Escherichia coli BL21(DE3)-BLBtysrt-06 and a method for fermenting high-purity 6'-sialyllactose and application thereof

Abstract

This invention relates to the field of human milk oligosaccharide production technology, specifically to an *Escherichia coli* BL21(DE3)-BLBtysrt-06 strain and its method for fermenting to produce high-purity 6'-sialylactose, as well as its application. *Escherichia coli* BL21(DE3)-BLBtysrt-06 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36810, deposited on November 28, 2025, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. This invention utilizes *Escherichia coli* BL21(DE3)-BLBtysrt-06 in the fermentation production of high-purity 6'-sialylactose (6'-SL). This invention improves the yield of 6'-sialic acid lactose and reduces the proportion of non-target products by using a multi-stage, multi-scale controlled fermentation technology. The fermentation broth is purified by integrated membrane separation and valve array continuous cross-linking technology, which improves the material yield. A high-purity 6'-sialic acid lactose product with a purity of 95.2% is prepared by controllable simulated moving bed chromatography, and the chromatographic separation solution is reused in the fermentation process, resulting in high raw material utilization.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of human milk oligosaccharide production, and in particular to an Escherichia coli BL21(DE3)-BLBtysrt-06, a method for fermentatively producing high-purity 6'-sialyllactose, and application thereof. BACKGROUND

[0002] Sialyllactose is a representative type of acidic oligosaccharide among human milk oligosaccharides (HMOs), and 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL) are the main existing forms. In the colostrum stage of breast milk, the content of 6'-SL is particularly rich, and it plays an important physiological role in promoting the neural development of infants, regulating the balance of intestinal microecology, enhancing the immune function of the body, and inhibiting the adhesion of pathogens. Therefore, 6'-SL has broad application prospects in the fields of infant formula, nutritional health products, and biological medicine, and has obtained the food application permission of the US Food and Drug Administration (FDA) and relevant agencies in the European Union.

[0003] The molecular structure of 6'-sialyllactose is that one sialic acid molecule (usually N-acetylneuraminic acid, Neu5Ac) is connected to the galactosyl group at the end of lactose (Gal-β1,4-Glc) through an α-2,6-glycosidic bond. At present, the preparation methods of this compound mainly include natural extraction method, chemical synthesis method, enzyme catalysis method, and microbial fermentation method. The natural extraction method can retain the natural conformation of 6'-SL and has high safety, but due to the limitation of human milk sources, it is difficult to realize large-scale production. Although the chemical synthesis method has the theoretical possibility of large-scale production, it faces problems such as difficulty in controlling the region and stereoselectivity, complex side reactions, use of toxic reagents, and large environmental burden in the reaction process, which restricts its application in food and medicine. The enzyme catalysis method depends on sialyltransferase and the relatively high-cost sugar nucleotide donor to convert lactose as the substrate, although the reaction selectivity is good, but the high cost of enzymes and substrates limits its industrial promotion.

[0004] In contrast, the microbial fermentation method uses metabolically engineered microorganisms as cell factories to synthesize 6'-SL in vivo using cheap carbon sources. This method has the advantages of wide raw material sources, environmentally friendly process flow, and easy industrial scale-up, and is therefore considered as the most potential technical path for realizing large-scale and commercial production of 6'-sialyllactose.

[0005] 6'-Sialyllactose is an important human milk oligosaccharide with significant prebiotic and antipathogenic adsorption functions. Currently, its production faces challenges. While microbial fermentation shows promise, it is often limited by low catalytic efficiency of host strains, numerous byproducts, and difficulties in downstream purification. Therefore, developing high-performance production strains and supporting efficient, low-cost integrated fermentation and purification processes is crucial for achieving large-scale production of 6'-sialic acid lactose. Summary of the Invention

[0006] This invention provides an Escherichia coli BL21(DE3)-BLBtysrt-06, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36810, deposited on November 28, 2025. The address of the depository is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0007] Furthermore, an ultraviolet (UV) mutagenesis method was employed, which is as follows: *E. coli* BL21(DE3)-tysrt was streaked onto a plate and incubated statically at 36-37°C. After single colonies grew, they were picked and inoculated into liquid culture medium, and cultured until OD (October Expiratory Time) was reached. 600 The concentration was 0.7-0.9. The bacterial culture to be mutated was placed on a magnetic stirrer in a clean bench. The UV lamp was turned on and irradiated at a vertical distance of 25-29 cm for 0, 10, 30, 50, 70, 90, and 100 seconds, followed by a gradient treatment of 10. -3 10 -4 10 -5 The culture medium was spread onto LB agar plates and incubated upside down at 37±0.5℃. Colony growth was observed, plate counts were performed, and the lethality at the appropriate dilution factor was calculated. The lethality calculation formula is as follows:

[0008] Mutagenesis was performed under mutagenesis conditions with a lethality rate of 72%-80%. The mutagenized bacterial solution was diluted appropriately and spread on solid plates for 8-15 hours. Single colonies with good growth were selected and inoculated into liquid culture medium and cultured at 37±0.5℃ for 6-12 hours. The culture was then transferred to shake flask fermentation medium and repeated several times until the superior strain BL21(DE3)-BLBtysrt-06 with the highest 6'-sialic acid lactose production was selected.

[0009] Furthermore, a method for producing high-purity 6'-sialic acid lactose by fermentation of Escherichia coli BL21(DE3)-BLBtysrt-06 includes the following steps: (1) Streak Escherichia coli BL21(DE3)-BLBtysrt-06 in three zones on solid medium and incubate upside down at 37±0.5℃ for 8-15 h to obtain single colonies; (2) Pick a single colony from step (1) and place it in the primary seed culture medium. Incubate at 37±0.5℃ for 6-10 h to obtain the primary seed culture. (3) Inoculate the primary seed culture from step (2) into the secondary seed culture medium and incubate at 37±0.5℃ for 5-7 h to obtain the secondary seed culture; (4) The secondary seed liquid obtained in step (3) is inoculated into a sterilized fermentation medium and fermented to obtain a fermentation broth containing 6'-sialic acid lactose. (5) The fermentation broth is first purified by an integrated membrane separation system and a continuous ion exchange system, and then a chromatographic extract is prepared by a controllable simulated moving bed chromatography separation technology. The chromatographic extract is concentrated and then vacuum spray dried to obtain 6'-sialic acid lactose solid product. The lactose separated by chromatography is reused in the fermentation.

[0010] Furthermore, in the method for fermenting to produce high-purity 6'-sialic acid lactose, in step (2), the seed culture medium is prepared with the following concentrations of components: yeast extract 3-7 g / L, peptone 7-14 g / L, and sodium chloride 7-12 g / L; Furthermore, in the method for fermenting to produce high-purity 6'-sialic acid lactose, in step (4), the fermentation medium is prepared with the following concentrations of components: glycerol 5-13 g / L, yeast extract 3-4 g / L, peptone 2-5 g / L, potassium dihydrogen phosphate 7-16 g / L, diammonium hydrogen phosphate 8-10 g / L, magnesium sulfate heptahydrate 0.5-3 g / L, citric acid monohydrate 0.5-4 g / L, and trace metal ion liquid element 8-16 mL / L; the trace metal element includes the following concentrations of components: ammonium molybdate tetrahydrate 0.05-0.3 g / L, zinc sulfate heptahydrate 1-3 g / L, manganese sulfate tetrahydrate 0.1-1 g / L, ferrous sulfate heptahydrate 8-12 g / L, copper sulfate pentahydrate 1-5 g / L, sodium tetraborate decahydrate 0.1-0.5 g / L, cobalt chloride hexahydrate 0.1-0.5 g / L, and anhydrous calcium chloride 1-5 g / L. g / L.

[0011] Furthermore, in the method for producing high-purity 6'-sialic acid lactose by fermentation, in step (4), the fermentation culture process conditions are as follows: the cultured secondary seed liquid is transferred to the sterilized fermentation culture medium at an inoculation rate of 3-15%, fermented at 37±0.5℃, and the pH is controlled at 6.5-7.0; after 12-18 hours, dissolved oxygen rebounds, and glycerol (600 g / L) is added in an exponential feeding manner, with the total glycerol accumulation not exceeding 1 g / L throughout the process, and dissolved oxygen controlled at around 30%; after OD 600 The temperature should be set to 30-50°C, then cooled to 25°C. The pH should be controlled at 7.0-7.3, and the lactose concentration should be maintained at 5-10 g / L.

[0012] Furthermore, in the method for producing high-purity 6'-sialic acid lactose by fermentation, in step (5), the membrane separation system includes a ceramic membrane, an ultrafiltration membrane, and a nanofiltration membrane. The pore size of the ceramic membrane is 0.1-1 μm, the pore size of the ultrafiltration membrane is 5K-10KDa, and the pore size of the nanofiltration membrane is 200-300Da. The washing water of the ceramic membrane (solids ≤0.5%) is used for sugar removal from the ultrafiltration membrane, while the washing water of the ultrafiltration membrane (solids ≤0.5%, discharged) is used as water in the nanofiltration membrane. The sugar in the washing water of the ceramic membrane and the ultrafiltration membrane can be recovered.

[0013] Furthermore, in the method for fermenting to produce high-purity 6'-sialic acid lactose, the continuous ion exchange in step (5) is a valve array ion exchange system, the resin uses a cation exchange resin D001-FD and anion exchange resin D354-FD with a volume ratio of 100:210, the ion exchange feed temperature is ≤40℃, the feed rate is 2~3 Bv / h, the conductivity of the effluent is <40us / cm, and the pH value of the effluent is 3.5~6.

[0014] Furthermore, in the method for producing high-purity 6'-sialic acid lactose by fermentation, in step (5), the simulated moving bed chromatographic separation conditions are as follows: the feed solid content is 25-40%, the column temperature is 55-60℃, and the water-to-solid ratio is 4-7:1; the purity of 6'-sialic acid lactose in the chromatographic extract is increased to over 90%, and the chromatographic separation liquid has a high lactose content and is reused in the fermentation process.

[0015] Furthermore, in the method for producing high-purity 6'-sialic acid lactose by fermentation, in step (5), the concentration temperature is ≤70℃, the chromatographic extract is concentrated to a solid content of 40-55%, and then spray-dried to obtain the finished product 6'-sialic acid lactose.

[0016] Furthermore, an application of Escherichia coli BL21(DE3)-BLBtysrt-06, specifically its application in the fermentation production of high-purity 6'-sialic acid lactose.

[0017] The beneficial effects of this invention are as follows: (1) The present invention obtained the excellent strain of Escherichia coli BL21(DE3)-BLBtysrt-06 by mutagenesis, and used the strain to produce 6'-sialic acid lactose by efficient fermentation; (2) The present invention adopts staged fermentation with precise control, which improves the production of the target product 6'-sialic acid lactose, reduces the dry matter ratio of by-products such as N-acetylglucosamine and sialic acid, and reduces the difficulty of subsequent extraction. (3) This invention uses integrated membrane separation and continuous ion exchange coupling technology for purification. The integrated membrane separation system removes bacteria, monosaccharides and small molecule salts. At the same time, there is a phenomenon in the industry where the solid content of the membrane separation and ion exchange process is <0.5% and the washing water is discharged. The integrated membrane separation and valve array continuous ion exchange system coupling solves the problem of washing water carrying material, reduces water use, has a high material yield, and the process is green. (4) The present invention prepares a high-purity 6'-sialic acid lactose product by chromatographic separation, with a purity of over 95%; the lactose content in the chromatographic separation liquid is high and can be reused in the fermentation process, resulting in low raw material consumption. (5) The present invention obtained the excellent strain of Escherichia coli BL21(DE3)-BLBtysrt-06 through mutagenesis, which can improve the yield of 6'-sialic acid lactose, reduce the proportion of non-target product dry matter, and has a simple separation and purification process, which is more conducive to high-purity industrial production. Detailed Implementation

[0018] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions in the embodiments of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0019] This invention provides a method for producing high-purity 6'-sialic acid lactose from Escherichia coli BL21(DE3)-BLBtysrt-06 and its fermentation, as well as its applications.

[0020] The Escherichia coli BL21(DE3)-BLBtysrt-06 used in this invention was obtained by treating the Escherichia coli BL21(DE3)-tysrt strain preserved by the applicant with ultraviolet mutagenesis and screening for excellent high-yield strains by measuring the fermentation yield of 6'-sialic acid lactose.

[0021] Example 1: Construction and screening of high-yielding strain Escherichia coli BL21(DE3)-BLBtysrt-06 The Escherichia coli BL21(DE3)-BLBtysrt-06 strain used in this invention was obtained by treating the Escherichia coli BL21(DE3)-tysrt strain preserved by the applicant with ultraviolet mutagenesis and screening for excellent high-yield strains by measuring the fermentation yield of 6'-sialic acid lactose.

[0022] This embodiment describes in detail the process of selecting a high-yielding strain BL21(DE3)-BLBtysrt-06 from the original host Escherichia coli BL21(DE3)-tysrt through ultraviolet mutagenesis and high-throughput screening.

[0023] 1. Starting strain and culture medium:

[0024] Starting strain: The high-yielding strain of Escherichia coli BL21(DE3)-tysrt preserved by the applicant was obtained by ultraviolet mutagenesis treatment and screening through fermentation yield determination of 6'-sialic acid lactose.

[0025] LB solid medium: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar powder 15 g / L.

[0026] LB liquid medium: Same as above, but without agar powder.

[0027] Fermentation screening medium: The composition is the same as the basic composition of the fermentation medium in Application Example 1 below (glycerol 15 g / L, yeast extract 2 g / L, peptone 4 g / L, potassium dihydrogen phosphate 8 g / L, diammonium hydrogen phosphate 8 g / L, magnesium sulfate heptahydrate 1 g / L, citric acid monohydrate 2 g / L, trace element solution 10 mL / L; the trace element solution includes the following components at the following concentrations: ammonium molybdate tetrahydrate 0.1 g / L, zinc sulfate heptahydrate 2 g / L, manganese sulfate tetrahydrate 1 g / L, ferrous sulfate heptahydrate 8 g / L, copper sulfate pentahydrate 2 g / L, sodium tetraborate decahydrate 0.2 g / L, cobalt chloride hexahydrate 0.2 g / L, anhydrous calcium chloride 2 g / L).

[0028] 2. Ultraviolet mutagenesis: The starting strain was streaked onto LB agar plates and incubated overnight at 37°C. A single colony was picked and inoculated into 5 mL of LB liquid medium, and cultured at 37°C with shaking at 220 rpm until the OD600 was approximately 0.8. 5 mL of the bacterial culture was transferred to a sterile Petri dish and placed on a magnetic stirrer. In a laminar flow hood, a 30 W UV lamp (main wavelength 253.7 nm) was turned on and preheated for 20 minutes. The Petri dish was placed directly below the lamp, with the vertical distance adjusted to 27 cm. Stirring was turned on, and the Petri dish was irradiated for 0, 10, 30, 50, 70, 90, and 100 seconds. The irradiated bacterial culture was immediately diluted appropriately (10-10). -3 10 -410 -5 100 μL of each sample was spread onto LB agar plates and incubated upside down at 37°C in the dark for 24-48 hours. Colony counts were performed, and the lethality rate was calculated using the formula: Lethality (%) = [(Number of colonies on unirradiated plate - Number of colonies on irradiated plate) / Number of colonies on unirradiated plate] × 100%. Experimental results showed that the lethality rate was approximately 75% after 70 seconds of irradiation, falling within the effective mutagenesis range of 72%-80%. Therefore, this condition was chosen for large-scale mutagenesis.

[0029] 3. High-throughput screening: The bacterial culture, after 70 seconds of UV mutagenesis treatment, was diluted and spread to obtain approximately 200 single colonies. Each single colony was picked up using a sterile toothpick and placed into a 96-well plate containing 200 μL of fermentation screening medium. The plates were incubated at 37°C and 220 rpm for 48 hours with shaking. After incubation, the fermentation supernatant was collected, and the 6'-sialic acid lactose content was rapidly determined using a pre-established micro-high performance liquid chromatography (HPLC) method. HPLC conditions: APS-2 HYPERSIL column (4.6 × 250 mm), mobile phase: acetonitrile:water (containing 2 mM MgCl2 and 10 mM H3PO4, adjusted to pH 4.5 with ammonia) = 50:50 (v / v), flow rate 0.6 mL / min, column temperature 35°C, and differential refractive index detector. The fermentation yield of 6'-sialic acid lactose for each clone was calculated.

[0030] 4. Secondary screening and strain identification: Clones with the top 20 transformation rates, exceeding the starting strain by more than 15%, were selected from the initial screening and transferred to 250 mL shake flasks containing 20 mL of fermentation screening medium. Three rounds of repeated fermentation (24 hours each) were performed at 37℃ and 220 rpm for verification. The product yield was precisely determined by HPLC after each round. Finally, a genetically stable strain with high product fermentation yield, significantly improved compared to the starting strain, was obtained and named *Escherichia coli* BL21(DE3)-BLBtysrt-06. This strain was deposited on November 28, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36810. The address of the depository is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0031] Application Example 1

[0032] A method for producing 6'-sialic acid lactose by fermentation of Escherichia coli BL21(DE3)-BLBtysrt-06 specifically includes the following steps:

[0033] (1) Escherichia coli BL21(DE3)-BLBtysrt-06 strain was inoculated onto primary seed culture medium and cultured at 37±0.5℃ and 220 r / min for 9 h; OD 600 When the OD value was 2.6, it was transferred to secondary seed culture medium at an inoculum rate of 1%, and cultured at 37±0.5℃ and 220 r / min for 7.5 h. 600 The culture medium was prepared at concentration 3.1 to obtain a secondary seed culture. The seed culture medium was prepared with the following components: yeast extract 4.5 g / L, peptone 9 g / L, and NaCl 9 g / L.

[0034] (2) Inoculate the secondary seed culture into the tertiary sterilized fermentation medium at an inoculation rate of 8%, and incubate at 37±0.5℃, with dissolved oxygen controlled at around 30% and pH controlled at 6.5-6.8. After 15 hours, when dissolved oxygen rebounds, feed the culture using an exponential feeding method, adding glycerol (600 g / L), ensuring that the cumulative amount of glycerol does not exceed 1 g / L throughout the process. When OD600 reaches 50, cool to 25℃, and control the pH at 7.0-7.2 to maintain the lactose concentration at 5-8 g / L. The fermentation medium is prepared with the following components at the following concentrations: glycerol 15 g / L, yeast extract 2 g / L, peptone 4 g / L, potassium dihydrogen phosphate 8 g / L, diammonium hydrogen phosphate 8 g / L, magnesium sulfate heptahydrate 1 g / L, citric acid monohydrate 2 g / L, and trace element solution 10 mL / L. The trace element solution includes the following components at the following concentrations: ammonium molybdate tetrahydrate 0.1 g / L, zinc sulfate heptahydrate 2 g / L, manganese sulfate tetrahydrate 1 g / L, ferrous sulfate heptahydrate 8 g / L, copper sulfate pentahydrate 2 g / L, sodium tetraborate decahydrate 0.2 g / L, cobalt chloride hexahydrate 0.2 g / L, and anhydrous calcium chloride 2 g / L.

[0035] (3) The fermentation broth obtained in step (2) is initially purified using an integrated membrane separation system. First, a ceramic membrane with a pore size of 0.1 μm is used to remove microbial cells and obtain supernatant. Then, a 5 kDa ultrafiltration membrane is used to remove macromolecular proteins. Finally, a nanofiltration membrane with a pore size of 200 Da is used to remove monosaccharides from the feed solution and reduce conductivity to obtain nanofiltration concentrate. In the integrated membrane separation process, the wash water of the ceramic membrane (solids ≤ 0.5%) is used for supernatant ...

[0036] (4) After decolorizing the nanofiltration concentrate prepared in step (3), it is fed into a valve array continuous ion exchange system. The volume ratio of cation resin D001-FD and anion resin D354-FD is 100:210. The feed temperature is 30℃, the feed rate is 2BV / h, the conductivity of the effluent is <50us / cm, and the pH of the mixed effluent is 4.2.

[0037] (5) The liquid after the separation in step (4) is concentrated to a solid content of 40%, and then simulated moving bed chromatography is performed. The simulated moving bed chromatography conditions are: the temperature inside the chromatographic column is 57~60℃, and the water-to-solid ratio is 5:1. After simulated moving bed chromatography, chromatographic extract and chromatographic separation are obtained. The purity of 6'-sialic acid lactose in the chromatographic extract is 90.5%. The chromatographic separation with high lactose content is reused in the fermentation process.

[0038] (6) Concentrate the chromatographic extract after chromatographic separation in step (5) to a solid content of 45%, and spray dry it to obtain the finished product 6'-sialic acid lactose.

[0039] Application Example 2 (1) Escherichia coli BL21(DE3)-BLBtysrt-06 strain was inoculated into primary seed culture medium and cultured at 37±0.5℃ and 220 r / min for 9.5 h. When the OD600 was 2.9, it was transferred to secondary seed culture medium at an inoculation rate of 1% and cultured at 37±0.5℃ and 220 r / min for 7.5 h. When the OD600 was 3.0, the secondary seed culture was obtained. The seed culture medium was prepared with the following concentrations of components: yeast extract 4.5 g / L, peptone 9 g / L, and NaCl 9 g / L.

[0040] (2) Inoculate the secondary seed culture into the tertiary fermentation medium at an inoculation rate of 10%, and incubate at 37±0.5℃, maintaining dissolved oxygen at approximately 30% and pH at 6.5–6.8. After 15 hours, once dissolved oxygen rebounds, feed the medium using an exponential feeding method, adding glycerol (600 g / L). The total cumulative glycerol content should not exceed 0.8 g / L throughout the process. Wait for OD... 600The temperature was raised to 40°C, then lowered to 25°C, with the pH controlled between 7.0 and 7.2 to maintain a lactose concentration of 8-10 g / L. The fermentation medium was prepared with the following components: glycerol 12 g / L, yeast extract 2 g / L, peptone 4 g / L, potassium dihydrogen phosphate 12 g / L, diammonium hydrogen phosphate 10 g / L, magnesium sulfate heptahydrate 2 g / L, citric acid monohydrate 3 g / L, and trace element solution 12 mL / L. The trace element solution contained the following components: ammonium molybdate tetrahydrate 0.1 g / L, zinc sulfate heptahydrate 2.5 g / L, manganese sulfate tetrahydrate 1 g / L, ferrous sulfate heptahydrate 8 g / L, copper sulfate pentahydrate 3 g / L, sodium tetraborate decahydrate 0.15 g / L, cobalt chloride hexahydrate 0.1 g / L, and anhydrous calcium chloride 1.5 g / L.

[0041] (3) The fermentation broth obtained in step (2) is initially purified by an integrated membrane separation system. First, a ceramic membrane with a pore size of 1.0 μm is used to filter out microbial cells to obtain supernatant. Then, the ultrafiltration membrane with a pore size of 10 kDa is used to filter out macromolecular proteins. Finally, the nanofiltration membrane with a pore size of 200 Da is used to remove monosaccharides in the broth and reduce conductivity to obtain nanofiltration concentrate.

[0042] (4) After decolorizing the nanofiltration concentrate prepared in step (3), it is fed into a valve array continuous ion exchange system. The volume ratio of cation resin D001-FD and anion resin D354-FD is 100:210. The feed temperature is 38℃, the feed rate is 3BV / h, the conductivity of the effluent is <40us / cm, and the pH of the mixed effluent is 4.1.

[0043] (5) Concentrate the liquid after separation in step (4) to a solid content of 30%, and then perform simulated moving bed chromatography separation. The simulated moving bed chromatography separation conditions are: the temperature inside the chromatographic separation column is 55-57℃, and the water-to-solid ratio is 6:1. After simulated moving bed chromatography separation, chromatographic extract and chromatographic separation liquid are obtained. The purity of 6'-sialic acid lactose in the chromatographic extract is 95.2%. The chromatographic separation liquid with high lactose content is reused in the fermentation process for further utilization.

[0044] (6) Concentrate the chromatographic extract obtained after chromatographic separation in step (5) to a solid content of 50%, and spray dry it to obtain the finished product 6'-sialic acid lactose.

[0045] Comparative Example 1 Unlike application examples 1-2, in step (2), the fermentation process conditions are as follows: glycerol is added intermittently to raise the concentration of glycerol to 15 g / L, then consumed to about 1 g / L, and then intermittently added again. The feeding is not carried out in an exponential feeding manner. Fermentation is carried out at 150~300 r / min for 70 h to obtain fermentation broth. The fermentation broth is then separated by an integrated membrane separation system coupled with valve array continuous cross-linking technology, and then separated by simulated moving bed chromatography to obtain 6'-sialic acid lactose with a purity of 89%. After spray drying, the finished product 6'-sialic acid lactose is obtained.

[0046] Comparative Example 2 Unlike Application Examples 1-2, in step (1), the original starting strain BL21(DE3)-tysrt was used, and the fermentation process employed intermittent feeding (adding glycerol to 15 g / L at a time when it was consumed to <1 g / L), without exponential feeding and precise control of lactose concentration. The purification process used conventional centrifugation, plate and frame filtration, batch cross-linking, and preparative chromatography.

[0047] Take 1 mL of each of the 6'-sialic acid lactose fermentation broths prepared in Application Examples 1-2 and Comparative Examples 1-2, centrifuge at 12000 rpm for 10 min, collect the supernatant, and use it for HPLC determination. Record the area ratio of each component in the fermentation broth, and calculate the fermentation yield of 6'-sialic acid lactose based on the determination results. The results are shown in Table 1 below.

[0048] HPLC detection conditions for glycerol, lactose, and acetic acid during fermentation: High-performance liquid chromatography (HPLC) system (Waters e2695); Column: HPX-87H column; Detector: Differential detector; Mobile phase: 5 mmol / L H₂SO₄ aqueous solution; Flow rate: 0.5 mL / min; Column temperature: 60℃; Injection volume: 20 μL. The results are shown in Table 1 below.

[0049] Detection: Differential refractive index detector, APS-2 HYPERSIL column, mobile phase 50% acetonitrile + 2mM MgCl2 + 10mM H3PO4, flow rate 0.6mL / min, column temperature 35℃, injection volume 20uL.

[0050]

[0051] Table 1 shows the area ratio of 6'-sialyl lactose and other sugar dry matter and the fermentation yield of 6'-sialyl lactose in the fermentation broths of Application Examples 1-2 and Comparative Examples 1-2.

[0052] Table 1

[0053] The high-yield strain of the present invention can significantly increase the yield of 6'-sialic acid lactose. When this strain is combined with the integrated process of the present invention, Application Examples 1 and 2 show significant synergistic enhancements in yield, product purity, and production efficiency, comprehensively surpassing the effects of single strain improvement or single process improvement.

[0054] In terms of industrial applicability, the high-yield bacterial strain and integrated production method provided by this invention have clearly defined process steps and controllable parameters. The method has been successfully validated on a 200L fermenter scale and has the potential to be directly scaled up to industrial production scale (such as ton-level fermenters). The entire process achieves material recycling (such as lactose and wash water), reduces waste emissions and production costs, and the purity of the obtained product meets the requirements for high-value-added applications. Therefore, this invention has excellent prospects for industrial application.

[0055] Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made to the embodiments of the present invention by those skilled in the art without departing from the spirit and essence of the invention, and such modifications or substitutions should all be within the scope of the present invention. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should also be covered within the protection scope of the present invention.

Claims

1. An Escherichia coli BL21(DE3)-BLBtysrt-06, characterized in that, The E. coli ( Escherichia coli BL21(DE3)-BLBtysrt-06 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36810, on November 28, 2025. The address of the depository is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. The *Escherichia coli* BL21(DE3)-BLBtysrt-06 as described in claim 1, characterized in that, The ultraviolet mutagenesis method is as follows: *Escherichia coli* BL21(DE3)-tysrt is streaked onto a plate and incubated statically at 36-37°C. After single colonies grow, they are picked and inoculated into liquid culture medium, and cultured until OD (October Expiratory Count). 600 The concentration was 0.7-0.

9. The bacterial culture to be mutated was placed on a magnetic stirrer in a clean bench. The UV lamp was turned on and irradiated at a vertical distance of 25-29 cm for 0, 10, 30, 50, 70, 90, and 100 seconds, followed by a gradient treatment of 10. -3 10 -4 10 -5 The culture medium was spread onto LB agar plates and incubated upside down at 37±0.5℃. Colony growth was observed, plate counts were performed, and the lethality at the appropriate dilution factor was calculated. The lethality calculation formula is as follows: ； Mutagenesis was performed under mutagenesis conditions with a lethality rate of 72%-80%. The mutagenized bacterial solution was diluted appropriately and spread on solid plates for 8-15 hours. Single colonies with good growth were selected and inoculated into liquid culture medium and cultured at 37±0.5℃ for 6-12 hours. The culture was then transferred to shake flask fermentation medium and repeated several times until the superior strain BL21(DE3)-BLBtysrt-06 with the highest 6'-sialic acid lactose production was selected.

3. The method for producing high-purity 6'-sialic acid lactose by fermentation of *Escherichia coli* BL21(DE3)-BLBtysrt-06 as described in claim 1, characterized in that... Includes the following steps: (1) Streak Escherichia coli BL21(DE3)-BLBtysrt-06 in three zones on solid medium and incubate upside down at 37±0.5℃ for 8-15 h to obtain single colonies; (2) Pick a single colony from step (1) and place it in the primary seed culture medium. Incubate at 37±0.5℃ for 6-10 h to obtain the primary seed culture. (3) Inoculate the primary seed culture from step (2) into the secondary seed culture medium and incubate at 37±0.5℃ for 5-7 h to obtain the secondary seed culture; (4) The secondary seed liquid obtained in step (3) is inoculated into a sterilized fermentation medium and fermented to obtain a fermentation broth containing 6'-sialic acid lactose. (5) The fermentation broth is first purified by an integrated membrane separation system and a continuous ion exchange system, and then a chromatographic extract is prepared by a controllable simulated moving bed chromatography separation technology. The chromatographic extract is concentrated and then vacuum spray dried to obtain 6'-sialic acid lactose solid product. The lactose separated by chromatography is reused in the fermentation.

4. The method for producing high-purity 6'-sialic acid lactose by fermentation as described in claim 3, characterized in that, In step (2), the seed culture medium is prepared with the following concentrations of components: yeast extract 3-7 g / L, peptone 7-14 g / L, and sodium chloride 7-12 g / L.

5. The method for producing high-purity 6'-sialic acid lactose by fermentation as described in claim 3, characterized in that, In step (4), the fermentation medium is prepared with the following concentrations of components: glycerol 5-13 g / L, yeast extract 3-4 g / L, peptone 2-5 g / L, potassium dihydrogen phosphate 7-16 g / L, diammonium hydrogen phosphate 8-10 g / L, magnesium sulfate heptahydrate 0.5-3 g / L, citric acid monohydrate 0.5-4 g / L, and trace metal ion liquid elements 8-16 mL / L; the trace metal elements include the following concentrations of components: ammonium molybdate tetrahydrate 0.05-0.3 g / L, zinc sulfate heptahydrate 1-3 g / L, manganese sulfate tetrahydrate 0.1-1 g / L, ferrous sulfate heptahydrate 8-12 g / L, copper sulfate pentahydrate 1-5 g / L, sodium tetraborate decahydrate 0.1-0.5 g / L, cobalt chloride hexahydrate 0.1-0.5 g / L, and anhydrous calcium chloride 1-5 g / L.

6. The method for producing high-purity 6'-sialic acid lactose by fermentation as described in claim 3, characterized in that, In step (4), the fermentation culture process conditions are as follows: the cultured secondary seed liquid is transferred to the sterilized fermentation medium at an inoculation rate of 3-15%, fermented at 37±0.5℃, and the pH is controlled at 6.5-7.0; after 12-18 hours, dissolved oxygen rebounds, and glycerol (600 g / L) is added in an exponential feeding manner, with the total glycerol accumulation not exceeding 1 g / L throughout the process, and dissolved oxygen controlled at around 30%; after OD... 600 The temperature should be set to 30-50°C, then cooled to 25°C. The pH should be controlled at 7.0-7.3, and the lactose concentration should be maintained at 5-10 g / L.

7. The method for producing high-purity 6'-sialic acid lactose by fermentation as described in claim 3, characterized in that, In step (5), the membrane separation system includes a ceramic membrane, an ultrafiltration membrane, and a nanofiltration membrane. The pore size of the ceramic membrane is 0.1-1 μm, the pore size of the ultrafiltration membrane is 5K-10KDa, and the pore size of the nanofiltration membrane is 200-300Da. The washing water of the ceramic membrane (solids ≤0.5%) is used for sugar removal from the ultrafiltration membrane, while the washing water of the ultrafiltration membrane (solids ≤0.5%, discharged) is used for water removal from the nanofiltration membrane. Sugar can be recovered from the washing water of the ceramic membrane and the ultrafiltration membrane.

8. The method for producing high-purity 6'-sialic acid lactose by fermentation as described in claim 3, characterized in that, The continuous ion exchange in step (5) is a valve array ion exchange system. The resin used is a cation exchange resin D001-FD and anion exchange resin D354-FD with a volume ratio of 100:

210. The ion exchange feed temperature is ≤40℃, the feed rate is 2~3 Bv / h, the conductivity of the effluent is <40us / cm, and the pH value of the effluent is 3.5~6.

9. The method for producing high-purity 6'-sialic acid lactose by fermentation as described in claim 3, characterized in that, In step (5), the simulated moving bed chromatography separation conditions are as follows: the feed solid content is 25-40%, the column temperature is 55-60℃, and the water-to-solid ratio is 4-7:1; the purity of 6'-sialic acid lactose in the chromatographic extract is increased to over 90%, and the chromatographic separation solution has a high lactose content and is reused in the fermentation process.

10. The method for producing high-purity 6'-sialic acid lactose by fermentation as described in claim 3, characterized in that, In step (5), the concentration temperature is ≤70℃, the chromatographic extract is concentrated to a solid content of 40-55%, and then spray-dried to obtain the finished product 6'-sialic acid lactose.

11. The application of Escherichia coli BL21(DE3)-BLBtysrt-06 as described in claim 1, characterized in that: The application of Escherichia coli BL21(DE3)-BLBtysrt-06 in the fermentation production of high-purity 6'-sialic acid lactose.

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