposed by heating the concentrate (retantate) and filtering it through an ultrafiltration membrane having a molecular weight cutoff of 1,000 to 3,000 Da
A method for purifying a lactone-type sophorolipid comprising a. The method for purifying lactone-type sophorolipids according to claim 1, comprising the step of pre-treating the sophoropyride mixture of the first step by filtration through an ultrafiltration membrane having a molecular weight cutoff of 5,000 to 10,000 Da. The method of claim 1, wherein the buffer has a pH of 6.5. The method of claim 1, wherein the heating in the fourth step is performed at a temperature of 50 °C or more and less than 120 °C.

Images