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

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

Technical Field

[0001] The 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. Background Art

[0002] 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 as follows Figure 3 shown.

[0003] according to Figure 3 The specific operation of producing human milk oligosaccharides by crystallization is as follows:

[0004] (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.

[0005] (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.

[0006] (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;

[0007] (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;

[0008] (5) The ion exchange liquid passes through a falling film evaporator to concentrate the concentration to about 55%;

[0009] (6) The concentrate is sterilized by passing through a precision filter;

[0010] (7) The material is spray-dried to obtain the finished human milk oligosaccharide product.

[0011] The above process has the following problems:

[0012] (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;

[0013] (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;

[0014] (3) It is necessary to recover the glacial acetic acid from the crystallized material;

[0015] (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.

[0016] 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 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.

[0017] 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

[0018] 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 or less 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.

[0019] The co-production process of lacto-N-difucotetraose and 2'-fucosyllactose based on chromatographic separation of the present invention comprises the following steps:

[0020] (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;

[0021] (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;

[0022] (3) The concentrated phase material is treated by fixed bed ion exchange to obtain ion exchange liquid;

[0023] (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;

[0024] (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;

[0025] (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.

[0026] in:

[0027] 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;

[0028] In step (2), the conductivity of the dense phase material is 7000-8000 us / cm, and the clear phase material includes monosaccharides and disaccharides;

[0029] In step (3), the conductivity of the ion exchange liquid is less than 50 us / cm;

[0030] In step (4), the ion exchange liquid is evaporated and concentrated to obtain a concentrated solution 1 with a concentration of 38-40 wt.%.

[0031] 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.

[0032] The eluent is fed into the water inlet pipe, and 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.

[0033] 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;

[0034] 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;

[0035] 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;

[0036] 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;

[0037] 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;

[0038] A recycling 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 recycling pipeline and the product pipeline are respectively provided with a third recycling valve and a third product valve;

[0039] 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;

[0040] 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;

[0041] 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.

[0042] In step (5), the second chromatographic separation system has the same structure as the first chromatographic separation system.

[0043] In step (4), the separation step of the concentrate 1 in the first chromatographic separation system includes:

[0044] (1) The concentrate enters the first chromatographic column and circulates in the circulation loop;

[0045] (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;

[0046] (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;

[0047] (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;

[0048] 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 1 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 2, which enters the second set of chromatographic separation system for separation.

[0049] In step (4), the separation step of the concentrate 1 in the first chromatographic separation system further comprises:

[0050] (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;

[0051] (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;

[0052] (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;

[0053] (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;

[0054] 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 1 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 2, which enters the second set of chromatographic separation system for separation.

[0055] In step (5), the separation step of the concentrated solution 2 in the second chromatographic separation system includes:

[0056] (1) The concentrated liquid 2 enters the first chromatographic column and circulates in the circulation loop;

[0057] (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;

[0058] (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;

[0059] (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;

[0060] 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.

[0061] In step (5), the separation step of the concentrated solution 2 in the second chromatographic separation system further comprises:

[0062] (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;

[0063] (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;

[0064] (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;

[0065] (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;

[0066] 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.

[0067] In the present invention, 2'-fucosyllactose (2'-FL) is referred to as trisaccharide; lacto-N-difucotetraose (DFL) is referred to as tetrasaccharide.

[0068] Beneficial effects of the present invention:

[0069] (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.

[0070] (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.

[0071] (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 range of selectivity for materials and can achieve good separation effects for substances with similar molecular weights and properties, thereby ensuring the stability of production.

[0072] (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.

[0073] 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

[0074] Figure 1 It is a schematic structural diagram of the first chromatographic separation system of the present invention;

[0075] Figure 2 It is a process flow chart of the present invention;

[0076] Figure 3 It is a process flow chart of the prior art.

[0077] 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 DESCRIPTION

[0078] The present invention is further described below with reference to the embodiments.

[0079] Example 1

[0080] The co-production process of lacto-N-difucotetraose and 2'-fucosyllactose based on chromatographic separation of the present invention comprises the following steps:

[0081] (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;

[0082] (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;

[0083] (3) The concentrated phase material is treated by fixed bed ion exchange to obtain ion exchange liquid;

[0084] (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;

[0085] (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;

[0086] (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.

[0087] 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;

[0088] In step (2), the conductivity of the dense phase material is 7000-8000 us / cm, and the clear phase material includes monosaccharides and disaccharides;

[0089] In step (3), the conductivity of the ion exchange liquid is less than 50 us / cm;

[0090] In step (4), the ion exchange liquid is evaporated and concentrated to obtain a concentrated solution 1 with a concentration of 38-40 wt.%.

[0091] 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.

[0092] The eluent is fed into the water inlet pipe, and 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.

[0093] 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;

[0094] 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;

[0095] 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;

[0096] 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;

[0097] The circulation loop between the second circulation pump 206 and the second circulation valve 203 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 second recovery valve 204 and a second product valve 205;

[0098] 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;

[0099] 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;

[0100] 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;

[0101] The recycling pipeline and the product pipeline are connected in sequence to the recycling loop between the sixth recycling pump 606 and the sixth recycling valve 603. The recycling pipeline and the product pipeline are respectively provided with a sixth recycling valve 604 and a sixth product valve 605.

[0102] In step (5), the second chromatographic separation system has the same structure as the first chromatographic separation system.

[0103] In step (4), the separation step of the concentrate 1 in the first chromatographic separation system includes:

[0104] (1) The concentrated liquid enters the first chromatographic column 1 and circulates in the circulation loop;

[0105] (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;

[0106] (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;

[0107] (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;

[0108] 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.

[0109] In step (4), the separation step of the concentrate 1 in the first chromatographic separation system further comprises:

[0110] (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;

[0111] (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;

[0112] (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;

[0113] (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;

[0114] 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.

[0115] In step (5), the separation step of the concentrated solution 2 in the second chromatographic separation system includes:

[0116] (1) The concentrated liquid 2 enters the first chromatographic column 1 and circulates in the circulation loop;

[0117] (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;

[0118] (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;

[0119] (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;

[0120] 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.

[0121] In step (5), the separation step of the concentrated solution 2 in the second chromatographic separation system further comprises:

[0122] (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 concentrate second fills the entire chromatographic separation system;

[0123] (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;

[0124] (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;

[0125] (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;

[0126] 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.

[0127] 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.

[0128] 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.

[0129]

Claims

1. A process for the co-production of lacto-N-difucotetraose and 2'-fucosyllactose based on chromatographic separation, characterized in that: The following steps are involved: (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, and a 2'-fucosyllactose product is obtained; 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, 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 pipeline, a feed pipeline, a recovery pipeline and a product pipeline, the water inlet pipeline is connected to all the chromatographic columns, the feed pipeline is connected to all the chromatographic columns, the recovery pipeline is connected to all the chromatographic columns, and the product pipeline 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; In step (5), the second chromatographic separation system has the same structure as the first chromatographic separation system; 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 evaporated and concentrated to obtain a concentrated solution 1 with a concentration of 38-40 wt.%; In step (4), the separation step of the concentrate 1 in the first chromatographic separation system includes: (a) the concentrate enters the first chromatography column (1) and circulates in the circulation loop; (b) 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 pipe; (c) 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 pipe; (d) 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 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 recovered liquid; the concentrated liquid 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 94 wt.%, 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 94 wt.%, 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 94 wt.% is obtained, and enters the third chromatographic column (3), and is separated into the lacto-N-difucotetraose product with a purity of more than 94 wt.% through the fourth chromatographic column (4); after completing one cycle of separation through the six chromatographic columns, the next cycle of separation is entered; after the separation of the recovered liquid and the lacto-N-difucotetraose product with a purity of more than 94 wt.% is completed, the recovered liquid is decolorized, fixed-bed ion exchanged, and evaporated to obtain a concentrated liquid 2, which enters the second set of chromatographic separation system for separation.

2. The process for co-production of lacto-N-difucotetraose and 2'-fucosyllactose based on chromatographic separation according to claim 1, characterized in that: In 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); A recovery pipeline and a product pipeline are sequentially connected to the circulation loop between the first circulation pump (106) and the first circulation valve (103), 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 to a recovery pipeline and a product pipeline in sequence, and the recovery pipeline and the product pipeline are provided with a third recovery valve (304) and a third product valve (305) respectively; The circulation loop between the fourth circulation pump (406) and the fourth circulation valve (403) is connected to 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 to 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 circulation loop between the sixth circulation pump (606) and the sixth circulation valve (603) 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 sixth recovery valve (604) and a sixth product valve (605).

3. The process for co-production of lacto-N-difucotetraose and 2'-fucosyllactose based on chromatographic separation according to claim 1, characterized in that: In step (4), the separation step of the concentrate 1 in the first chromatographic separation system includes: (a) 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 concentrated liquid fills the entire chromatographic separation system; (b) opening the first water inlet valve (102) to add the eluent, and opening 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), a lacto-N-difucotetraose product with a purity of more than 94 wt.% is separated and enters the product pipeline; (c) opening the first water inlet valve (102) to add the eluent, and opening the first circulation pump (106) and the first recovery valve (104) to separate the recovery liquid from the first chromatographic column (1); opening the fourth feed valve (401) to add the concentrate 1, and opening 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), a lacto-N-difucotetraose product with a purity of more than 94 wt.% is separated and enters the product pipeline; (d) opening the second water inlet valve (202) to add the eluent, and opening the second circulation pump (206) and the second recovery valve (204) to separate the recovery liquid from the second chromatographic column (2); opening the fifth feed valve (501) to add the concentrate 1, and opening 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), a 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 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 94 wt.%, 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 94 wt.%, 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 94 wt.% is obtained, and enters the third chromatographic column (3), and is separated into the lacto-N-difucotetraose product with a purity of more than 94 wt.% through the fourth chromatographic column (4); after completing one cycle of separation through the six chromatographic columns, the next cycle of separation is entered; after the separation of the recovered liquid and the lacto-N-difucotetraose product with a purity of more than 94 wt.% is completed, the recovered liquid is decolorized, fixed-bed ion exchanged, and evaporated to obtain a concentrated liquid 2, which enters the second set of chromatographic separation system for separation.

4. The process for co-production of lacto-N-difucotetraose and 2'-fucosyllactose based on chromatographic separation according to claim 1, characterized in that: 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 pipe; 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 97 wt.%, enters the first chromatographic column (1), and passes through the second chromatographic column (2) to separate 2'-fucosyllactose with a purity of more than 97 wt.%, enters the second chromatographic column (2), and passes through the third chromatographic column (3) to separate 2'-fucosyllactose with a purity of more than 97 wt.%, enters the third chromatographic column (3), and passes through the fourth chromatographic column (4) to separate 2'-fucosyllactose with a purity of more than 97 wt.%; after completing one cycle of separation of the six chromatographic columns, the next cycle of separation is entered; after the separation of the residual liquid and the 2'-fucosyllactose with a purity of more than 97 wt.% is completed, the residual liquid is subjected to nanofiltration for recycling.

5. The process for co-production of lacto-N-difucotetraose and 2'-fucosyllactose based on chromatographic separation according to claim 4, characterized in that: In step (5), the separation step of the concentrated solution 2 in the second chromatographic separation system includes: 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 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), separate 2'-fucosyllactose with a purity of more than 97 wt.% and enter 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 97 wt.%, enters the first chromatographic column (1), and passes through the second chromatographic column (2) to separate 2'-fucosyllactose with a purity of more than 97 wt.%, enters the second chromatographic column (2), and passes through the third chromatographic column (3) to separate 2'-fucosyllactose with a purity of more than 97 wt.%, enters the third chromatographic column (3), and passes through the fourth chromatographic column (4) to separate 2'-fucosyllactose with a purity of more than 97 wt.%; after completing one cycle of separation of the six chromatographic columns, the next cycle of separation is entered; after the separation of the residual liquid and the 2'-fucosyllactose with a purity of more than 97 wt.% is completed, the residual liquid is subjected to nanofiltration for recycling.

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