CN114032261A

CN114032261A - Method for obtaining high-purity fucosyllactose by degrading lactose in fermentation liquor

Info

Publication number: CN114032261A
Application number: CN202111179170.7A
Authority: CN
Inventors: 袁丽霞; 陈祥松; 吴金勇; 李翔宇; 李忠奎; 姚建铭; 孙立洁; 王刚; 郑家妹; 费贤春
Original assignee: Hefei Institutes of Physical Science of CAS; Cabio Biotech Wuhan Co Ltd
Current assignee: Hefei Institutes of Physical Science of CAS; Cabio Biotech Wuhan Co Ltd
Priority date: 2021-10-09
Filing date: 2021-10-09
Publication date: 2022-02-11

Abstract

The invention belongs to the field of separation and purification of fucosyllactose, and particularly relates to a method for obtaining high-purity fucosyllactose by degrading lactose in fermentation liquor, which is based on analysis of components in the fucosyllactose fermentation liquor, constructs a purification method with stronger pertinence, sequentially removes thalli in the fermentation liquor, carries out enzymolysis on the lactose in the fermentation liquor, carries out heat treatment and activated carbon adsorption to remove macromolecular proteins, removes micromolecular substances such as galactose, glucose and partial salts in the fermentation liquor through filter membranes with different pore diameters, further removes salts through anion-cation exchange resin, and is dried and dehydrated, and the purity of the fucosyllactose treated by the steps is not lower than 95%. The method for purifying the fermentation liquor has simple process, does not depend on expensive import equipment, can achieve higher purity based on conventional equipment, has low cost and good purification effect, and has great popularization and application values.

Description

Method for obtaining high-purity fucosyllactose by degrading lactose in fermentation liquor

Technical Field

The invention belongs to the field of separation and purification of fucosyllactose, and particularly relates to a method for obtaining high-purity fucosyllactose by degrading lactose in fermentation liquor.

Background

2 '-fucosyllactose (2' -FL) is the oligosaccharide with the highest content in human milk. The biological function of the compound is a hot spot of international nutrition research at present. A plurality of research results suggest that the 2' -fucosyllactose has a plurality of functional activities of regulating intestinal flora, resisting the adhesion of pathogenic bacteria, regulating immunity, promoting the development and repair of a nervous system and the like.

The separation and purification of high purity 2 '-fucosyllactose from 2' -fucosyllactose fermentation broth is a key problem. The existing method for separating and purifying fucosyllactose from fermentation liquor is complex, depends on expensive equipment and has high cost, and the industrial cost is extremely high when monosaccharide and disaccharide in the fermentation liquor are removed by using a simulated moving chromatographic bed technology (AU2014331091A1, EP2857410A 1).

Furthermore, as in several of the JENNEWEIN incorporated patents: patent AU2014331091A1 uses simulated moving bed chromatography technology to separate and purify, and then carries out spray drying to obtain neutral human milk oligosaccharide; EP2857410a1 uses simulated moving bed chromatography to purify 2' -FL, followed by spray drying; EP2896628B1, neutral HMOs are purified, wherein biomass is removed by adopting an ultrafiltration membrane capable of intercepting 1-10 million daltons, desalting is carried out by ion exchange, filtering is carried out by adopting a membrane of 3K daltons, and then, sterilization and endotoxin removal are carried out, and then, concentration and spray drying are carried out; the overall process is cross-flow filtration, cation and anion exchanger treatment, concentration, electrodialysis, concentration, activated carbon treatment, 3k dalton membrane filtration, and finally spray drying. Mainly adopts ultrafiltration membrane technology, ion exchange technology, spray drying technology and the like, and has extremely complicated treatment process.

Glycom corporation for 2 ' -fucosyllactose crystallization patents such as WO2016095924A1, through ion exchange desalination, 5 ~ 50KD membrane ultrafiltration pretreatment, and the requirements of the concentrated solution of carbohydrate content is preferably greater than 50%, 2 ' -FL/DFL ratio is greater than 2:1, the final product 2 ' -FL purity is greater than 92%, DFL content < 3%, acetic acid content < 3%.

Aiming at the problems of complex purification process and high cost of the fucosyl lactose at present, a purification method which has simple working procedures and low cost and can obtain higher fucosyl lactose purity is needed to be provided.

Disclosure of Invention

Aiming at the problems of high cost, complex process and difficult further improvement of product purity of fucosyllactose separation and purification in the prior art, the invention aims to provide a method for obtaining high-purity fucosyllactose by degrading lactose in fermentation liquor, the process is simple, the efficiency is high, the cost is low, and the purity of the fucosyllactose prepared by the method is not lower than 95%.

Based on the above purpose, the technical scheme adopted by the invention is as follows:

in a first aspect, the present invention provides a method for obtaining high-purity fucosyllactose by degrading lactose in a fermentation broth, comprising the following steps:

s1: filtering fucosyl lactose fermentation liquor by a 30-200 nm filter membrane to remove thalli, and collecting the aseptic fermentation liquor;

s2: adding lactase into S1 sterile fermentation liquid, degrading lactose in the fermentation liquid under the conditions that the pH is 6-7 and the temperature is 32-45 ℃ until no free lactose is detected in the sterile fermentation liquid;

s3: adjusting the pH of the fermentation liquor treated by the S2 to 9.5-10.5, performing heat treatment to denature and precipitate protein in the fermentation liquor, and adjusting the pH of the fermentation liquor to 6-7 after the heat treatment is finished; then adding activated carbon into the fermentation liquor, stirring for 30-60 min, and filtering through a filter membrane of 0.22-0.45 μm to remove the activated carbon and denatured protein;

s4: filtering the fermentation liquor treated by S3 through a 1000-3000Da membrane to collect filtrate, filtering the filtrate through a 500-600 Da membrane, and collecting trapped concentrated solution;

s5: and adsorbing and desalting the trapped concentrated solution treated by the S4 by cation exchange resin and anion exchange resin, and drying to obtain the high-purity fucosyllactose.

The invention is based on analyzing the main components of fucosyl lactose fermentation liquor, constructing a high-efficiency purification treatment mode aiming at the fermentation liquor, preferentially removing thalli in the fermentation liquor, further performing enzymolysis on lactose in sterile fermentation liquor into micromolecular galactose and glucose, simultaneously performing denaturation precipitation on macromolecular proteins in the fermentation liquor by heat, adsorbing the denatured proteins by using activated carbon, decoloring the fermentation liquor, filtering to remove the activated carbon and the denatured proteins, removing micromolecular impurities such as galactose, glucose and partial salt by using filtering membranes with different pore diameters, performing interception and concentration, further removing salt in concentrated solution by using cation and anion exchange resin, and drying, wherein the purity of the fucosyl lactose treated by the steps is not lower than 95%.

The fucosyl lactose fermentation liquor is purified by the method, the process is simple, the used equipment is conventional equipment, the cost is low, the highly targeted fermentation liquor purification treatment method is constructed by analyzing the components in the fermentation liquor, the fermentation liquor can be purified based on the conventional equipment, and the purity of the purified fucosyl lactose is higher.

Further, the lactase added in step S2 is calculated by the weight percentage of the sterile fermentation liquid, specifically, 0.5 wt% to 1 wt% of the weight of the sterile fermentation liquid.

According to the invention, through adding a proper amount of lactase into the sterile fermentation broth, the problems that the lactase with low addition amount is not thoroughly decomposed on lactose in the fermentation broth, the time consumption is long, the efficiency is low, the lactase with high addition amount is prevented from remaining in the fermentation broth, and new impurities are introduced into the fermentation broth are avoided, so that multiple experiments show that the addition amount of the lactase is more proper, namely 0.5-1 wt%, and the problems can be effectively avoided.

Further, the specific process of performing the heat treatment on the fermentation broth in step S3 is as follows: heating the fermentation liquor to 80-90 ℃, keeping the temperature for reaction for 0.5-1 h, and cooling to 40-45 ℃.

The method adjusts the pH value of lactase to be alkaline after the lactase enzymolysis treatment, carries out heat treatment, is not only used for carrying out denaturation precipitation treatment on the original protein in the fermentation liquor, but also carries out denaturation precipitation treatment on the residual lactase added into the system in the step S2, and effectively removes macromolecular organic matters in the fermentation liquor.

Further, the adding amount of the activated carbon in the fermentation liquid obtained in the step S3 is, in terms of the weight percentage of the fermentation liquid treated with S2, specifically 0.5 wt% to 4.0 wt% of the weight of the fermentation liquid.

The purpose of adding activated carbon into the system is to adsorb denatured protein and decolorize the fermentation liquor, and the activated carbon can be filtered and removed by a filter membrane with a proper pore size, so that the activated carbon introduced into the system has no residue, and the denatured protein and colored substances in the fermentation liquor are removed together.

Further, step S4 is to pass the filtrate through a 500 Da-600 Da membrane, and the specific process of collecting the trapped concentrated solution is as follows: adding water with the weight of 4-6 times of that of the filtrate filtered by the 1000-ion 3000Da membrane, and collecting the trapped concentrated solution through the 500-600 Da membrane.

Filtering the filtrate by a 1000-3000Da membrane to further remove macromolecular impurities and small molecular impurities, collecting the filtrate, adding sufficient water into the filtrate, dissolving water-soluble substances such as micromolecular galactose, glucose and partial salt in the filtrate by using excessive water during the filtration by a 500-600 Da membrane, and filtering the water-soluble substances by the 500-600 Da membrane to remove the water-soluble substances such as micromolecular galactose, glucose and partial salt, and collecting concentrated solution intercepted on the membrane, wherein the removal rate of the glucose and the galactose in the filtrate in the treatment process is more than 95%.

Further, step S5 is to desalt the trapped concentrated solution with cation exchange resin and anion exchange resin until the conductivity of the trapped concentrated solution is lower than 400 μ S/cm.

Further, the anion exchange resin is a polyacrylic acid skeleton macroporous weak base anion exchange resin; the cation exchange resin is strong-acid styrene cation exchange resin.

Furthermore, the fucosyllactose fermentation liquid is prepared by fermenting Escherichia coli with a preservation number of CCTCC NO: M2020027 as a fermentation strain.

Based on fucosyllactose fermentation liquor prepared by taking escherichia coli constructed earlier by the inventor as a fermentation strain as a basis (see CN111808790A escherichia coli and application thereof in fucosylation oligosaccharide synthesis), fucosyllactose in the fermentation liquor is further separated and purified to prepare high-purity fucosyllactose.

In a second aspect, the present invention provides a high purity fucosyllactose prepared by the above method.

Further, the purity of the high-purity fucosyllactose is not less than 95%.

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

the invention constructs a purification method with stronger pertinence based on the analysis of components in fucosyl lactose fermentation liquor, sequentially removes thalli in the fermentation liquor, carries out enzymolysis on lactose in the fermentation liquor, carries out heat treatment and active carbon adsorption to remove macromolecular proteins, removes micromolecular substances such as galactose, glucose and partial salts in the fermentation liquor through filter membranes with different pore diameters, further removes salt through anion and cation exchange resin, and is dried and dehydrated, and the purity of the fucosyl lactose treated by the steps is not lower than 95%. The method for purifying the fermentation liquor has simple process, does not depend on expensive import equipment, can achieve higher purity based on conventional equipment, has low cost and good purification effect, and has great popularization and application values.

Drawings

FIG. 1 is a schematic flow diagram of the process of the present invention.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples. It will be understood by those skilled in the art that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The test methods used in the examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available unless otherwise specified.

Example 1

This example provides a method for obtaining high-purity fucosyllactose by degrading lactose in a fermentation broth, the process is shown in fig. 1, and the method comprises the following steps:

s1 membrane-passing sterilized body: filtering fucosyllactose fermentation liquor by a 30nm ceramic membrane, intercepting thalli, and collecting the bacteria-free fermentation liquor, wherein the fucosyllactose fermentation liquor is prepared by fermenting escherichia coli with a preservation number of CCTCC NO: M2020027 as a fermentation strain constructed by the inventor in the earlier stage, and the fucosyllactose fermentation liquor is specifically shown in the embodiment 2 in CN111808790A escherichia coli and application thereof in synthesizing fucosyloligosaccharide.

S2 degrades lactose: 0.5 wt% lactase is added into S1 sterile fermentation liquor, and lactose in the fermentation liquor is degraded under the condition that the pH value is 6.7 and the temperature is 37 ℃, until no free lactose is detected in the sterile fermentation liquor.

S3 deproteinization and decolorization: adding a sodium hydroxide solution into the fermentation liquor treated in the step S2, adjusting the pH of the fermentation liquor to 10, then heating the fermentation liquor to 90 ℃, keeping the temperature for reaction for 0.5h to denature and precipitate protein, then cooling to 45 ℃, and adding hydrochloric acid into the cooled fermentation liquor to adjust the pH of the fermentation liquor to 7; then, adding 0.5 wt% of bamboo stream 305 activated carbon into the cooled fermentation liquor, fully stirring for 60min, fully adsorbing and precipitating protein by using the activated carbon, carrying out decoloration treatment on the fermentation liquor, and filtering by using a 0.45-micron filter membrane to remove the activated carbon and denatured protein.

S4 fine filtration and concentration: collecting the filtrate by passing the fermentation liquor treated by S3 through a 1000-3000Da membrane, further removing macromolecular impurities and micromolecular impurities in the filtrate, adding water with the weight 5 times of that of the filtrate, and passing through a 600Da membrane to collect trapped concentrated solution; the water-soluble substances such as small molecular galactose, glucose and partial salt in the filtrate are dissolved by using excessive water and are further filtered by a 600Da membrane, and the removal rate of glucose and galactose in the filtrate is more than 95%.

S5 desalting: and (4) sequentially adsorbing and desalting the trapped concentrated solution treated by the S4 by anion and cation exchange resins until the conductivity of the trapped concentrated solution is lower than 400 mu S/cm. Wherein the anion exchange resin is polyacrylic acid skeleton macroporous weak base anion exchange resin D315 (Anhui Samsung resin Co.); the cation exchange resin is strong acid styrene cation exchange resin 001X7 (Sanxing resin Co., Anhui).

S6 drying and dehydrating: spray drying the filtrate treated with S5 at 120 deg.C to obtain white solid powder, i.e. high purity fucosyllactose, with yield of 80% and purity of 95%.

Example 2

s1 membrane-passing sterilized body: filtering fucosyllactose fermentation liquor by a ceramic membrane of 200nm, intercepting thalli, and collecting the bacteria-free fermentation liquor, wherein the fucosyllactose fermentation liquor is prepared by fermenting escherichia coli with a preservation number of CCTCC NO: M2020027 as a fermentation strain constructed by the inventor in the earlier stage, and the fucosyllactose fermentation liquor is specifically shown in the embodiment 2 in CN111808790A escherichia coli and application thereof in synthesizing fucosyloligosaccharide.

S2 degrades lactose: adding 1 wt% lactase into S1 sterile fermentation liquid, degrading lactose in the fermentation liquid at pH 6.7 and 37 deg.C until no free lactose is detected in the fermentation liquid.

S3 deproteinization and decolorization: adding a sodium hydroxide solution into the fermentation liquor treated in the step S2, adjusting the pH of the fermentation liquor to 10, then heating the fermentation liquor to 80 ℃, carrying out heat preservation reaction for 1h to denature and precipitate protein, then cooling to 45 ℃, and adding hydrochloric acid into the cooled fermentation liquor to adjust the pH of the fermentation liquor to 7; then, adding 4.0 wt% of bamboo stream 305 activated carbon into the cooled fermentation liquor, fully stirring for 60min, fully adsorbing and precipitating protein by using the activated carbon, carrying out decoloration treatment on the fermentation liquor, and filtering by using a 0.45-micron filter membrane to remove the activated carbon and denatured protein.

S4 fine filtration and concentration: collecting the filtrate by passing the fermentation liquor treated by S3 through a 1000-3000Da membrane, further removing macromolecular impurities and micromolecular impurities in the filtrate, adding water with the weight 5 times of that of the filtrate, and passing through a 500Da membrane to collect trapped concentrated solution; the water-soluble substances such as small molecular galactose, glucose and partial salt in the filtrate are dissolved by using excessive water and are further filtered by a 500Da membrane, and the removal rate of glucose and galactose in the filtrate is more than 95%.

S6 drying and dehydrating: spray drying the filtrate treated with S5 at 150 deg.C to obtain white solid powder, i.e. high purity fucosyllactose, with yield of 61.6% and purity of 95.3%.

Compared with example 1, the addition amount of the activated carbon in this example is relatively high, the activated carbon of the filtering membrane forms an activated carbon filter cake, the filter cake contains feed liquid which cannot be collected, so that the net loss of the feed liquid is caused, and the yield of the final product is reduced, therefore, the addition amount of the activated carbon is not suitable to be too high.

Example 3

The present example is intended to investigate the influence of the addition of lactase on the yield and purity of fucosyllactose finally obtained, the specific experimental process refers to example 1, the addition of lactase of each sample in this example is shown in table 1, and the corresponding product yield and purity are also shown in table 1.

TABLE 1 influence of lactase addition on fucosyllactose yield and purity

As can be seen from Table 1, when the addition amount of lactase is in the range of 0 wt% -1.2 wt%, the purity of fucosyl lactose in the final dried sample is gradually improved along with the increasing of the addition amount of lactase, and when the addition amount exceeds 0.5 wt%, the addition amount of lactase is increased, so that the purity of fucosyl lactose is not obviously improved, and therefore, the addition amount of lactase is preferably selected to be 0.5 wt% -1 wt%. In addition, the test found that lactase addition amount has little influence on the yield of fucosyllactose.

Example 4

In this example, the influence of the added amount of activated carbon on the yield and purity of finally prepared fucosyllactose is studied, the specific experimental process refers to example 1, the added amount of lactase of each sample in this example refers to table 2, and the corresponding product yield and purity are recorded in table 2.

TABLE 2 influence of the amount of activated carbon added on the yield and purity of fucosyllactose

Test specimen	Adding amount of activated carbon	Fucosyllactose yield	Fucosyllactose purity
				Sample No. 1	0wt％	84％	89.4％
Sample No. 2	0.5wt％	80％	95％
				Sample No. 3	1.0wt％	77.3％	95.2％
Sample No. 4	1.5wt％	74.5％	95.3％
				Sample No. 5	2.0wt％	69.8％	95.3％
Sample No. 6	2.5wt％	66.5％	95.3％
				Sample 7	3.0wt％	64.9％	95.3％
Sample 8	3.5wt％	63.2％	95.3％
				Sample 9	4.0wt％	61.6％	95.3％

As can be seen from table 2, when the amount of the activated carbon is in the range of 0 wt% to 2.5 wt%, the yield of fucosyllactose gradually decreases with the increase of the amount of the activated carbon, and the purity of fucosyllactose does not change significantly after the amount of the activated carbon exceeds 0.5 wt%. Therefore, the optimum amount of the activated carbon to be added is 0.5 wt%.

Comparative example 1

This comparative example differs from example 1 only in step S4, the procedure of step S4 of this comparative example is as follows:

and (3) directly passing the fermentation liquor treated by the S3 through a 600Da membrane, adding water which is 5 times of the weight of the fermentation liquor treated by the S3 into the membrane concentrated liquor, and collecting trapped concentrated liquor.

The product obtained by the process described in this comparative example had a yield of 83% and a purity of 92%.

The comparative example only differs from example 1 in that the comparative example has not undergone the 1000-3000Da ultrafiltration treatment, the yield of the product obtained in the comparative example is higher than that of example 1, and the purity is significantly lower than that of example 1.

The ultrafiltration treatment can separate water-soluble substances such as galactose, glucose and partial salt of small molecules and macromolecular protein in the filtrate from fucosyllactose through a 1000-3000Da membrane, and comparative example 1 is not subjected to ultrafiltration treatment, so that partial protein is trapped in the concentrated solution together with fucosyllactose, thereby reducing the purity of the final product.

Comparative example 2

The present comparative example differs from example 1 only in step S4, the present comparative example step S4 is not subjected to the concentration treatment process, and the present comparative example S4 is specifically as follows: the fermentation liquid treated by S3 is filtered through a 1000-3000Da membrane to collect filtrate.

The product obtained by the process described in this comparative example had a yield of 84% and a purity of 89%.

The present comparative example differs from example 1 only in that it does not undergo 600Da membrane filtration concentration treatment, and cannot further separate galactose, glucose and partial salts from fucosyllactose, resulting in a reduction in the purity of the final product.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for obtaining high-purity fucosyllactose by degrading lactose in fermentation liquor is characterized by comprising the following steps:

2. The method according to claim 1, wherein the lactase of step S2 is added in an amount of 0.5 wt% to 1 wt% based on the weight of the fermentation broth.

3. The method according to claim 1, wherein the step S3 comprises the following steps: heating the fermentation liquor to 80-90 ℃, keeping the temperature for reaction for 0.5-1 h, and cooling to 40-45 ℃.

4. The method of claim 1, wherein the activated carbon is added to the fermentation broth in step S3 in an amount of 0.5 wt% to 4.0 wt% based on the weight of the fermentation broth after the fermentation treatment in step S2.

5. The method according to claim 1, wherein the step S4 is to pass the filtrate through a 500 Da-600 Da membrane, and the specific process of collecting the trapped concentrated solution is as follows: adding water with the weight of 4-6 times of that of the filtrate filtered by the 1000-ion 3000Da membrane, and collecting the trapped concentrated solution through the 500-600 Da membrane.

6. The method according to claim 1, wherein the step S5 is to desalt the retentate with cation exchange resin or anion exchange resin until the conductivity of the retentate is less than 400 μ S/cm.

7. The method of claim 1, wherein the anion exchange resin is a polyacrylic acid-based macroporous weakly basic anion exchange resin; the cation exchange resin is strong-acid styrene cation exchange resin.

8. The method of claim 1, wherein the fucosyllactose fermentation broth is prepared by fermentation using Escherichia coli with a preservation number of CCTCC NO: M2020027 as a fermentation strain.

9. A high purity fucosyllactose produced by the method of any one of claims 1 to 8.

10. The high purity fucosyllactose as claimed in claim 9, wherein the high purity fucosyllactose has a purity of not less than 95%.