CN118286872A - Co-production process of lacto-N-difucose tetraose and 2' -fucosyl lactose based on chromatographic separation - Google Patents

Abstract

The present invention belongs to the technical field of chromatographic separation, and specifically relates to a co-production process of lacto-N-difucotetraose and 2'-fucosyllactose based on chromatographic separation, comprising the following steps: removing bacterial cells and performing nanofiltration; nanofiltration separation of dense phase materials and clear phase materials; ion exchange of dense phase materials through a fixed bed; concentrated solution one enters a first set of chromatographic separation system for separation; continued evaporation and concentration to obtain concentrated solution two, and then enters a second set of chromatographic separation system for chromatographic separation; post-processing to obtain a 2'-fucosyllactose product. The present invention utilizes an aqueous solution of 7.0 < pH ≤ 10.0 as an eluent, which can stabilize the product properties; and the chromatographic separation has high sensitivity, and the separated tetrasaccharide products and trisaccharides have high purity; the combination of two sets of chromatograms can separate the tetrasaccharide products and trisaccharides in the fermentation broth, respectively, to ensure the stability and yield of the tetrasaccharide products and trisaccharides.

Description

Co-production process of lacto-N-difucotetraose and 2'-fucosyllactose based on chromatographic separation

Technical Field

The present invention belongs to the technical field of chromatographic separation, and in particular relates to a co-production process of lacto-N-difucotetraose and 2'-fucosyllactose based on chromatographic separation.

Background technique

Human milk oligosaccharides are the third most abundant solid component in breast milk. They have the functions of regulating immunity, helping brain development and regulating intestinal flora, and are helpful for the growth and development of infants and young children. In traditional industrial production, acetic acid is often used for extraction, crystallization, centrifugation, decolorization and other processes to prepare high-purity human milk oligosaccharides, namely 2'-fucosyllactose. The process flow chart is shown in Figure 3.

According to the process flow in Figure 3, the specific operation of producing human milk oligosaccharides using the crystallization method is as follows:

(1) After fermentation, the fermentation liquid is filtered through a ceramic membrane to remove bacteria, and a nanofiltration membrane is used to remove salt and a small amount of small molecular monosaccharides, disaccharides and other miscellaneous sugars in the material.

(2) The concentrated phase material after nanofiltration is concentrated from about 10% to about 40% using a falling film evaporator, and then cooled and crystallized. According to the dry basis amount of the material in the crystallization tank, 0.2wt.% of glacial acetic acid is added as an extractant, and the crystallization cycle is 100-120 hours.

(3) After crystallization, use a centrifuge to remove impurities, inject the supernatant into the pre-decolorization tank, and add 0.3wt.% of powdered activated carbon on a dry basis for decolorization at 60°C, keep warm and stir for 30 minutes; after plate and frame filtration, enter the pre-ionization tank;

(4) The ion exchange system is a yin and yang column series mode, two in use and one in reserve, and the material is finally discharged from the yin column; the discharge pH value is 4.5-5.5, and the discharge conductivity is 30-50us/cm;

(5) The ion exchange liquid passes through a falling film evaporator to concentrate the concentration to about 55%;

(6) The concentrated liquid is sterilized by passing through a precision filter;

(7) The material is spray-dried to obtain the finished human milk oligosaccharide product.

The above process has the following problems:

(1) The crystallization yield is only about 70%, resulting in a large amount of human milk oligosaccharides in the mother liquor that cannot be recycled, resulting in high production costs;

(2) During the crystallization process, glacial acetic acid is added as an extractant, which poses a great safety hazard due to its volatility and irritation. At the same time, glacial acetic acid is corrosive, so the requirements for crystallization equipment are relatively high;

(3) It is necessary to recover the glacial acetic acid from the crystallized material;

(4) Due to the large number of variable factors in fermentation, the content of human milk oligosaccharides is very unstable, so it is difficult to guarantee the crystallization yield and product quality.

Chinese patent CN113004347A discloses a method for separating and purifying 2'-fucosyllactose, comprising the following steps: S1, removing bacteria through a membrane to obtain a membrane clear solution; S2, electrodialysis desalination: the membrane clear solution prepared in step S1 is subjected to electrodialysis desalination, with salt electrolytes as electrolytes, the membrane clear solution is filled in the dilute chamber, and the deionized water is filled in the concentrated chamber, to obtain an electroosmotic desalted concentrated solution with a solute concentration of 20-100 g/L and a conductivity of 400-2000 us/cm; S3, filtering to remove protein, decolorizing, filtering, and obtaining a filtrate; S4, concentrating, and crystallizing to obtain 2'-fucosyllactose.

In summary, the prior art uses acetic acid for extraction and separation, but the crystallization yield is low and the cost is high; acetic acid is irritating and volatile, which may cause operational risks during the extraction process; acetic acid is also corrosive to a certain extent, and has high requirements for crystallization equipment, and the acetic acid needs to be recovered after preparation; due to the large number of variable factors in fermentation and the unstable content of human milk oligosaccharides, the yield and quality of the product prepared by crystallization extraction are uncontrollable.

Summary of the invention

The purpose of the present invention is to provide a co-production process of lacto-N-difucotetraose and 2'-fucosyllactose based on chromatographic separation, using an aqueous solution with a pH value of 7.0 < 10.0 as an eluent, and performing secondary chromatographic separation on a fermentation broth to obtain a lacto-N-difucotetraose product and 2'-fucosyllactose, wherein the separated lacto-N-difucotetraose product and 2'-fucosyllactose have high purity and yield, stable properties, and low production cost.

The co-production process of lacto-N-difucotetraose and 2'-fucosyllactose based on chromatographic separation of the present invention comprises the following steps:

(1) Removing the bacterial cells from the fermentation broth containing monosaccharides and polysaccharides to obtain a clear liquid and bacterial residue, and subjecting the clear liquid to nanofiltration;

(2) After nanofiltration, the product is separated into a dense phase and a clear phase, and the clear phase is discharged as the mother liquor;

(3) The concentrated phase material is treated by fixed bed ion exchange to obtain ion exchange liquid;

(4) The ion exchange liquid is evaporated and concentrated to obtain a concentrated liquid 1, which enters the first chromatographic separation system to separate a lacto-N-difucotetraose product with a purity of more than 94 wt.% and a recovered liquid;

(5) The recovered liquid is decolorized, subjected to fixed bed ion exchange, and concentrated by evaporation to obtain concentrated liquid 2, which enters the second chromatographic separation system to separate 2'-fucosyllactose with a purity of more than 97 wt.% and the residual liquid;

(6) After decolorization, fixed bed ion exchange, evaporation concentration, sterilization, and spray drying, 2'-fucosyllactose with a purity of more than 97 wt.% is obtained.

in:

In step (1), a ceramic membrane is used to remove the bacterial cells in the fermentation liquid containing monosaccharides and polysaccharides, and the bacterial residue is recycled as protein;

In step (2), the conductivity of the dense phase material is 7000-8000 us/cm, and the clear phase material includes monosaccharides and disaccharides;

In step (3), the conductivity of the ion exchange liquid is less than 50 us/cm;

In step (4), the ion exchange liquid is concentrated by evaporation to obtain a concentrated solution 1 with a concentration of 38-40 wt.%.

In step (4), the first chromatographic separation system includes a first chromatographic column, a second chromatographic column, a third chromatographic column, a fourth chromatographic column, a fifth chromatographic column and a sixth chromatographic column connected in sequence, the sixth chromatographic column is also connected to the first chromatographic column, and the six chromatographic columns form a circulation loop; the first chromatographic separation system also includes a water inlet pipe, a feed pipe, a recovery pipe and a product pipe, the water inlet pipe is connected to all the chromatographic columns, the feed pipe is connected to all the chromatographic columns, the recovery pipe is connected to all the chromatographic columns, and the product pipe is connected to all the chromatographic columns.

The eluent is fed into the water inlet pipe, the eluent is an aqueous solution, and the pH value of the aqueous solution is 7.0＜pH≤10.0, preferably 8.0≤pH≤10.0. The preparation process of the eluent is to adjust the pH value of water using alkali solutions such as sodium carbonate, sodium hydroxide, potassium carbonate, potassium hydroxide, etc.

On the circulation loop, the first chromatographic column, the first circulation pump, the first circulation valve, the second chromatographic column, the second circulation pump, the second circulation valve, the third chromatographic column, the third circulation pump, the third circulation valve, the fourth chromatographic column, the fourth circulation pump, the fourth circulation valve, the fifth chromatographic column, the fifth circulation pump, the fifth circulation valve, the sixth chromatographic column, the sixth circulation pump, the sixth circulation valve and the first chromatographic column are connected in sequence;

A first water inlet valve, a second water inlet valve, a third water inlet valve, a fourth water inlet valve, a fifth water inlet valve and a sixth water inlet valve are respectively arranged between the water inlet pipeline and the first chromatographic column, between the water inlet pipeline and the second chromatographic column, between the water inlet pipeline and the third chromatographic column, between the water inlet pipeline and the fourth chromatographic column, between the water inlet pipeline and the fifth chromatographic column, and between the water inlet pipeline and the sixth chromatographic column;

A first feed valve, a second feed valve, a third feed valve, a fourth feed valve, a fifth feed valve and a sixth feed valve are respectively arranged between the feed pipeline and the first chromatographic column, between the feed pipeline and the second chromatographic column, between the feed pipeline and the third chromatographic column, between the feed pipeline and the fourth chromatographic column, between the feed pipeline and the fifth chromatographic column and between the feed pipeline and the sixth chromatographic column;

A recovery pipeline and a product pipeline are connected in sequence to the circulation loop between the first circulation pump and the first circulation valve, and the recovery pipeline and the product pipeline are respectively provided with a first recovery valve and a first product valve;

A recovery pipeline and a product pipeline are connected in sequence to the circulation loop between the second circulation pump and the second circulation valve, and the recovery pipeline and the product pipeline are respectively provided with a second recovery valve and a second product valve;

A recovery pipeline and a product pipeline are connected in sequence to the circulation loop between the third circulation pump and the third circulation valve, and the recovery pipeline and the product pipeline are respectively provided with a third recovery valve and a third product valve;

The circulation loop between the fourth circulation pump and the fourth circulation valve is connected with a recovery pipeline and a product pipeline in sequence, and the recovery pipeline and the product pipeline are respectively provided with a fourth recovery valve and a fourth product valve;

The circulation loop between the fifth circulation pump and the fifth circulation valve is connected with a recovery pipeline and a product pipeline in sequence, and the recovery pipeline and the product pipeline are respectively provided with a fifth recovery valve and a fifth product valve;

The recycling loop between the sixth recycling pump and the sixth recycling valve is connected with a recycling pipeline and a product pipeline in sequence, and the recycling pipeline and the product pipeline are respectively provided with a sixth recycling valve and a sixth product valve.

In step (5), the second chromatographic separation system has the same structure as the first chromatographic separation system.

In step (4), the separation step of the concentrate 1 in the first chromatographic separation system includes:

(1) The concentrate enters the first chromatographic column and circulates in the circulation loop;

(2) The eluent enters the first chromatographic column from the water inlet pipe, circulates to the fifth chromatographic column, separates the lacto-N-difucotetraose product with a purity of more than 94 wt.%, and enters the product pipeline;

(3) The eluent enters the first chromatographic column from the water inlet pipe, and the recovery liquid is separated from the first chromatographic column; the concentrated liquid enters the fourth chromatographic column from the feed pipe and circulates to the fifth chromatographic column, and the lacto-N-difucotetraose product with a purity of more than 94wt.% is separated from the fifth chromatographic column and enters the product pipeline;

(4) The eluent enters the second chromatographic column from the water inlet pipe, and the recovered liquid is separated from the second chromatographic column; the concentrated liquid enters the fifth chromatographic column from the feed pipe and circulates to the sixth chromatographic column, and the lacto-N-difucotetraose product with a purity of more than 94 wt.% is separated from the sixth chromatographic column and enters the product pipeline;

Afterwards, the eluent enters the third chromatographic column, the fourth chromatographic column, the fifth chromatographic column, and the sixth chromatographic column in sequence to separate the recovered liquid; the concentrated liquid one enters the sixth chromatographic column in sequence, and is separated into a lacto-N-difucotetraose product with a purity of more than 94wt.% through the first chromatographic column, enters the first chromatographic column, and is separated into a lacto-N-difucotetraose product with a purity of more than 94wt.% through the second chromatographic column, enters the second chromatographic column, and is separated into a lacto-N-difucotetraose product with a purity of more than 94wt.% through the third chromatographic column, enters the third chromatographic column, and is separated into a lacto-N-difucotetraose product with a purity of more than 94wt.% through the fourth chromatographic column; after completing one cycle of separation of the six chromatographic columns, enter the next cycle of separation; after the separation of the recovered liquid and the lacto-N-difucotetraose product with a purity of more than 94wt.% is completed, the recovered liquid is decolorized, fixed-bed ion exchanged, and evaporated and concentrated to obtain a concentrated liquid two, which enters the second set of chromatographic separation system for separation.

In step (4), the separation step of the concentrate 1 in the first chromatographic separation system further comprises:

(1) Open the first circulation valve, the second circulation valve, the third circulation valve, the fourth circulation valve, the fifth circulation valve, the sixth circulation valve, the first circulation pump, the second circulation pump, the third circulation pump, the fifth circulation pump and the sixth circulation pump to allow the concentrate to fill the entire chromatographic separation system;

(2) Open the first water inlet valve to add the eluent, and open the first circulation pump, the second circulation pump, the third circulation pump, the fourth circulation pump, the fifth circulation pump, the first circulation valve, the second circulation valve, the third circulation valve, the fourth circulation valve and the fifth product valve. After passing through the fifth product valve, the lacto-N-difucotetraose product with a purity of more than 94 wt.% is separated and enters the product pipeline;

(3) Open the first water inlet valve to add the eluent, and open the first circulation pump and the first recovery valve to separate the recovery liquid from the first chromatographic column; open the fourth feed valve to add the concentrate 1, and open the fourth circulation pump, the fifth circulation pump, the fourth circulation valve and the fifth product valve, and separate the lacto-N-difucotetraose product with a purity of more than 94 wt.% through the fifth product valve and enter the product pipeline;

(4) Open the second water inlet valve to add the eluent, and open the second circulation pump and the second recovery valve to separate the recovery liquid from the second chromatographic column; open the fifth feed valve to add the concentrate 1, and open the fifth circulation pump, the sixth circulation pump, the fifth circulation valve and the sixth product valve. After passing through the sixth product valve, separate the lacto-N-difucotetraose product with a purity of more than 94 wt.% and enter the product pipeline;

Afterwards, the eluent enters the third chromatographic column, the fourth chromatographic column, the fifth chromatographic column, and the sixth chromatographic column in sequence to separate the recovered liquid; the concentrated liquid one enters the sixth chromatographic column in sequence, and is separated into a lacto-N-difucotetraose product with a purity of more than 94wt.% through the first chromatographic column, enters the first chromatographic column, and is separated into a lacto-N-difucotetraose product with a purity of more than 94wt.% through the second chromatographic column, enters the second chromatographic column, and is separated into a lacto-N-difucotetraose product with a purity of more than 94wt.% through the third chromatographic column, enters the third chromatographic column, and is separated into a lacto-N-difucotetraose product with a purity of more than 94wt.% through the fourth chromatographic column; after completing one cycle of separation of the six chromatographic columns, enter the next cycle of separation; after the separation of the recovered liquid and the lacto-N-difucotetraose product with a purity of more than 94wt.% is completed, the recovered liquid is decolorized, fixed-bed ion exchanged, and evaporated and concentrated to obtain a concentrated liquid two, which enters the second set of chromatographic separation system for separation.

In step (5), the separation step of the concentrated solution 2 in the second chromatographic separation system includes:

(1) The concentrated liquid 2 enters the first chromatographic column and circulates in the circulation loop;

(2) The eluent enters the first chromatographic column from the water inlet pipe, circulates to the fifth chromatographic column, separates 2'-fucosyllactose with a purity of more than 97 wt.%, and enters the product pipeline;

(3) The eluent enters the first chromatographic column from the water inlet pipe, and the residual liquid is separated from the first chromatographic column; the concentrated liquid 2 enters the fourth chromatographic column from the feed pipe and circulates to the fifth chromatographic column, and 2'-fucosyllactose with a purity of more than 97 wt.% is separated from the fifth chromatographic column and enters the product pipeline;

(4) The eluent enters the second chromatographic column from the water inlet pipe, and the residual liquid is separated from the second chromatographic column; the concentrated liquid 2 enters the fifth chromatographic column from the feed pipe and circulates to the sixth chromatographic column, and 2'-fucosyllactose with a purity of more than 97 wt.% is separated from the sixth chromatographic column and enters the product pipeline;

Afterwards, the eluent enters the third chromatographic column, the fourth chromatographic column, the fifth chromatographic column, and the sixth chromatographic column in sequence to separate the residual liquid; the concentrated liquid 2 enters the sixth chromatographic column in sequence, and passes through the first chromatographic column to separate 2'-fucosyllactose with a purity of more than 97wt.%, enters the first chromatographic column, and passes through the second chromatographic column to separate 2'-fucosyllactose with a purity of more than 97wt.%, enters the second chromatographic column, and passes through the third chromatographic column to separate 2'-fucosyllactose with a purity of more than 97wt.%, enters the third chromatographic column, and passes through the fourth chromatographic column to separate 2'-fucosyllactose with a purity of more than 97wt.%; after completing one cycle of separation of the six chromatographic columns, enter the next cycle of separation; after the separation of the residual liquid and 2'-fucosyllactose with a purity of more than 97wt.% is completed, the residual liquid is recycled by nanofiltration.

In step (5), the separation step of the concentrated solution 2 in the second chromatographic separation system further comprises:

(1) Open the first circulation valve, the second circulation valve, the third circulation valve, the fourth circulation valve, the fifth circulation valve, the sixth circulation valve, the first circulation pump, the second circulation pump, the third circulation pump, the fifth circulation pump and the sixth circulation pump to allow the concentrate to fill the entire chromatographic separation system;

(2) Open the first water inlet valve to add the eluent, and open the first circulation pump, the second circulation pump, the third circulation pump, the fourth circulation pump, the fifth circulation pump, the first circulation valve, the second circulation valve, the third circulation valve, the fourth circulation valve and the fifth product valve. After passing through the fifth product valve, 2'-fucosyllactose with a purity of more than 97 wt.% is separated and enters the product pipeline;

(3) Open the first water inlet valve to add the eluent, and open the first circulation pump and the first recovery valve to separate the residual liquid from the first chromatographic column; open the fourth feed valve to add the concentrated liquid 2, and open the fourth circulation pump, the fifth circulation pump, the fourth circulation valve and the fifth product valve. After passing through the fifth product valve, 2'-fucosyllactose with a purity of more than 97 wt.% is separated and enters the product pipeline;

(4) Open the second water inlet valve to add the eluent, and open the second circulation pump and the second recovery valve to separate the residual liquid from the second chromatographic column; open the fifth feed valve to add the concentrated liquid 2, and open the fifth circulation pump, the sixth circulation pump, the fifth circulation valve and the sixth product valve. After passing through the sixth product valve, separate 2'-fucosyllactose with a purity of more than 97 wt.% and enter the product pipeline;

Afterwards, the eluent enters the third chromatographic column, the fourth chromatographic column, the fifth chromatographic column, and the sixth chromatographic column in sequence to separate the residual liquid; the concentrated liquid 2 enters the sixth chromatographic column in sequence, and passes through the first chromatographic column to separate 2'-fucosyllactose with a purity of more than 97wt.%, enters the first chromatographic column, and passes through the second chromatographic column to separate 2'-fucosyllactose with a purity of more than 97wt.%, enters the second chromatographic column, and passes through the third chromatographic column to separate 2'-fucosyllactose with a purity of more than 97wt.%, enters the third chromatographic column, and passes through the fourth chromatographic column to separate 2'-fucosyllactose with a purity of more than 97wt.%; after completing one cycle of separation of the six chromatographic columns, enter the next cycle of separation; after the separation of the residual liquid and 2'-fucosyllactose with a purity of more than 97wt.% is completed, the residual liquid is recycled by nanofiltration.

In the present invention, 2'-fucosyllactose (2'-FL) is referred to as trisaccharide; lacto-N-difucotetraose (DFL) is referred to as tetrasaccharide.

Beneficial effects of the present invention:

(1) In the chromatographic separation process, the present invention uses alkaline solutions such as sodium carbonate, sodium hydroxide, potassium carbonate, potassium hydroxide, etc. to control the pH value of the eluent at 7.0＜pH≤10.0; the purpose is to prevent trisaccharides from decomposing into monosaccharides and disaccharides under high temperature and acidic conditions, and from converting into other miscellaneous sugars, thereby affecting the purity and quality of the product. The pH control range is particularly important. If the pH value is too low, it will not have any effect, and if the pH value is too high, the color of the product will be aggravated. Therefore, the regulation of pH value becomes the key to the production of human milk oligosaccharides.

(2) The combination of the two sets of chromatograms in the present invention can not only obtain two different products, trisaccharide and tetrasaccharide products, but also ensure the purity and yield of the trisaccharide and tetrasaccharide products as well as the stability of the trisaccharide and tetrasaccharide products.

(3) In industrial production, crystallization and chromatographic separation are both important means to improve product purity. Crystallization has high requirements for the purity of the material before entering the crystal. If the purity is low, the product is difficult to crystallize and requires multiple crystallizations or the use of organic solvents to increase its solubility, which brings great uncertainty to production. The pre-process of human milk oligosaccharides uses a fermentation process. Due to the many variable factors in the fermentation process, the purity of the material is difficult to control. Chromatographic separation has a wide selectivity for materials and can achieve good separation effects for substances with similar molecular weights and properties, thereby ensuring the stability of production.

(4) Chromatographic separation uses water as the eluent. The separation process is a physical change and no chemical change occurs. No excess impurities are generated during the separation process. Therefore, it is more suitable for the food industry and infant formula industry.

In summary, the present invention uses water with a pH value controlled between 7.0＜pH≤10.0 as an eluent, which can stabilize the properties of the product; and the chromatographic separation has high sensitivity and is not affected by the fermentation effect of the fermentation liquid, and the separated tetrasaccharide product and trisaccharide have high purity; the combination of two sets of chromatograms can separate the tetrasaccharide product and trisaccharide in the fermentation liquid respectively, and the separation processes do not interfere with each other, thereby ensuring the stability and yield of the tetrasaccharide product and trisaccharide; the chromatographic production process is simple, the operation is more convenient, and the residual liquid after separation can be recycled again.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic diagram of the structure of a first chromatographic separation system of the present invention;

Fig. 2 is a process flow chart of the present invention;

FIG. 3 is a process flow chart of the prior art.

In the figure: 1, first chromatographic column; 101, first feed valve; 102, first water inlet valve; 103, first circulation valve; 104, first recovery valve; 105, first product valve; 106, first circulation pump; 2, second chromatographic column; 201, second feed valve; 202, second water inlet valve; 203, second circulation valve; 204, second recovery valve; 205, second product valve; 206, second circulation pump; 3, third chromatographic column; 301, third feed valve; 302, third water inlet valve; 303, third circulation valve; 304, third recovery valve; 305, third product valve; 306, third circulation pump pump; 4, fourth chromatographic column; 401, fourth feed valve; 402, fourth water inlet valve; 403, fourth circulation valve; 404, fourth recovery valve; 405, fourth product valve; 406, fourth circulation pump; 5, fifth chromatographic column; 501, fifth feed valve; 502, fifth water inlet valve; 503, fifth circulation valve; 504, fifth recovery valve; 505, fifth product valve; 506, fifth circulation pump; 6, sixth chromatographic column; 601, sixth feed valve; 602, sixth water inlet valve; 603, sixth circulation valve; 604, sixth recovery valve; 605, sixth product valve; 606, sixth circulation pump.

Detailed ways

The present invention is further described below with reference to the embodiments.

Example 1

The co-production process of lacto-N-difucotetraose and 2'-fucosyllactose based on chromatographic separation of the present invention comprises the following steps:

(1) Removing the bacterial cells from the fermentation broth containing monosaccharides and polysaccharides to obtain a clear liquid and bacterial residue, and subjecting the clear liquid to nanofiltration;

(2) After nanofiltration, the product is separated into a dense phase and a clear phase, and the clear phase is discharged as the mother liquor;

(3) The concentrated phase material is treated by fixed bed ion exchange to obtain ion exchange liquid;

(4) The ion exchange liquid is evaporated and concentrated to obtain a concentrated liquid 1, which enters the first chromatographic separation system to separate a lacto-N-difucotetraose product with a purity of more than 94 wt.% and a recovered liquid;

(5) The recovered liquid is decolorized, subjected to fixed bed ion exchange, and concentrated by evaporation to obtain concentrated liquid 2, which enters the second chromatographic separation system to separate 2'-fucosyllactose with a purity of more than 97 wt.% and the residual liquid;

(6) After decolorization, fixed bed ion exchange, evaporation concentration, sterilization, and spray drying, 2'-fucosyllactose with a purity of more than 97 wt.% is obtained.

In step (1), a ceramic membrane is used to remove the bacterial cells in the fermentation liquid containing monosaccharides and polysaccharides, and the bacterial residue is recycled as protein;

In step (2), the conductivity of the dense phase material is 7000-8000 us/cm, and the clear phase material includes monosaccharides and disaccharides;

In step (3), the conductivity of the ion exchange liquid is less than 50 us/cm;

In step (4), the ion exchange liquid is concentrated by evaporation to obtain a concentrated solution 1 with a concentration of 38-40 wt.%.

In step (4), the first chromatographic separation system comprises a first chromatographic column 1, a second chromatographic column 2, a third chromatographic column 3, a fourth chromatographic column 4, a fifth chromatographic column 5 and a sixth chromatographic column 6 which are connected in sequence, and the sixth chromatographic column 6 is also connected to the first chromatographic column 1, and the six chromatographic columns form a circulation loop; the first chromatographic separation system also comprises a water inlet pipe, a feed pipe, a recovery pipe and a product pipe, the water inlet pipe is connected to all the chromatographic columns, the feed pipe is connected to all the chromatographic columns, the recovery pipe is connected to all the chromatographic columns, and the product pipe is connected to all the chromatographic columns.

The eluent is fed into the water inlet pipe, the eluent is an aqueous solution, and the pH value of the aqueous solution is 7.0＜pH≤10.0, preferably 8.0≤pH≤10.0. The preparation process of the eluent is to adjust the pH value of water using alkali solutions such as sodium carbonate, sodium hydroxide, potassium carbonate, potassium hydroxide, etc.

On the circulation loop, the first chromatographic column 1, the first circulation pump 106, the first circulation valve 103, the second chromatographic column 2, the second circulation pump 206, the second circulation valve 203, the third chromatographic column 3, the third circulation pump 306, the third circulation valve 303, the fourth chromatographic column 4, the fourth circulation pump 406, the fourth circulation valve 403, the fifth chromatographic column 5, the fifth circulation pump 506, the fifth circulation valve 503, the sixth chromatographic column 6, the sixth circulation pump 606, the sixth circulation valve 603 and the first chromatographic column 1 are connected in sequence;

A first water inlet valve 102, a second water inlet valve 202, a third water inlet valve 302, a fourth water inlet valve 402, a fifth water inlet valve 502 and a sixth water inlet valve 602 are respectively arranged between the water inlet pipeline and the first chromatographic column 1, between the water inlet pipeline and the second chromatographic column 2, between the water inlet pipeline and the third chromatographic column 3, between the water inlet pipeline and the fourth chromatographic column 4, between the water inlet pipeline and the fifth chromatographic column 5, and between the water inlet pipeline and the sixth chromatographic column 6;

A first feed valve 101, a second feed valve 201, a third feed valve 301, a fourth feed valve 401, a fifth feed valve 501 and a sixth feed valve 601 are respectively arranged between the feed pipeline and the first chromatographic column 1, between the feed pipeline and the second chromatographic column 2, between the feed pipeline and the third chromatographic column 3, between the feed pipeline and the fourth chromatographic column 4, between the feed pipeline and the fifth chromatographic column 5, and between the feed pipeline and the sixth chromatographic column 6;

The circulation loop between the first circulation pump 106 and the first circulation valve 103 is connected with a recovery pipeline and a product pipeline in sequence, and the recovery pipeline and the product pipeline are respectively provided with a first recovery valve 104 and a first product valve 105;

The circulation loop between the second circulation pump 206 and the second circulation valve 203 is connected in sequence to a recovery pipeline and a product pipeline, and the recovery pipeline and the product pipeline are respectively provided with a second recovery valve 204 and a second product valve 205;

The circulation loop between the third circulation pump 306 and the third circulation valve 303 is connected with a recovery pipeline and a product pipeline in sequence, and the recovery pipeline and the product pipeline are respectively provided with a third recovery valve 304 and a third product valve 305;

The circulation loop between the fourth circulation pump 406 and the fourth circulation valve 403 is connected with a recovery pipeline and a product pipeline in sequence, and the recovery pipeline and the product pipeline are respectively provided with a fourth recovery valve 404 and a fourth product valve 405;

The circulation loop between the fifth circulation pump 506 and the fifth circulation valve 503 is connected with a recovery pipeline and a product pipeline in sequence, and the recovery pipeline and the product pipeline are respectively provided with a fifth recovery valve 504 and a fifth product valve 505;

The recycling loop between the sixth recycling pump 606 and the sixth recycling valve 603 is connected with a recovery pipeline and a product pipeline in sequence, and the recovery pipeline and the product pipeline are provided with a sixth recovery valve 604 and a sixth product valve 605 respectively.

In step (5), the second chromatographic separation system has the same structure as the first chromatographic separation system.

In step (4), the separation step of the concentrate 1 in the first chromatographic separation system includes:

(1) The concentrated liquid enters the first chromatographic column 1 and circulates in the circulation loop;

(2) The eluent enters the first chromatographic column 1 from the water inlet pipe, circulates to the fifth chromatographic column 5, separates the lacto-N-difucotetraose product with a purity of more than 94 wt.%, and enters the product pipeline;

(3) The eluent enters the first chromatographic column 1 from the water inlet pipe, and the recovery liquid is separated from the first chromatographic column 1; the concentrated liquid enters the fourth chromatographic column 4 from the feed pipe and circulates to the fifth chromatographic column 5, and the lacto-N-difucotetraose product with a purity of more than 94 wt.% is separated from the fifth chromatographic column 5 and enters the product pipeline;

(4) The eluent enters the second chromatographic column 2 from the water inlet pipe, and the recovered liquid is separated from the second chromatographic column 2; the concentrated liquid enters the fifth chromatographic column 5 from the feed pipe and circulates to the sixth chromatographic column 6, and the lacto-N-difucotetraose product with a purity of more than 94 wt.% is separated from the sixth chromatographic column 6 and enters the product pipeline;

Afterwards, the eluent enters the third chromatographic column 3, the fourth chromatographic column 4, the fifth chromatographic column 5, and the sixth chromatographic column 6 in sequence to separate the recovered liquid; the concentrated liquid 1 enters the sixth chromatographic column 6 in sequence, and passes through the first chromatographic column 1 to separate the lacto-N-difucotetraose product with a purity of more than 94wt.%, enters the first chromatographic column 1, and passes through the second chromatographic column 2 to separate the lacto-N-difucotetraose product with a purity of more than 94wt.%, enters the second chromatographic column 2, and passes through the third chromatographic column 3 to separate the lacto-N-difucotetraose product with a purity of more than 94wt.%, enters the third chromatographic column 3, and passes through the fourth chromatographic column 4 to separate the lacto-N-difucotetraose product with a purity of more than 94wt.%; after completing one cycle of separation of the six chromatographic columns, enter the next cycle of separation; after the separation of the recovered liquid and the lacto-N-difucotetraose product with a purity of more than 94wt.% is completed, the recovered liquid is decolorized, fixed-bed ion exchanged, and evaporated and concentrated to obtain the concentrated liquid 2, which enters the second set of chromatographic separation system for separation.

In step (4), the separation step of the concentrate 1 in the first chromatographic separation system further comprises:

(1) opening the first circulation valve 103, the second circulation valve 203, the third circulation valve 303, the fourth circulation valve 403, the fifth circulation valve 503, the sixth circulation valve 603, the first circulation pump 106, the second circulation pump 206, the third circulation pump 306, the fifth circulation pump 506 and the sixth circulation pump 606, so that the concentrate fills the entire chromatographic separation system;

(2) Open the first water inlet valve 102 to add the eluent, and open the first circulation pump 106, the second circulation pump 206, the third circulation pump 306, the fourth circulation pump 406, the fifth circulation pump 506, the first circulation valve 103, the second circulation valve 203, the third circulation valve 303, the fourth circulation valve 403 and the fifth product valve 505. After passing through the fifth product valve 505, the lacto-N-difucotetraose product with a purity of more than 94 wt.% is separated and enters the product pipeline;

(3) Open the first water inlet valve 102 to add the eluent, and open the first circulation pump 106 and the first recovery valve 104 to separate the recovery liquid from the first chromatographic column 1; open the fourth feed valve 401 to add the concentrate 1, and open the fourth circulation pump 406, the fifth circulation pump 506, the fourth circulation valve 403 and the fifth product valve 505. After passing through the fifth product valve 505, the lacto-N-difucotetraose product with a purity of more than 94 wt.% is separated and enters the product pipeline;

(4) Open the second water inlet valve 202 to add the eluent, and open the second circulation pump 206 and the second recovery valve 204 to separate the recovery liquid from the second chromatographic column 2; open the fifth feed valve 501 to add the concentrate 1, and open the fifth circulation pump 506, the sixth circulation pump 606, the fifth circulation valve 503 and the sixth product valve 605. After passing through the sixth product valve 605, the lacto-N-difucotetraose product with a purity of more than 94 wt.% is separated and enters the product pipeline;

Afterwards, the eluent enters the third chromatographic column 3, the fourth chromatographic column 4, the fifth chromatographic column 5, and the sixth chromatographic column 6 in sequence to separate the recovered liquid; the concentrated liquid 1 enters the sixth chromatographic column 6 in sequence, and passes through the first chromatographic column 1 to separate the lacto-N-difucotetraose product with a purity of more than 94wt.%, enters the first chromatographic column 1, and passes through the second chromatographic column 2 to separate the lacto-N-difucotetraose product with a purity of more than 94wt.%, enters the second chromatographic column 2, and passes through the third chromatographic column 3 to separate the lacto-N-difucotetraose product with a purity of more than 94wt.%, enters the third chromatographic column 3, and passes through the fourth chromatographic column 4 to separate the lacto-N-difucotetraose product with a purity of more than 94wt.%; after completing one cycle of separation of the six chromatographic columns, enter the next cycle of separation; after the separation of the recovered liquid and the lacto-N-difucotetraose product with a purity of more than 94wt.% is completed, the recovered liquid is decolorized, fixed-bed ion exchanged, and evaporated and concentrated to obtain the concentrated liquid 2, which enters the second set of chromatographic separation system for separation.

In step (5), the separation step of the concentrated solution 2 in the second chromatographic separation system includes:

(1) The concentrated liquid 2 enters the first chromatographic column 1 and circulates in the circulation loop;

(2) The eluent enters the first chromatographic column 1 from the water inlet pipe, circulates to the fifth chromatographic column 5, separates 2'-fucosyllactose with a purity of more than 97 wt.%, and enters the product pipeline;

(3) The eluent enters the first chromatographic column 1 from the water inlet pipe, and the residual liquid is separated from the first chromatographic column 1; the concentrated liquid 2 enters the fourth chromatographic column 4 from the feed pipe and circulates to the fifth chromatographic column 5, and 2'-fucosyllactose with a purity of more than 97 wt.% is separated from the fifth chromatographic column 5 and enters the product pipe;

(4) The eluent enters the second chromatographic column 2 from the water inlet pipe, and the residual liquid is separated from the second chromatographic column 2; the concentrated liquid 2 enters the fifth chromatographic column 5 from the feed pipe and circulates to the sixth chromatographic column 6, and 2'-fucosyllactose with a purity of more than 97 wt.% is separated from the sixth chromatographic column 6 and enters the product pipe;

Afterwards, the eluent enters the third chromatographic column 3, the fourth chromatographic column 4, the fifth chromatographic column 5, and the sixth chromatographic column 6 in sequence to separate the residual liquid; the concentrated liquid 2 enters the sixth chromatographic column 6 in sequence, and passes through the first chromatographic column 1 to separate 2'-fucosyllactose with a purity of more than 97wt.%, enters the first chromatographic column 1, and passes through the second chromatographic column 2 to separate 2'-fucosyllactose with a purity of more than 97wt.%, enters the second chromatographic column 2, and passes through the third chromatographic column 3 to separate 2'-fucosyllactose with a purity of more than 97wt.%, enters the third chromatographic column 3, and passes through the fourth chromatographic column 4 to separate 2'-fucosyllactose with a purity of more than 97wt.%; after completing one cycle of separation of the six chromatographic columns, enter the next cycle of separation; after the separation of the residual liquid and 2'-fucosyllactose with a purity of more than 97wt.% is completed, the residual liquid is recycled by nanofiltration.

In step (5), the separation step of the concentrated solution 2 in the second chromatographic separation system further comprises:

(1) Open the first circulation valve 103, the second circulation valve 203, the third circulation valve 303, the fourth circulation valve 403, the fifth circulation valve 503, the sixth circulation valve 603, the first circulation pump 106, the second circulation pump 206, the third circulation pump 306, the fifth circulation pump 506 and the sixth circulation pump 606, so that the concentrated liquid 2 fills the entire chromatographic separation system;

(2) Open the first water inlet valve 102 to add the eluent, and open the first circulation pump 106, the second circulation pump 206, the third circulation pump 306, the fourth circulation pump 406, the fifth circulation pump 506, the first circulation valve 103, the second circulation valve 203, the third circulation valve 303, the fourth circulation valve 403 and the fifth product valve 505. After passing through the fifth product valve 505, 2'-fucosyllactose with a purity of more than 97 wt.% is separated and enters the product pipeline;

(3) Open the first water inlet valve 102 to add the eluent, and open the first circulation pump 106 and the first recovery valve 104 to separate the residual liquid from the first chromatographic column 1; open the fourth feed valve 401 to add the concentrated liquid 2, and open the fourth circulation pump 406, the fifth circulation pump 506, the fourth circulation valve 403 and the fifth product valve 505. After passing through the fifth product valve 505, 2'-fucosyllactose with a purity of more than 97 wt.% is separated and enters the product pipeline;

(4) Open the second water inlet valve 202 to add the eluent, and open the second circulation pump 206 and the second recovery valve 204 to separate the residual liquid from the second chromatographic column 2; open the fifth feed valve 501 to add the concentrated liquid 2, and open the fifth circulation pump 506, the sixth circulation pump 606, the fifth circulation valve 503 and the sixth product valve 605. After passing through the sixth product valve 605, 2'-fucosyllactose with a purity of more than 97 wt.% is separated and enters the product pipeline;

Afterwards, the eluent enters the third chromatographic column 3, the fourth chromatographic column 4, the fifth chromatographic column 5, and the sixth chromatographic column 6 in sequence to separate the residual liquid; the concentrated liquid 2 enters the sixth chromatographic column 6 in sequence, and passes through the first chromatographic column 1 to separate 2'-fucosyllactose with a purity of more than 97wt.%, enters the first chromatographic column 1, and passes through the second chromatographic column 2 to separate 2'-fucosyllactose with a purity of more than 97wt.%, enters the second chromatographic column 2, and passes through the third chromatographic column 3 to separate 2'-fucosyllactose with a purity of more than 97wt.%, enters the third chromatographic column 3, and passes through the fourth chromatographic column 4 to separate 2'-fucosyllactose with a purity of more than 97wt.%; after completing one cycle of separation of the six chromatographic columns, enter the next cycle of separation; after the separation of the residual liquid and 2'-fucosyllactose with a purity of more than 97wt.% is completed, the residual liquid is recycled by nanofiltration.

The fermentation broth of the present invention is a prior art, which is prepared by fermenting lactose and glucose as substrates with conventional bacteria such as Escherichia coli.

The process of the present invention was run for 6 months, producing 105 tons of trisaccharide and 25 tons of tetrasaccharide. After testing, the product indicators are as shown in Table 1. The product indicators of the glacial acetic acid crystallization method are also attached.

Claims (10)

1. A process for co-production of lacto-N-difucose tetraose and 2' -fucosyllactose based on chromatographic separation, characterized in that it comprises the following steps:

(1) Removing thalli in fermentation liquor containing monosaccharide and polysaccharide to obtain clear liquid and fungus residues, and carrying out nanofiltration on the clear liquid;

(2) After nanofiltration, the mixture is separated into a concentrated phase material and a clear phase material, and the clear phase material is discharged as mother liquor;

(3) Carrying out fixed bed ion exchange treatment on the concentrated phase material to obtain ion exchange liquid;

(4) Evaporating and concentrating the ion exchange liquid to obtain concentrated liquid I, and separating the concentrated liquid I in a first chromatographic separation system to obtain a milk-N-difucose tetraose product with the purity of more than 94wt.% and a recovery liquid;

(5) Decolorizing the recovered liquid, carrying out fixed bed ion exchange, evaporating and concentrating to obtain a concentrated liquid II, and allowing the concentrated liquid II to enter a second chromatographic separation system to obtain 2' -fucosyllactose with the purity of more than 97wt.% and residual liquid;

(6) The 2 '-fucosyllactose with the purity of more than 97wt.% is decolorized, subjected to fixed bed ion exchange, evaporated and concentrated, sterilized and spray-dried to obtain the 2' -fucosyllactose product.

2. A co-production process of milk-N-disaccharide based on chromatographic separation and 2' -fucosyllactose according to claim 1, characterized in that,

In the step (1), a ceramic membrane is adopted to remove thalli in fermentation liquor containing monosaccharide and polysaccharide, and bacterial residues are used as proteins for recycling;

In the step (2), the conductivity of the concentrated phase material is 7000-8000us/cm, and the clear phase material comprises monosaccharide and disaccharide;

In the step (3), the conductivity of the ion exchange liquid is below 50 us/cm;

In the step (4), the ion exchange liquid is evaporated and concentrated to obtain concentrated solution I with the concentration of 38-40 wt.%.

3. The process for co-production of milk-N-disaccharide and 2' -fucosyllactose based on chromatographic separation according to claim 1, characterized in that in step (4), the first set of chromatographic separation systems comprises a first chromatographic column (1), a second chromatographic column (2), a third chromatographic column (3), a fourth chromatographic column (4), a fifth chromatographic column (5) and a sixth chromatographic column (6) connected in sequence, the sixth chromatographic column (6) being further connected to the first chromatographic column (1), the six chromatographic columns forming a circulation loop; the first chromatographic separation system further comprises a water inlet pipeline, a feeding pipeline, a recovery pipeline and a product pipeline, wherein the water inlet pipeline is communicated with all chromatographic columns, the feeding pipeline is communicated with all chromatographic columns, the recovery pipeline is communicated with all chromatographic columns, and the product pipeline is communicated with all chromatographic columns.

4. A co-production process of milk-N-difucose tetraose and 2' -fucosyllactose based on chromatographic separation according to claim 3, characterized in that the eluent is fed from the water inlet pipe, the eluent is aqueous solution, and the pH value of the aqueous solution is 7.0 < ph.ltoreq.10.0.

5. The process for co-production of lacto-N-disaccharide and 2' -fucosyllactose based on chromatographic separation according to claim 3, characterized in that on the circulation loop, the first chromatographic column (1), the first circulation pump (106), the first circulation valve (103), the second chromatographic column (2), the second circulation pump (206), the second circulation valve (203), the third chromatographic column (3), the third circulation pump (306), the third circulation valve (303), the fourth chromatographic column (4), the fourth circulation pump (406), the fourth circulation valve (403), the fifth chromatographic column (5), the fifth circulation pump (506), the fifth circulation valve (503), the sixth chromatographic column (6), the sixth circulation pump (606), the sixth circulation valve (603) and the first chromatographic column (1) are connected in this order;

A first water inlet valve (102), a second water inlet valve (202), a third water inlet valve (302), a fourth water inlet valve (402), a fifth water inlet valve (502) and a sixth water inlet valve (602) are respectively arranged between the water inlet pipeline and the first chromatographic column (1), between the water inlet pipeline and the second chromatographic column (2), between the water inlet pipeline and the third chromatographic column (3), between the water inlet pipeline and the fourth chromatographic column (4), between the water inlet pipeline and the fifth chromatographic column (5) and between the water inlet pipeline and the sixth chromatographic column (6);

a first feeding valve (101), a second feeding valve (201), a third feeding valve (301), a fourth feeding valve (401), a fifth feeding valve (501) and a sixth feeding valve (601) are respectively arranged between the feeding pipeline and the first chromatographic column (1), between the feeding pipeline and the second chromatographic column (2), between the feeding pipeline and the third chromatographic column (3), between the feeding pipeline and the fourth chromatographic column (4), between the feeding pipeline and the fifth chromatographic column (5) and between the feeding pipeline and the sixth chromatographic column (6);

a recycling pipeline and a product pipeline are sequentially connected to a recycling loop between the first circulating pump (106) and the first circulating valve (103), and the recycling pipeline and the product pipeline are respectively provided with a first recycling valve (104) and a first product valve (105);

A recycling pipeline and a product pipeline are sequentially connected to a recycling loop between the second circulating pump (206) and the second circulating valve (203), and the recycling pipeline and the product pipeline are respectively provided with a second recycling valve (204) and a second product valve (205);

a recycling pipeline and a product pipeline are sequentially connected to a recycling loop between the third circulating pump (306) and the third circulating valve (303), and the recycling pipeline and the product pipeline are respectively provided with a third recycling valve (304) and a third product valve (305);

A recycling pipeline and a product pipeline are sequentially connected to a recycling loop between the fourth circulating pump (406) and the fourth circulating valve (403), and the recycling pipeline and the product pipeline are respectively provided with a fourth recycling valve (404) and a fourth product valve (405);

a recycling pipeline and a product pipeline are sequentially connected to a recycling loop between the fifth recycling pump (506) and the fifth recycling valve (503), and the recycling pipeline and the product pipeline are respectively provided with a fifth recycling valve (504) and a fifth product valve (505);

And a recycling pipeline and a product pipeline are sequentially connected to a recycling loop between the sixth recycling pump (606) and the sixth recycling valve (603), and the recycling pipeline and the product pipeline are respectively provided with a sixth recycling valve (604) and a sixth product valve (605).

6. The chromatography separation-based co-production process of lacto-N-difucosa and 2' -fucosyllactose according to claim 5, wherein in step (5), the second set of chromatography separation systems is identical in structure to the first set of chromatography separation systems.

7. A co-production process of milk-N-difiuorotetraose and 2' -fucosyllactose based on chromatographic separation according to claim 3, characterized in that in step (4), the separation step of the concentrate one in the first chromatographic separation system comprises:

(1) The concentrated solution enters the first chromatographic column (1) and circulates in a circulation loop;

(2) The eluent enters the first chromatographic column (1) from a water inlet pipeline, circulates to the fifth chromatographic column (5), separates milk-N-difucose tetraose product with purity of more than 94wt.% and enters a product pipeline;

(3) The eluent enters the first chromatographic column (1) from a water inlet pipeline, and the recovery liquid is separated from the first chromatographic column (1); the concentrated solution I enters a fourth chromatographic column (4) from a feeding pipeline and is recycled to a fifth chromatographic column (5), and milk-N-difucose tetraose product with the purity of more than 94wt.% is separated from the fifth chromatographic column (5) and enters a product pipeline;

(4) The eluent enters the second chromatographic column (2) from a water inlet pipeline, and the recovery liquid is separated from the second chromatographic column (2); the concentrated solution I enters a fifth chromatographic column (5) from a feeding pipeline and is recycled to a sixth chromatographic column (6), and milk-N-difucose tetraose product with the purity of more than 94wt.% is separated from the sixth chromatographic column (6) and enters a product pipeline;

Then, the eluent sequentially enters a third chromatographic column (3), a fourth chromatographic column (4), a fifth chromatographic column (5) and a sixth chromatographic column (6), and the recovery liquid is separated; sequentially introducing the concentrated solution into a sixth chromatographic column (6), separating milk-N-difucose tetraose products with the purity of more than 94wt.% by a first chromatographic column (1), introducing into the first chromatographic column (1), separating milk-N-difucose tetraose products with the purity of more than 94wt.% by a second chromatographic column (2), introducing into the second chromatographic column (2), separating milk-N-difucose tetraose products with the purity of more than 94wt.% by a third chromatographic column (3), introducing into the third chromatographic column (3), and separating milk-N-difucose tetraose products with the purity of more than 94wt.% by a fourth chromatographic column (4); after completing the primary cycle separation period of six chromatographic columns, entering the next cycle separation period; after the recovery liquid and the milk-N-difucose tetraose product with the purity of more than 94wt.% are completely separated, the recovery liquid is decolorized, subjected to fixed bed ion exchange and evaporated concentration to obtain a concentrated solution II, and the concentrated solution II enters a second chromatographic separation system for separation.

8. The chromatography separation-based co-production process of lacto-N-difiuorotetraose and 2' -fucosyllactose according to claim 7, wherein in step (4), the separation step of the concentrate one in the first chromatography separation system comprises:

(1) Opening a first circulation valve (103), a second circulation valve (203), a third circulation valve (303), a fourth circulation valve (403), a fifth circulation valve (503), a sixth circulation valve (603), a first circulation pump (106), a second circulation pump (206), a third circulation pump (306), a fifth circulation pump (506) and a sixth circulation pump (606) to enable the concentrated solution to fill the whole chromatographic separation system;

(2) Opening a first water inlet valve (102), adding eluent, opening a first circulating pump (106), a second circulating pump (206), a third circulating pump (306), a fourth circulating pump (406), a fifth circulating pump (506), a first circulating valve (103), a second circulating valve (203), a third circulating valve (303), a fourth circulating valve (403) and a fifth product valve (505), and separating milk-N-difucose tetrasaccharide products with the purity of 94wt.% or more from the products after passing through the fifth product valve (505), and entering a product pipeline;

(3) Opening a first water inlet valve (102) to add eluent, and opening a first circulating pump (106) and a first recovery valve (104) to separate recovery liquid from the first chromatographic column (1); starting a fourth feeding valve (401) to add concentrated solution I, starting a fourth circulating pump (406), a fifth circulating pump (506), a fourth circulating valve (403) and a fifth product valve (505), separating milk-N-difucose tetraose products with purity of more than 94wt.% after passing through the fifth product valve (505), and entering a product pipeline;

(4) Opening a second water inlet valve (202) to add eluent, and opening a second circulating pump (206) and a second recovery valve (204) to separate recovery liquid from the second chromatographic column (2); starting a fifth feed valve (501) to add concentrated solution I, starting a fifth circulating pump (506), a sixth circulating pump (606), a fifth circulating valve (503) and a sixth product valve (605), separating milk-N-difucose tetraose products with purity of more than 94wt.% after passing through the sixth product valve (605), and entering a product pipeline;

Then, the eluent sequentially enters a third chromatographic column (3), a fourth chromatographic column (4), a fifth chromatographic column (5) and a sixth chromatographic column (6), and the recovery liquid is separated; sequentially introducing the concentrated solution into a sixth chromatographic column (6), separating milk-N-difucose tetraose products with the purity of more than 94wt.% by a first chromatographic column (1), introducing into the first chromatographic column (1), separating milk-N-difucose tetraose products with the purity of more than 94wt.% by a second chromatographic column (2), introducing into the second chromatographic column (2), separating milk-N-difucose tetraose products with the purity of more than 94wt.% by a third chromatographic column (3), introducing into the third chromatographic column (3), and separating milk-N-difucose tetraose products with the purity of more than 94wt.% by a fourth chromatographic column (4); after completing the primary cycle separation period of six chromatographic columns, entering the next cycle separation period; after the recovery liquid and the milk-N-difucose tetraose product with the purity of more than 94wt.% are completely separated, the recovery liquid is decolorized, subjected to fixed bed ion exchange and evaporated concentration to obtain a concentrated solution II, and the concentrated solution II enters a second chromatographic separation system for separation.

9. The chromatographic separation based co-production process of lacto-N-difiuorotetraose and 2' -fucosyllactose according to claim 6, wherein in step (5), the separation step of the concentrate two in the second chromatographic separation system comprises:

(1) The concentrated solution II enters a first chromatographic column (1) and circulates in a circulation loop;

(2) The eluent enters the first chromatographic column (1) from a water inlet pipeline, is circulated into the fifth chromatographic column (5), and is separated into 2' -fucosyllactose with the purity of more than 97wt.% and enters a product pipeline;

(3) The eluent enters the first chromatographic column (1) from a water inlet pipeline, and residual liquid is separated from the first chromatographic column (1); the concentrated solution II enters a fourth chromatographic column (4) from a feeding pipeline and is recycled to a fifth chromatographic column (5), 2' -fucosyllactose with the purity of more than 97wt.% is separated from the fifth chromatographic column (5), and enters a product pipeline;

(4) The eluent enters the second chromatographic column (2) from a water inlet pipeline, and residual liquid is separated from the second chromatographic column (2); the concentrated solution II enters a fifth chromatographic column (5) from a feeding pipeline and is recycled to a sixth chromatographic column (6), 2' -fucosyllactose with the purity of more than 97wt.% is separated from the sixth chromatographic column (6), and enters a product pipeline;

Then, the eluent sequentially enters a third chromatographic column (3), a fourth chromatographic column (4), a fifth chromatographic column (5) and a sixth chromatographic column (6) to separate residual liquid; sequentially introducing the concentrated solution II into a sixth chromatographic column (6), separating 2 '-fucosyllactose with the purity of more than 97wt.% through a first chromatographic column (1), introducing into the first chromatographic column (1), separating 2' -fucosyllactose with the purity of more than 97wt.% through a second chromatographic column (2), introducing into the second chromatographic column (2), separating 2 '-fucosyllactose with the purity of more than 97wt.% through a third chromatographic column (3), introducing into the third chromatographic column (3), and separating 2' -fucosyllactose with the purity of more than 97wt.% through a fourth chromatographic column (4); after completing the primary cycle separation period of six chromatographic columns, entering the next cycle separation period; after the separation of the residual liquid and the 2' -fucosyllactose with the purity of more than 97wt.% is completed, the residual liquid is subjected to nanofiltration and recycling.

10. The chromatographic separation based co-production process of lacto-N-difiuorotetraose and 2' -fucosyllactose according to claim 9, wherein in step (5), the separation step of the concentrate two in the second chromatography separation system comprises:

(1) Opening a first circulation valve (103), a second circulation valve (203), a third circulation valve (303), a fourth circulation valve (403), a fifth circulation valve (503), a sixth circulation valve (603), a first circulation pump (106), a second circulation pump (206), a third circulation pump (306), a fifth circulation pump (506) and a sixth circulation pump (606) to enable the concentrated solution II to fill the whole chromatographic separation system;

(2) Opening a first water inlet valve (102) to add eluent, and opening a first circulating pump (106), a second circulating pump (206), a third circulating pump (306), a fourth circulating pump (406), a fifth circulating pump (506), a first circulating valve (103), a second circulating valve (203), a third circulating valve (303), a fourth circulating valve (403) and a fifth product valve (505), wherein after passing through the fifth product valve (505), 2' -fucosyllactose with the purity of more than 97wt.% is separated, and enters a product pipeline;

(3) Opening a first water inlet valve (102) to add eluent, and opening a first circulating pump (106) and a first recovery valve (104) to separate residual liquid from the first chromatographic column (1); starting a fourth feeding valve (401) to add concentrated solution II, starting a fourth circulating pump (406), a fifth circulating pump (506), a fourth circulating valve (403) and a fifth product valve (505), separating 2' -fucosyllactose with the purity of more than 97wt.% after passing through the fifth product valve (505), and entering a product pipeline;

(4) Opening a second water inlet valve (202) to add eluent, and opening a second circulating pump (206) and a second recovery valve (204) to separate residual liquid from the second chromatographic column (2); starting a fifth feed valve (501) to add concentrated solution II, starting a fifth circulating pump (506), a sixth circulating pump (606), a fifth circulating valve (503) and a sixth product valve (605), separating 2' -fucosyllactose with purity of more than 97wt.% after passing through the sixth product valve (605), and entering a product pipeline;

Then, the eluent sequentially enters a third chromatographic column (3), a fourth chromatographic column (4), a fifth chromatographic column (5) and a sixth chromatographic column (6) to separate residual liquid; sequentially introducing the concentrated solution II into a sixth chromatographic column (6), separating 2 '-fucosyllactose with the purity of more than 97wt.% through a first chromatographic column (1), introducing into the first chromatographic column (1), separating 2' -fucosyllactose with the purity of more than 97wt.% through a second chromatographic column (2), introducing into the second chromatographic column (2), separating 2 '-fucosyllactose with the purity of more than 97wt.% through a third chromatographic column (3), introducing into the third chromatographic column (3), and separating 2' -fucosyllactose with the purity of more than 97wt.% through a fourth chromatographic column (4); after completing the primary cycle separation period of six chromatographic columns, entering the next cycle separation period; after the separation of the residual liquid and the 2' -fucosyllactose with the purity of more than 97wt.% is completed, the residual liquid is subjected to nanofiltration and recycling.