Escherichia coli BL21 star (DE3) BLBRTXST3 and method for producing high-purity lactose-N-neotetraose by fermentation and application thereof

Abstract

A method and application of Escherichia coli BL21star (DE3) BLBRTXST3 and its fermentation method for producing high-purity lactose-N-neotetraose, the invention relates to the technical field of human milk oligosaccharide production, Escherichia coli )BL21 star (DE3) BLBRTXST3 is deposited in the General Microbiology Center of China Microorganism Culture Collection Committee, with the deposit number CGMCC No. 32521 and the deposit date November 6, 2024. The address of the deposit institution is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; Escherichia coli ( Escherichia coli )BL21 star (DE3) BLBRTXST3 is used in the fermentation production of lactose-N-neotetraose (LNnT). The present invention improves the yield of lactose-N-neotetraose, uses a chromatographic simulated moving bed to produce high-purity lactose-N-neotetraose with a purity greater than 94%, replaces the traditional solvent crystallization method, has simple separation and purification, is conducive to industrial production, and simultaneously produces linear lactose-N-neohexaose with a purity greater than 95%.

Description

Technical Field

[0001] The invention relates to the technical field of human milk oligosaccharide production, and in particular to Escherichia coli BL21star (DE3) BLBRTXST3 and a method and application thereof for producing high-purity lactose-N-neotetraose by fermentation. Background Art

[0002] Lacto-N-neotetraose (LNnT) is a naturally occurring tetrasaccharide that can be detected in the milk of some mammals. It is found in human milk at the highest concentration, so it is often referred to as human milk oligosaccharide (HMO). LNnT is a chemically defined linear tetrasaccharide that is composed of D-galactose, N-acetylglucosamine, D-galactose, and D-glucose, and is a specific conformational isomer. LNnT is one of the important components of HMOs and has important biological functions such as enhancing human immunity, regulating intestinal flora, and promoting cell maturation. It can be added to infant formula as a nutritional enhancer.

[0003] At present, the research on LNnT at home and abroad mainly focuses on the construction of strains for producing LNnT by microbial fermentation. There is no report on the fermentation production of lactose-N-neotetraose and linear lactose-N-neohexose in the prior art. Summary of the invention

[0004] The invention provides an Escherichia coli BL21star (DE3) BLBRTXST3 and a method and application thereof for producing high-purity lactose-N-neotetraose by fermentation. The separation and purification process is simple and more conducive to industrial production.

[0005] An Escherichia coli BL21star (DE3) BLBRTXST3, wherein the Escherichia coli (Escherichiacoli) is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with a deposit number of CGMCC No.32521, a deposit date of November 6, 2024, and an address of the deposit institution: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0006] Furthermore, the Escherichia coli BL21star (DE3) BLBRTXST3 was subjected to UV mutagenesis: a streak plate of Escherichia coli BL21 (DE3) was cultured at 30°C, a single colony was picked and inoculated into a seed culture medium, and cultured until OD 600 The mutagenic bacterial solution was placed on a magnetic stirrer under an ultraviolet lamp at a vertical distance of 20-30 cm, and stirred and irradiated; the prepared mutagenic bacterial solution was treated with ultraviolet light for 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 s, and then subjected to gradient 10-3 , 10 -4 , 10 -5 , spread on the seed culture medium agar plate, invert and culture at 30-37℃, select the plate with appropriate dilution multiple to count and calculate the lethality; the formula for calculating the lethality is as follows:

[0007] ;

[0008] Select a mutagenesis time with a lethality rate of 70%-80% for mutagenesis, pick colonies with better growth on the resistance plate and inoculate them into the seed culture medium for 8-10 hours, transfer them to the fermentation medium, screen out the strain with the highest transformation rate, repeat several times until the excellent strain BL21 star (DE3) BLBRTXST3 is screened.

[0009] A further method for producing high-purity lactose-N-neotetraose by fermentation of Escherichia coli BL21star (DE3) BLBRTXST3 comprises the following steps:

[0010] (1) Inoculate Escherichia coli BL21 star (DE3) BLBRTXST3 into seed liquid culture medium to obtain primary seed liquid;

[0011] (2) inoculating the first-level seed solution into the seed solution culture medium to obtain the second-level seed solution;

[0012] (3) inoculating the secondary seed liquid into a fermentation medium for fermentation to obtain a fermentation liquid;

[0013] (4) The fermentation liquid is filtered through a ceramic membrane, an ultrafiltration membrane, and a nanofiltration membrane to obtain a supernatant, the supernatant is decolorized by activated carbon to obtain a feed liquid, the feed liquid is subjected to ion exchange to reduce conductivity, and then concentrated, chromatographically separated, decolorized for a second time, deionized for a second time, concentrated, and dried to obtain high-purity lactose-N-neotetraose with a purity greater than 94%;

[0014] (5) The by-product linear lactose-N-neohexose is separated by chromatography to obtain linear lactose-N-neohexose with a purity greater than 95%, and then decolorized, ion exchanged, concentrated and dried to obtain high-purity linear lactose-N-neohexose with a purity greater than 95%.

[0015] In the further step (3), the fermentation medium is prepared according to the following concentrations: 8-12 g / L glycerol, 1-5 g / L yeast powder, 4-8 g / L peptone, 2-6 g / L ammonium sulfate, 8-10 g / L dipotassium hydrogen phosphate, 8-10 g / L potassium dihydrogen phosphate, 1-5 g / L magnesium sulfate heptahydrate, 0.1-1 g / L citric acid, 0.01-0.05 g / L calcium chloride; 8-10 mL / L trace metal ion liquid elements; trace metal elements include the following concentrations of components, 0.01-0.2 g / L ammonium heptamolybdate, 1-3 g / L zinc sulfate heptahydrate, 0.1-1 g / L manganese sulfate tetrahydrate, 8-12 g / L ferrous sulfate heptahydrate, 1-5 g / L copper sulfate pentahydrate, and 0.1-0.3 g / L sodium tetraborate.

[0016] In the further step (3), the fermentation process conditions are as follows: the secondary seed liquid is transferred to the fermentation tank at an inoculation rate of 8-12%, chloramphenicol is added at a final concentration of 20-30 ug / mL, cultured at 37°C, the dissolved oxygen is controlled at about 30%, and the pH is controlled at 6.5-7.0; after 5-8 hours, the dissolved oxygen rebounds, and feeding is performed in an exponential flow addition manner, the feeding glycerol is 600 g / L, and the cumulative amount of glycerol does not exceed 1 g / L; after OD600 reaches 30-40, the temperature is lowered to 25-27°C, 0.1 mM IPTG is added for induction, and then lactose is continuously fed at 200 g / L to maintain the lactose concentration at 5-10 g / L.

[0017] In a further step (4), the fermentation broth is centrifuged and then filtered through a ceramic membrane and an ultrafiltration membrane to remove bacteria, and the nanofiltration membrane removes inorganic salts and small molecular sugars.

[0018] In the further step (4), the ion exchange adopts a mixed bed ion exchange of cation and anion resins, wherein the cation resin and the anion resin are D001 resin and D301-F resin in a volume ratio of 100:210.

[0019] Furthermore, the ion exchange feed temperature is 30-40°C, the feed rate is 2-3 Bv / h, the discharge conductivity is less than 50 us / cm, and the discharge pH value is 4-6; the concentration temperature is 60-75°C, and the solid content is concentrated to 20-50%;

[0020] Furthermore, the chromatographic separation conditions in step (4) are as follows: for the first chromatographic separation of small molecular sugars except trisaccharides and disaccharides, the feed mass concentration is 30-50%, the temperature is 60-65°C, and the feed-water ratio is 1:4-8; for the second chromatographic separation of large molecular sugars except hexasaccharides, the feed mass concentration is 20-50%, the column temperature is 60-65°C, and the feed-water ratio is 1:4-8.

[0021] Furthermore, in step (4), the concentration temperature is ≤75°C, the fermentation broth is concentrated to a solid content of 40-60%, and spray-dried to obtain lactose-N-neotetraose powder with a purity greater than 94%.

[0022] Furthermore, the chromatographic separation conditions in step (5) are as follows: for chromatographic separation of small molecular sugars except tetrasaccharides and trisaccharides, the feed mass concentration is 20-50%, the temperature is 60-65°C, and the feed-water ratio is 1:4-8 to obtain high-purity linear lactose-N-neohexose with a purity greater than 95%, and then the linear lactose-N-neohexose powder is obtained by decolorization, desorption, concentration and drying.

[0023] Furthermore, the preparation method of the first-level seed liquid in step (1) is: inoculating the strain on the seed liquid culture medium, adding chloramphenicol with a final concentration of 20-30 μg / mL, and fermenting and culturing for 8-10 hours at a temperature of 37°C and a stirring rate of 150-250 r / min; the seed liquid culture medium is prepared according to the following concentrations: tryptone 8-16 g / L, yeast powder 4-8 g / L, and sodium chloride 8-16 g / L.

[0024] Furthermore, the Escherichia coli BL21star (DE3) BLBRTXST3 is used in the fermentation production of high-purity lactose-N-neotetraose.

[0025] The beneficial effects are as follows:

[0026] (1) The present invention uses Escherichia coli ( Escherichia coli )BL21 star (DE3) BLBRTXST3 strain efficiently ferments and produces high-purity lactose-N-neotetraose and co-produces linear lactose-N-neohexose. The fermentation is precisely controlled, including temperature, pressure, pH, dissolved oxygen, etc. The temperature, pressure and pH of the first stage fermentation process are the most suitable growth conditions for the strain, which enables the strain to multiply rapidly and quickly reach the condition of OD600 of 30-40, laying a good foundation for the second stage inducing the bacteria to produce the target product; in the second stage fermentation process, the temperature is lowered to the optimal production temperature of the strain, and the pressure and pH are adjusted at the same time, and glycerol and lactose are added to increase the yield of lactose-N-neotetraose. At the same time, the linear lactose-N-neohexose produced by fermentation is purified by chromatography to obtain a high-purity product;

[0027] (2) The present invention adopts a multi-stage membrane coupling method to remove bacteria and small molecule salts, reduce the burden of ion exchange, and reduce the acid and alkali consumption of ion exchange treatment;

[0028] (3) The present invention replaces solvent crystallization technology with chromatographic separation technology to achieve the co-production of lactose-N-neotetraose and linear lactose-N-neohexaose, with the purity reaching more than 94%, thereby improving the utilization rate of raw materials. DETAILED DESCRIPTION

[0029] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0030] The invention provides an Escherichia coli BL21star (DE3) BLBRTXST3 and a method and application thereof for producing high-purity lactose-N-neotetraose by fermentation.

[0031] The Escherichia coli ( Escherichia coli )BL21 star (DE3) BLBRTXST3 is obtained as follows:

[0032] The present invention uses the Escherichia coli BL21 star (DE3) strain purchased by the applicant, and the β-1,3-N-acetylglucosamine transferase donor source of the Escherichia coli BL21 star (DE3) BLBRTXST3 strain is Neisseria ( Neisseria species ).

[0033] Example 1: Escherichia coli used in the present invention ( Escherichia coli )BL21 star (DE3)BLBRTXST3 The Escherichia coli BL21 star (DE3) strain preserved by the applicant is subjected to ultraviolet mutagenesis treatment, and an excellent high-yield strain is screened by measuring the conversion rate of lactose-N-neotetraose production, which specifically comprises the following steps:

[0034] UV mutagenesis: Streak E. coli BL21 (DE3) plates, incubate at 30°C, pick a single colony and inoculate it into the seed medium, and culture until OD 600 The mutagenic bacterial solution is about 0.8, and the mutagenic bacterial solution is placed on a magnetic stirrer and placed under an ultraviolet lamp. The ultraviolet lamp parameters are 30 W, 253.7 nm, and a vertical distance of 20-30 cm. Stir and irradiate. The prepared mutagenic bacterial solution is treated with ultraviolet light for 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 s, and then a gradient of 10-3, 10-4, and 10-5 is performed. It is spread on a seed culture medium agar plate and inverted at 30°C. The plate count of the appropriate dilution multiple is selected to calculate the lethality. The formula for calculating the lethality is as follows:

[0035] ;

[0036] Select a mutagenesis time with a lethality rate of 70%-80% for mutagenesis, pick colonies with better growth on the resistance plate and inoculate them into the seed culture medium for 8-10 hours, transfer them to the fermentation medium, screen out the strain with the highest transformation rate, repeat several times until the excellent strain BL21 star (DE3) BLBRTXST3 is screened.

[0037] Example 2: The Escherichia coli BL21 star (DE3) BLBRTXST3 used in the present invention is a strain of Escherichia coli BL21 star (DE3) preserved by the applicant, which is subjected to ultraviolet mutagenesis treatment and screened to obtain an excellent high-yield strain by measuring the conversion rate of lactose-N-neotetraose production, which specifically comprises the following steps:

[0038] UV mutagenesis: Streak E. coli BL21 (DE3) plates, incubate at 35°C, pick a single colony and inoculate it into the seed medium, and culture until OD 600 The mutagenic bacterial solution is about 0.9, and the mutagenic bacterial solution is placed on a magnetic stirrer and placed under an ultraviolet lamp. The ultraviolet lamp parameters are 40 W, 268.7 nm, and a vertical distance of 20-30 cm. Stir and irradiate. The prepared mutagenic bacterial solution is treated with ultraviolet light for 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 s, and then a gradient of 10-3, 10-4, and 10-5 is performed. It is spread on a seed culture medium agar plate and inverted at 35°C. The plate count of the appropriate dilution multiple is selected to calculate the lethality. The lethality calculation formula is as follows:

[0039] ;

[0040] Select a mutagenesis time with a lethality rate of 70%-80% for mutagenesis, pick colonies with better growth on the resistance plate and inoculate them into the seed culture medium for 8-10 hours, transfer them to the fermentation medium, screen out the strain with the highest transformation rate, repeat several times until the excellent strain BL21 star (DE3) BLBRTXST3 is screened.

[0041] Application Example 1: Application of Escherichia coli BL21 star (DE3) BLBRTXST3 in the fermentation production of high-purity lactose-N-neotetraose and co-production of linear lactose-N-neohexaose.

[0042] The specific steps include:

[0043] (1) Escherichia coli BL21 star (DE3) BLBRTXST3 strain was inoculated into the seed liquid culture medium, chloramphenicol was added at a final concentration of 20 μg / mL, and fermented at a temperature of 37°C and a stirring rate of 200 r / min for 8 h to obtain the first-level seed liquid;

[0044] The seed liquid medium was prepared with the following concentrations: tryptone 8 g / L, yeast powder 4 g / L, sodium chloride 8 g / L;

[0045] The first-level seed solution is inoculated into the seed solution culture medium to obtain the second-level seed solution.

[0046] (2) The secondary seed liquid was transferred to the fermentation tank at an inoculation rate of 8%, and chloramphenicol was added at a final concentration of 20 ug / mL. The fermentation was cultured at 37°C, the dissolved oxygen was controlled at about 30%, and the pH was controlled at 6.5-6.8. After 5-8 hours, the dissolved oxygen rebounded and the feed was added in an exponential manner. The feed was glycerol, which was prepared by adding 600 g / L of glycerol to 3 g / L of magnesium sulfate heptahydrate. The cumulative amount of glycerol in the whole process did not exceed 1 g / L. 600 After reaching 30-40°C, the temperature was lowered to 25°C, and IPTG with a final concentration of 0.1 mM was added for induction. After 2 hours, lactose was intermittently fed, and the lactose concentration was maintained at 5-8 g / L.

[0047] The fermentation medium of the fermenter is prepared according to the following concentrations: 8 g / L glycerol, 2 g / L yeast powder, 8 g / L peptone, 3 g / L ammonium sulfate, 8 g / L dipotassium hydrogen phosphate, 10 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate heptahydrate, 0.2 g / L citric acid, 0.01 g / L calcium chloride, and 8 mL / L trace metal ion liquid elements; the trace metal elements include the following concentrations: 0.2 g / L ammonium heptamolybdate, 1 g / L zinc sulfate heptahydrate, 0.5 g / L manganese sulfate tetrahydrate, 8 g / L ferrous sulfate heptahydrate, 1 g / L copper sulfate pentahydrate, and 0.1 g / L sodium tetraborate.

[0048] (3) The fermentation liquid obtained in step (2) is filtered through a ceramic membrane, an ultrafiltration membrane, and a nanofiltration membrane to obtain a supernatant, wherein the ultrafiltration membrane is used to remove bacterial cells, and the nanofiltration membrane is used to remove inorganic salts and small molecular sugars.

[0049] (4) The supernatant obtained in step (3) is decolorized with activated carbon to obtain a feed liquid, wherein the amount of activated carbon used is 3% of the dry matter content of the feed liquid, the decolorization temperature is 60°C, the decolorization time is 50 min, and the light transmittance of the feed liquid is greater than 85%.

[0050] (5) The decolorized liquid in step (4) is subjected to ion exchange to reduce conductivity. The cation resin and the anion resin are D001 resin and D301-F resin in a volume ratio of 100:210. The ion exchange feed temperature is 30°C, the feed rate is 2Bv / h, the discharge conductivity is <50us / cm, and the discharge pH value is 4-6.

[0051] (6) Concentrating the feed liquid after the cross-linking in step (5), with a feed mass concentration of 30%, a concentration temperature of 60-67°C, and concentrating to a solid content of 50%, removing small molecular sugars such as trisaccharides and disaccharides by primary chromatography, with an in-column temperature of 60-62°C and a feed-water ratio of 1:8;

[0052] In the second chromatographic separation, macromolecular sugars such as hexasaccharides were separated with a feed mass concentration of 20%, a column temperature of 60-65 °C, and a feed-water ratio of 1:4.

[0053] (7) The lactose-N-neotetraose liquid after chromatographic separation in step (6) is subjected to secondary decolorization, cross-linking, and concentration to a solid content of 50-75%, and then spray-dried to obtain a finished lactose-N-neotetraose powder, wherein the purity of the finished lactose-N-neotetraose powder is greater than 94%.

[0054] (8) Concentrating the linear lactose-N-neohexose liquid after chromatographic separation in step (6), and then chromatographically separating it to obtain high-purity linear lactose-N-neohexose, concentrating it to a solid content of 50%, and spray drying it to obtain high-purity linear lactose-N-neohexose powder.

[0055] Application Example 2: A method for producing lactose-N-neotetraose by fermentation of Escherichia coli BL21 star (DE3) BLBRTXST3, comprising the following steps:

[0056] (1) Escherichia coli BL21 star (DE3) BLBRTXST3 strain was inoculated into seed liquid medium, chloramphenicol was added at a final concentration of 25 μg / mL, and fermented at 37°C and a stirring rate of 200 r / min for 8 h; the seed liquid medium was prepared with the following ingredients: tryptone 10 g / L, yeast powder 5 g / L, and sodium chloride 10 g / L. The first-level seed liquid was inoculated into the seed liquid medium to obtain the second-level seed liquid.

[0057] (2) Transfer the secondary seed liquid to the fermentation tank at a 12% inoculation rate, add chloramphenicol at a final concentration of 30ug / mL, culture at 37°C, control the dissolved oxygen at about 30%, and control the pH at 6.5-7.0. After 5-8 hours, the dissolved oxygen rebounds, and feed is added in an exponential manner. Glycerol is added. The glycerol is 600 g / L and 5 g / L of magnesium sulfate heptahydrate is added. The cumulative amount of glycerol in the whole process does not exceed 1g / L. After OD 600 After reaching 30-40°C, the temperature was lowered to 25°C, IPTG was added at a final concentration of 0.1 mM for induction, and lactose was intermittently fed after 2 hours, the lactose was 200 g / L, and the lactose concentration was maintained at 5-10 g / L;

[0058] The fermentation medium of the fermentation tank is prepared according to the following concentrations of ingredients: 8 g / L glycerol, 2 g / L yeast powder, 8 g / L peptone, 3 g / L ammonium sulfate, 8 g / L dipotassium hydrogen phosphate, 10 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate heptahydrate, 0.2 g / L citric acid, 0.01 g / L calcium chloride, and 8 mL / L trace metal ion liquid elements; the trace metal elements include the following concentrations of ingredients: 0.2 g / L ammonium heptamolybdate, 1 g / L zinc sulfate heptahydrate, 0.5 g / L manganese sulfate tetrahydrate, 8 g / L ferrous sulfate heptahydrate, 1 g / L copper sulfate pentahydrate, and 0.1 g / L sodium tetraborate.

[0059] (3) The fermentation liquid obtained in step (2) is filtered through a ceramic membrane, an ultrafiltration membrane, and a nanofiltration membrane to obtain a supernatant, wherein the ultrafiltration membrane is used to remove bacterial cells, and the nanofiltration membrane is used to remove inorganic salts and small molecular sugars.

[0060] (4) The supernatant obtained in step (3) is decolorized with activated carbon, the amount of activated carbon used is 4% of the dry matter content of the feed liquid, the decolorization temperature is 65°C, the decolorization time is 60 minutes, and the light transmittance of the feed liquid is greater than 85%.

[0061] (5) The decolorized liquid in step (4) is subjected to ion exchange to reduce conductivity. The cation resin and the anion resin are D001 resin and D301-F resin in a volume ratio of 100:210. The ion exchange feed temperature is 40°C, the feed rate is 3Bv / h, the discharge conductivity is <50us / cm, and the discharge pH value is 4-6.

[0062] (6) Decolorizing, decolorizing, and concentrating the feed liquid after the decolorization in step (5), with the feed mass concentration being 50%, the concentration temperature being 67-75°C, and the solid content being concentrated to 20%, and the small molecular weight sugars such as trisaccharides and disaccharides being separated by a single chromatography, the temperature in the column being 60-65°C, and the feed-water ratio being 1:4;

[0063] In the second chromatographic separation, macromolecular sugars such as hexasaccharides were separated, with a feed mass concentration of 50%, a column temperature of 60-65 °C, and a feed-water ratio of 1:8.

[0064] (7) The slurry after chromatographic separation in step (6) is subjected to secondary decolorization, cross-linking, and concentration to a solid content of 55%, and then spray-dried to obtain lactose-N-neotetraose powder, wherein the purity of the finished lactose-N-neotetraose powder is greater than 94%.

[0065] (8) Concentrating the linear lactose-N-neohexose liquid after chromatographic separation in step (6), and then chromatographically separating it to obtain high-purity linear lactose-N-neohexose, concentrating it to a solid content of 50%, and spray drying it to obtain high-purity linear lactose-N-neohexose powder.

[0066] .

[0067] .

[0068] .

[0069] The techniques, shapes, and structural parts not described in detail in the present invention are all well-known techniques.

[0070] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An Escherichia coli BL21star (DE3) BLBRTXST3, characterized in that: The Escherichia coli ( Escherichia coli )BL21star(DE3)BLBRTXST3, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No. 32521, the deposit date is November 6, 2024, and the address of the deposit institution is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. A method for producing high-purity lactose-N-neotetraose and linear lactose-N-neohexose by fermentation of an Escherichia coli BL21star (DE3) BLBRTXST3 according to claim 1, characterized in that: The steps include: (1) Inoculate Escherichia coli BL21 star (DE3) BLBRTXST3 into seed liquid culture medium to obtain primary seed liquid; (2) inoculating the first-level seed solution into the seed solution culture medium to obtain the second-level seed solution; (3) inoculating the secondary seed liquid into a fermentation medium for fermentation to obtain a fermentation liquid; (4) The fermentation liquid is filtered through a ceramic membrane, an ultrafiltration membrane, and a nanofiltration membrane to obtain a supernatant, the supernatant is decolorized by activated carbon to obtain a feed liquid, the feed liquid is subjected to ion exchange to reduce conductivity, and then concentrated, chromatographically separated, decolorized for a second time, deionized for a second time, concentrated, and dried to obtain high-purity lactose-N-neotetraose with a purity greater than 94%; (5) The by-product linear lactose-N-neohexose is separated by chromatography to obtain linear lactose-N-neohexose with a purity greater than 95%, and then decolorized, ion exchanged, concentrated and dried to obtain high-purity linear lactose-N-neohexose with a purity greater than 95%.

3. The method for producing high-purity lactose-N-neotetraose and linear lactose-N-neohexose by fermentation of Escherichia coli BL21star (DE3) BLBRTXST3 as claimed in claim 2, characterized in that: The fermentation medium in step (3) is prepared according to the following concentrations: 8-12 g / L glycerol, 1-5 g / L yeast powder, 4-8 g / L peptone, 2-6 g / L ammonium sulfate, 8-10 g / L dipotassium hydrogen phosphate, 8-10 g / L potassium dihydrogen phosphate, 1-5 g / L magnesium sulfate heptahydrate, 0.1-1 g / L citric acid, 0.01-0.05 g / L calcium chloride; 8-10 mL / L trace metal ion liquid elements; trace metal elements include the following concentrations of components, 0.01-0.2 g / L ammonium heptamolybdate, 1-3 g / L zinc sulfate heptahydrate, 0.1-1 g / L manganese sulfate tetrahydrate, 8-12 g / L ferrous sulfate heptahydrate, 1-5 g / L copper sulfate pentahydrate, and 0.1-0.3 g / L sodium tetraborate.

4. The method for producing high-purity lactose-N-neotetraose and linear lactose-N-neohexose by fermentation of Escherichia coli BL21star (DE3) BLBRTXST3 as claimed in claim 2, characterized in that: In step (3), the fermentation process conditions are as follows: the secondary seed liquid is transferred to the fermentation tank at an inoculation rate of 8-12%, chloramphenicol is added at a final concentration of 20-30 ug / mL, cultured at 37°C, the dissolved oxygen is controlled at about 30%, and the pH is controlled at 6.5-7.0; after 5-8 hours, the dissolved oxygen rebounds, and feeding is performed in an exponential flow manner, and glycerol is fed, the glycerol is 600 g / L, and the cumulative amount of glycerol does not exceed 1 g / L; after OD600 reaches 30-40, the temperature is lowered to 25-27°C, 0.1 mM IPTG is added for induction, and then lactose is continuously fed, the lactose is 200 g / L, and the lactose concentration is maintained at 5-10 g / L.

5. The method for producing high-purity lactose-N-neotetraose and linear lactose-N-neohexose by fermentation of Escherichia coli BL21star (DE3) BLBRTXST3 as claimed in claim 2, characterized in that: In step (4), the fermentation liquid is centrifuged and then filtered through a ceramic membrane and an ultrafiltration membrane to remove the bacteria, and the nanofiltration membrane removes inorganic salts and small molecular sugars.

6. The method for producing high-purity lactose-N-neotetraose and linear lactose-N-neohexose by fermentation of Escherichia coli BL21star (DE3) BLBRTXST3 as claimed in claim 2, characterized in that: In step (4), the ion exchange adopts a mixed bed ion exchange of cation and anion resins, wherein the cation resin and the anion resin are D001 resin and D301-F resin in a volume ratio of 100:

210.

7. The method for producing high-purity lactose-N-neotetraose and linear lactose-N-neohexose by fermentation of Escherichia coli BL21star (DE3) BLBRTXST3 as claimed in claim 6, characterized in that: The ion exchange feed temperature is 30-40°C, the feed speed is 2-3Bv / h, the discharge conductivity is less than 50us / cm, and the discharge pH value is 4-6; the concentration temperature is 60-75°C, and the solid content is concentrated to 20-50%.

8. The method for producing high-purity lactose-N-neotetraose and linear lactose-N-neohexose by fermentation of Escherichia coli BL21star (DE3) BLBRTXST3 as claimed in claim 2, characterized in that: The chromatographic separation conditions in step (4) are as follows: for the first chromatographic separation of small molecular sugars except trisaccharides and disaccharides, the feed mass concentration is 30-50%, the temperature is 60-65°C, and the feed-water ratio is 1:4-8; for the second chromatographic separation of large molecular sugars except hexasaccharides, the feed mass concentration is 20-50%, the column temperature is 60-65°C, and the feed-water ratio is 1:4-8.

9. The method for producing high-purity lactose-N-neotetraose and linear lactose-N-neohexose by fermentation of Escherichia coli BL21star (DE3) BLBRTXST3 as claimed in claim 2, characterized in that: The concentration temperature in step (4) is ≤75°C, the fermentation broth is concentrated to a solid content of 40-60%, and spray-dried to obtain lactose-N-neotetraose powder with a purity greater than 94%.

10. The method for producing high-purity lactose-N-neotetraose and linear lactose-N-neohexose by fermentation of Escherichia coli BL21star (DE3) BLBRTXST3 as claimed in claim 2, characterized in that: The chromatographic separation conditions in step (5) are as follows: for chromatographic separation of small molecular sugars except tetrasaccharides and trisaccharides, the feed mass concentration is 20-50%, the temperature is 60-65°C, and the feed-water ratio is 1:4-8 to obtain high-purity linear lactose-N-neohexose with a purity greater than 95%, and then the linear lactose-N-neohexose powder is obtained by decolorization, desorption, concentration and drying.

11. A method for producing high-purity lactose-N-neotetraose and linear lactose-N-neohexaose by fermentation of an Escherichia coli BL21star (DE3) BLBRTXST3 as claimed in claim 2, characterized in that: The preparation method of the first-level seed liquid in step (1) is as follows: inoculate the strain on the seed liquid culture medium, add chloramphenicol with a final concentration of 20-30 μg / mL, and ferment and culture for 8-10 hours at a temperature of 37°C and a stirring rate of 150-250 r / min; the seed liquid culture medium is prepared according to the following concentrations: tryptone 8-16 g / L, yeast powder 4-8 g / L, and sodium chloride 8-16 g / L.

12. A use of the Escherichia coli BL21star (DE3) BLBRTXST3 according to claim 1, characterized in that: The Escherichia coli BL21star (DE3) BLBRTXST3 is used in fermentation to produce high-purity lactose-N-neotetraose.