A medium composition for fermentation of a strain expressing 2'-fl, and application and large-scale preparation method thereof

Abstract

The application discloses a medium composition for fermentation of a strain expressing 2'-FL and application and a large-scale preparation method, and belongs to the technical field of microbial culture medium. The technical problem to be solved is that the existing 2'-FL fermentation medium formula is not accurate, and the process regulation is extensive, resulting in low strain fermentation efficiency, insufficient product yield and purity, and difficulty in meeting the demand of large-scale production. The technical solution points are as follows: the medium composition includes seed, initial and feeding medium, the initial medium contains specific inorganic salt, nutrient component and glycerol and malt dextrin compound carbon source; the large-scale preparation takes E. coli BLCY-019 as the strain, and the OD 600 The node segmentation regulation parameter and the staged addition of IPTG are used, and the product is obtained by purifying and crystallizing the fermentation liquor. The application solves the existing problems, improves the product yield and purity, adapts to large-scale production, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbial culture medium technology, specifically relating to a culture medium composition for fermentation of 2'-FL engineered strains, its application, and a method for large-scale preparation of 2'-FL. Background Technology

[0002] 2'-Fucose-based lactose (2'-FL) is the most abundant core active component in human breast milk oligosaccharides. It possesses important physiological functions such as regulating intestinal microecology, enhancing immunity, inhibiting pathogenic microorganism adhesion, and promoting infant nervous system development. It is a key nutritional fortifier in infant formula and functional foods, and the market demand for its high-yield, high-purity, large-scale production is increasingly urgent. Microbial fermentation has become the mainstream technology for 2'-FL preparation due to its readily available raw materials, controllable production process, environmental friendliness, and ease of industrial scale-up. Among these methods, using genetically engineered E. coli strains as the fermentation substrate is currently the most promising technology for industrialization. The core of this technology lies in providing a foundation for strain growth and product synthesis through a suitable fermentation culture medium composition, achieving efficient 2'-FL synthesis through scientific fermentation process control, and obtaining high-quality 2'-FL crystalline products through optimized purification and crystallization processes. The synergy of these three factors determines the efficiency and quality of 2'-FL industrial production.

[0003] Relevant patent literature retrieved: Publication country: China, Publication number: CN113186142A, Publication date: 2021.07.30. This literature discloses an engineered strain of *E. coli* that efficiently produces 2'-FL. By knocking out genes related to substrate degradation and intermediate product diversion, heterologously expressing key enzymes and fucosyltransferases in the GDP-L-fucose salvage pathway, and assembling and strengthening the cofactor regeneration pathway using short peptide-mediated enzyme complexes, a 2'-FL yield of 30.5 g / L was achieved in a 3L fermenter using glycerol as the carbon source and lactose and L-fucose as substrates. The fermentation medium used pure glycerol as the carbon source, combined with conventional inorganic salts and trace amounts of metal liquid. During the feeding stage, a high-concentration glycerol solution was used for constant feeding. The carbon source system, precursor supply, and fermentation parameters were not dynamically optimized. The document, published in China (CN115073539B) on June 16, 2023, discloses a method for the separation and purification of 2'-FL. It involves removing monosaccharides, disaccharides, and tetrasaccharide byproducts through nanofiltration, ion exchange resin decolorization and desalting, and two-dimensional chromatography. The 2'-FL is then concentrated and crystallized in ethanol to obtain a purity of over 90%. However, this technique focuses solely on the post-processing of the fermentation broth and does not address upstream aspects such as fermentation medium design and fermentation process control. The crystallization process is a simple hot ethanol crystallization, without any targeted optimization for crystal quality.

[0004] Relevant non-patent literature retrieved: Journal article: Difan Qian, Chunyue Zhang, Chen Deng, et al. De novo biosynthesis of 2'-fucosyllactose in engineered Pichiapastoris [J]. SpringerLink, 2025. This article uses Pichia pastoris GS115 as the chassis strain and achieves de novo synthesis of 2'-FL by integrating lactose transporter protein and key enzyme genes for 2'-FL synthesis. After optimization in a 5L bioreactor, the yield only reached 0.276 g / L, which confirms the limitations of 2'-FL production by non-E. coli chassis strains and reflects the common technical challenge of low yield in the production of 2'-FL by microbial fermentation.

[0005] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: 1. Unreasonable carbon source system: CN113186142A uses a single glycerol carbon source, which leads to a sharp increase in oxygen consumption and insufficient dissolved oxygen in the early stage of fermentation, resulting in anaerobic metabolism and the production of byproducts such as acetic acid. In addition, the high concentration of glycerol causes the carbon source to be biased towards cell proliferation, and the yield of a 3L fermenter is only 30.5g / L. The yield of Pichia pastoris fermentation in non-patent literature is only 0.276g / L, both of which are far below the industrial requirements, confirming the common problem of low yield in existing fermentation systems.

[0006] 2. Defective precursor supply: Existing patents all use lactose as a single precursor. In the later stages of high-density fermentation, when dissolved oxygen is limited, lactose is easily metabolized anaerobically to produce byproducts, resulting in raw material waste and environmental degradation. Furthermore, the lack of a continuous supply of galactosyl groups restricts the synthesis efficiency of 2'-FL.

[0007] 3. Lack of targeted formulation of trace metal ions: The trace metal solution in CN113186142A does not optimize the concentrations of manganese, calcium, and nickel and is missing nickel ions. However, these ions are activators / structural stabilizers of enzymes related to 2'-FL synthesis. Improper formulation can lead to a decrease in enzyme activity of more than 30%, affecting the utilization rate of carbon source and precursor.

[0008] 4. Inefficient fermentation control: Existing technologies use constant glycerol and lactose concentrations for control, without dynamic adjustment according to cell growth (OD600 value) and product synthesis stages. This easily leads to competition between growth and synthesis, and an imbalance in precursor supply, resulting in fermentation broth purity below 85% and the production of byproducts such as difucosyl-D-lactose.

[0009] 5. Insufficient purification and crystallization process: CN115073539B relies on two-dimensional chromatography, which has high equipment costs and is difficult to scale up; conventional crystallization processes are simple, resulting in crystal impurities, uneven particle size, repose angle greater than 40°, and hygroscopic weight gain exceeding 0.1% after 8 hours, which cannot meet food-grade requirements.

[0010] 6. Incomplete technical system: Existing technologies only focus on improving a single link (strain modification, fermentation parameters or purification process), without forming a complete chain optimization of "fermentation medium - process control - purification and crystallization", making it difficult to achieve synergistic improvement of high yield, high purity and high quality of 2'-FL.

[0011] In solving the above problems or overcoming the above defects, the present invention encountered the following difficulties and obstacles: 1. In the initial stage of carbon source adaptation, when using glycerol, glucose or glycerol-glucose compound system alone, the oxygen consumption rate of the system increases sharply, accompanied by the generation of a large amount of acetic acid byproduct, and the crystallization performance of yeast broth is extremely poor, which cannot meet the requirements of subsequent processes. After finally determining to use glycerol-maltodextrin compound carbon source, the precise matching of the two ratios still requires a lot of condition exploration.

[0012] 2. During the optimization of lactose-galactomannan complex precursors, it is difficult to synchronously control the rate of rapid lactose supply and the rate of continuous galactomannan decomposition and release of galactose. Imbalance in the ratio can easily lead to insufficient precursor supply or trigger anaerobic lactose metabolism and the production of byproducts.

[0013] 3. When using a conventional trace metal ion system, the utilization efficiency of galactomannan by the strain is significantly low. Furthermore, adjusting the concentration of a single metal ion can directly alter the osmotic pressure of the culture medium, interfering with the normal growth of the bacteria. There are obvious interactions between the ions, making it impossible to achieve system balance through simple adjustments. System optimization and verification have shown that nickel ions are the key component for ensuring the efficient utilization of polysaccharides.

[0014] 4. After optimizing each fermentation parameter individually, the combined application effect is not ideal. The cell growth and product synthesis are prone to significant imbalance. The response of different cell concentration stages to dissolved oxygen, feeding, pH and other conditions is significantly different. It is necessary to match the optimal multi-parameter coupling scheme for different OD600 key nodes one by one. Summary of the Invention

[0015] The purpose of this invention is to provide: A culture medium composition for 2'-fucosylated lactose fermentation, its application in the fermentation production of 2'-fucosylated lactose, and a method for the large-scale preparation of 2'-fucosylated lactose are disclosed to address the technical problems in existing technologies, such as unreasonable carbon source system, defective precursor supply, non-targeted micro-metal ion ratio, crude fermentation control, insufficient purification and crystallization process, and incomplete technical system, which lead to low 2'-FL fermentation yield, insufficient product purity, poor crystal quality, and difficulty in meeting the needs of large-scale production.

[0016] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0017] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0018] Unless otherwise stated, conventional methods within the scope of the art shall be used.

[0019] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0020] The terms “optional / arbitrary” or “optionally / arbitrarily” mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0021] 2'-Fucosyllactose (2'-FL): 2'-fucosyllactose is a human milk oligosaccharide (HMO) formed by fucose, glucose and galactose linked by glycosidic bonds. It is the most abundant core active component in breast milk and has physiological functions such as regulating intestinal microecology and enhancing the body's immunity. It is the target product of this invention.

[0022] Compound carbon source: refers to a fermentation carbon source system formed by mixing two or more carbon sources in a specific ratio. In this invention, it specifically refers to a carbon source formed by mixing glycerol and maltodextrin in a mass ratio of (15-20):1, which is used to provide energy and carbon skeleton for cell growth and product synthesis.

[0023] Feeding medium: refers to the nutrient system added to the fermenter during fermentation according to the needs of cell growth and product synthesis. In this invention, it includes feeding medium one (containing magnesium sulfate heptahydrate, industrial yeast powder, glycerol, and porcine bone peptone) and feeding medium two (lactose-galactomannan mixture).

[0024] IPTG (isopropyl-β-D-thiogalactopyranoside): A commonly used gene expression inducer that activates the expression of target genes (such as 2'-FL synthesis-related enzyme genes) by binding to the repressor protein in the E. coli lactose operon, thereby relieving the repression effect.

[0025] Angle of repose: refers to the angle between the generatrix of the cone formed by the natural accumulation of granular materials under the action of gravity and the horizontal plane. It is a key indicator for measuring the flowability of granules. The smaller the angle of repose, the better the flowability. The product in this invention has an angle of repose of 30°-33°, which is excellent flowability.

[0026] Hygroscopicity: refers to the ability of a substance to absorb moisture from the environment. In this invention, the weight gain of the product is measured by placing it in an environment with room temperature and relative humidity of 35%-45% for 8 hours. The weight gain of the product is only 0.03%-0.07%, indicating extremely low hygroscopicity.

[0027] Mother liquor recycling: refers to the process of returning the mother liquor separated by the first evaporation and crystallization to the crystallization process for reuse. In this invention, the recycling of the batch of mother liquor is stopped when the amount of crystallization drops to below 15% to avoid the accumulation of impurities.

[0028] The %m / m mentioned in this article refers to: mass percentage content.

[0029] The %m / v mentioned in this article refers to the volume percentage of the substance, for example: x %m / v means that each 100mL of solution contains xg of the substance.

[0030] The ‰m / v mentioned in this article refers to the volume fraction of mass, for example: x ‰m / v means that each 1L of solution contains xg of the substance.

[0031] The %v / v mentioned in this article refers to volume percentage content.

[0032] In a first aspect, the present invention provides a culture medium for the fermentation of 2'-fucosylated lactose, comprising a seed culture medium, an initial culture medium, and a fed culture medium; wherein the initial culture medium uses water as a solvent and comprises: potassium dihydrogen phosphate 5‰-8‰ m / v, magnesium sulfate heptahydrate 1‰-2‰ m / v, diammonium hydrogen phosphate 2‰-3‰ m / v, citric acid 1‰-2‰ m / v, industrial yeast powder 5‰-6‰ m / v, porcine bone peptone 2‰-3‰ m / v, a compound carbon source 3%-4% m / v, and trace metal solution 8‰-1% m / v; wherein the compound carbon source is composed of glycerol and maltodextrin in a mass ratio of 15-20:1, and the DE value of the maltodextrin is 6-10.

[0033] Specifically, in some embodiments, the initial culture medium uses water as a solvent and specifically comprises: potassium dihydrogen phosphate 6.5‰ m / v, magnesium sulfate heptahydrate 1.5‰ m / v, diammonium hydrogen phosphate 2.5‰ m / v, citric acid 1.5‰ m / v, industrial yeast powder 5.5‰ m / v, porcine bone peptone 2.5‰ m / v, compound carbon source 3.5% m / v, and trace metal solution 9‰ m / v; the compound carbon source is composed of glycerol and maltodextrin in a mass ratio of 18:1, and the DE value of maltodextrin is 8.36.

[0034] Specifically, in some other embodiments, the initial culture medium uses water as a solvent and specifically comprises: potassium dihydrogen phosphate 5‰ m / v, magnesium sulfate heptahydrate 1‰ m / v, diammonium hydrogen phosphate 2‰ m / v, citric acid 1‰ m / v, industrial yeast powder 5‰ m / v, porcine bone peptone 2‰ m / v, compound carbon source 3% m / v, and trace metal solution 8‰ m / v; the compound carbon source is composed of glycerol and maltodextrin in a mass ratio of 15:1, and the maltodextrin has a DE value of 6.71.

[0035] Specifically, in some other embodiments, the initial culture medium uses water as a solvent and specifically comprises: potassium dihydrogen phosphate 8‰ m / v, magnesium sulfate heptahydrate 2‰ m / v, diammonium hydrogen phosphate 3‰ m / v, citric acid 2‰ m / v, industrial yeast powder 6‰ m / v, porcine bone peptone 3‰ m / v, compound carbon source 4% m / v, and trace metal solution 1% m / v; the compound carbon source is composed of glycerol and maltodextrin in a mass ratio of 20:1, and the maltodextrin has a DE value of 9.84.

[0036] Specifically, the composition of the trace metal liquid is as follows: Na2EDTA 0.8-1.2‰ m / v, zinc sulfate heptahydrate 0.3-0.5‰ m / v, manganese chloride dihydrate 0.3-0.5‰ m / v, cobalt chloride hexahydrate 0.01-0.03‰ m / v, copper sulfate pentahydrate 0.01-0.03‰ m / v, sodium molybdate dihydrate 0.03-0.05‰ m / v, calcium chloride dihydrate 0.4-0.6‰ m / v, ferrous sulfate heptahydrate 0.1-0.3‰ m / v, nickel chloride hexahydrate 0.01-0.03‰ m / v, and boric acid 0.05-0.1‰ m / v.

[0037] Preferably, the trace metal liquid composition is: Na2EDTA 1.0 ‰ m / v, zinc sulfate heptahydrate 0.45 ‰ m / v, manganese chloride dihydrate 0.5 ‰ m / v, cobalt chloride hexahydrate 0.03 ‰ m / v, copper sulfate pentahydrate 0.03 ‰ m / v, sodium molybdate dihydrate 0.04 ‰ m / v, calcium chloride dihydrate 0.6 ‰ m / v, ferrous sulfate heptahydrate 0.3 ‰ m / v, nickel chloride hexahydrate 0.03 ‰ m / v, and boric acid 0.1 ‰ m / v.

[0038] Specifically, the feed culture medium includes feed culture medium one and feed culture medium two; feed culture medium one comprises, by mass percentage: magnesium sulfate heptahydrate 20%-24% m / m, industrial yeast powder 5%-7% m / m, glycerol 40%-55% m / m, and porcine bone peptone 2.5%-3.5% m / m.

[0039] Preferably, in some embodiments, the supplemental culture medium specifically comprises, by mass percentage: 22% magnesium sulfate heptahydrate, 6% industrial yeast powder, 47% glycerol, and 3% porcine bone peptone.

[0040] Preferably, in other embodiments, the supplemental culture medium specifically comprises, by mass percentage: 20% magnesium sulfate heptahydrate, 5% industrial yeast powder, 40% glycerol, and 2.5% porcine bone peptone.

[0041] Preferably, in some other embodiments, the supplemental culture medium specifically comprises, by mass percentage: 24% magnesium sulfate heptahydrate, 7% industrial yeast powder, 55% glycerol, and 3.5% porcine bone peptone.

[0042] Specifically, the supplemental culture medium 2 is a lactose-galactomannan mixture with a mass concentration of 18%-24% m / v, and the mass ratio of lactose to galactomannan is 9-19:1.

[0043] Preferably, in some embodiments, the supplemental culture medium 2 is a lactose-galactomannan mixture with a mass concentration of 21% m / v, and the mass ratio of lactose to galactomannan is 14:1.

[0044] Preferably, in other embodiments, the supplemental culture medium 2 is a lactose-galactomannan mixture with a mass concentration of 18% m / v, and the mass ratio of lactose to galactomannan is 9:1.

[0045] Preferably, in some other embodiments, the supplemental culture medium 2 is a lactose-galactomannan mixture with a mass concentration of 24% m / v, and the mass ratio of lactose to galactomannan is 19:1.

[0046] Specifically, the seed culture medium includes shake flask seed culture medium and seed tank culture medium; the shake flask seed culture medium contains 8‰-10‰ m / v peptone, 4‰-6‰ m / v yeast extract, and 8‰-10‰ m / v sodium chloride; the seed tank culture medium contains 8‰-10‰ m / v peptone, 5‰-7‰ m / v corn steep liquor extract, and 8‰-10‰ m / v sodium chloride, with water as the solvent.

[0047] Preferably, in some embodiments, the shake flask seed culture medium specifically comprises 9‰ m / v peptone, 5‰ m / v yeast extract, and 9‰ m / v sodium chloride; the seed tank culture medium specifically comprises 9‰ m / v peptone, 6‰ m / v corn steep liquor, and 9‰ m / v sodium chloride, with water as the solvent.

[0048] Preferably, in some other embodiments, the shake flask seed culture medium specifically comprises 8‰ m / v peptone, 4‰ m / v yeast extract, and 8‰ m / v sodium chloride; the seed tank culture medium specifically comprises 8‰ m / v peptone, 5‰ m / v corn steep liquor, and 8‰ m / v sodium chloride, with water as the solvent.

[0049] Preferably, in some other embodiments, the shake flask seed culture medium specifically comprises 10‰ m / v peptone, 6‰ m / v yeast extract, and 10‰ m / v sodium chloride; the seed tank culture medium specifically comprises 10‰ m / v peptone, 7‰ m / v corn steep liquor, and 10‰ m / v sodium chloride, with water as the solvent.

[0050] Secondly, this invention provides a method for fermenting and producing 2'-FL, using the aforementioned culture medium and Escherichia coli BLCY-019 with preservation number CCTCC NO: M 2026303 as the strain, and controlling the concentration in segments according to the OD600 node: when OD600=10-15, the temperature is adjusted to 22-25℃, the glycerol concentration is 15-20 g / L, and the lactose concentration is 3-6 g / L; when OD600=50-60, the glycerol concentration is 12-17 g / L, and the lactose concentration is 5-8 g / L; when OD600=80-100, the glycerol concentration is 10-15 g / L, and the lactose concentration is 6-9 g / L; when OD600=130-150, the glycerol concentration is 7-10 g / L, and the lactose concentration is 8-10 g / L.

[0051] Preferably, in some embodiments, according to OD 600 Node segmented control specifically refers to: OD 600 When OD600 = 13, the temperature was adjusted to 23℃, the glycerol concentration was 17.5 g / L and the lactose concentration was 4.5 g / L; when OD600 = 55, the glycerol concentration was 15 g / L and the lactose concentration was 6.5 g / L; when OD600 = 90, the glycerol concentration was 12.5 g / L and the lactose concentration was 7.5 g / L. 600 At a concentration of 140, the glycerol concentration was 8.5 g / L and the lactose concentration was 9 g / L.

[0052] Preferably, in other embodiments, according to OD 600 Node segmented control specifically refers to: OD 600 When the concentration is 10, the temperature is adjusted to 22℃, the glycerol concentration is 15 g / L, and the lactose concentration is 3 g / L; OD 600 At 50, the glycerol concentration was 12 g / L and the lactose concentration was 5 g / L; OD 600 At 80, the glycerol concentration was 10 g / L and the lactose concentration was 6 g / L; OD 600 At a concentration of 130, the glycerol concentration is 7 g / L and the lactose concentration is 8 g / L.

[0053] Preferably, in some other embodiments, according to OD 600 Node segmented control specifically refers to: OD 600 At =15, the temperature was adjusted to 25℃, the glycerol concentration was 20 g / L, and the lactose concentration was 6 g / L; OD 600 At 60, the glycerol concentration was 17 g / L and the lactose concentration was 8 g / L; OD 600 At a concentration of 100, the glycerol concentration was 15 g / L and the lactose concentration was 9 g / L; OD 600At a concentration of 150, the glycerol concentration is 10 g / L and the lactose concentration is 10 g / L.

[0054] Specifically, IPTG adds OD in stages. 600 =10-15, add to a final concentration of 0.1-0.2 mM; OD 600 When the concentration is 50-60, 80-100, or 130-150, add more water to bring the final concentration to 0.08-0.1 mM.

[0055] Preferably, in some embodiments, the phased addition of IPTG specifically involves: OD 600 =13, add to a final concentration of 0.15 mM; OD 600 At concentrations of 55, 90, and 140, additional solutions were added to bring the final concentration to 0.09 mM.

[0056] Preferably, in other embodiments, the phased addition of IPTG specifically involves: OD 600 =10, add to a final concentration of 0.1 mM; OD 600 When the concentration is 50, 80, and 130, add more to the final concentration of 0.08 mM.

[0057] Preferably, in some other embodiments, the phased addition of IPTG specifically involves: OD 600= Add at 15:00 to a final concentration of 0.2 mM; OD 600 At concentrations of 60, 100, and 150, replenish to a final concentration of 0.1 mM.

[0058] Specifically, the aeration ratio and stirring speed are controlled in segments: OD 600 When OD600 = 10-15, the aeration ratio is 1.0-1.2 vvm and the stirring speed is 150-200 rpm; when OD600 = 50-150, the aeration ratio is 1.0-1.5 vvm and the stirring speed is 150-400 rpm.

[0059] Preferably, in some embodiments, the segmented control of the aeration ratio and stirring speed is specifically as follows: OD 600 At a concentration of 13, the aeration ratio is 1.1 vvm, and the stirring speed is 180 rpm; OD 600 At a concentration of 55, the aeration ratio is 1.3 vvm, and the stirring speed is 250 rpm; OD 600 At 90°C, the aeration ratio is 1.4 vvm, and the stirring speed is 350 rpm; OD 600 When the aeration rate is 140, the ventilation ratio is 1.4 vvm and the stirring speed is 350 rpm.

[0060] Preferably, in other embodiments, the aeration ratio and stirring speed are controlled in segments as follows: OD 600 When the aeration ratio is 10, the ventilation ratio is 1.0 vvm, and the stirring speed is 150 rpm; OD 600 At a concentration of 50, the aeration ratio is 1.2 vvm, and the stirring speed is 200 rpm; OD 600 At 80, the aeration ratio is 1.3 vvm, and the stirring speed is 300 rpm; OD 600 When the aeration rate is 130, the ventilation ratio is 1.3 vvm and the stirring speed is 300 rpm.

[0061] Preferably, in some other embodiments, the aeration ratio and stirring speed are controlled in segments as follows: OD 600 At a concentration of 15, the aeration ratio is 1.2 vvm, and the stirring speed is 200 rpm; OD 600 At a concentration of 60, the aeration ratio is 1.5 vvm, and the stirring speed is 300 rpm; OD 600 At a concentration of 100, the aeration ratio is 1.5 vvm, and the stirring speed is 400 rpm; OD 600 When the aeration ratio is 150, the ventilation ratio is 1.5 vvm, and the stirring speed is 400 rpm.

[0062] Specifically, the fermentation broth is sterilized, centrifuged to remove bacteria, finely filtered through a ceramic membrane, decolorized, desalted by electrodialysis and ion exchange, concentrated to a sugar concentration of 72-75%, crystallized by primary evaporation, crystallized by secondary gradient cooling, and dried to obtain the 2'-FL product. The secondary crystallization adopts a gradient cooling method: cooling at 0.5-0.8℃ / h at 40-50℃, cooling at 0.3-0.5℃ / h at 30-40℃, and cooling to 25℃ at 0.2-0.3℃ / h at 25-30℃.

[0063] Preferably, the sugar concentration is concentrated to 74%, the single evaporation crystallization temperature is 49°C, and the vacuum degree is -0.09 MPa.

[0064] Preferably, the sugar concentration is concentrated to 75%, the single evaporation crystallization temperature is 50°C, and the vacuum degree is -0.1 MPa.

[0065] Preferably, the sugar concentration is concentrated to 72%, the single evaporation crystallization temperature is 48°C, and the vacuum degree is -0.08 MPa.

[0066] Preferably, the secondary crystallization adopts a gradient cooling method: cooling at 0.6℃ / h for 40-50℃, cooling at 0.4℃ / h for 30-40℃, and cooling to 25℃ at 0.2℃ / h for 25-30℃.

[0067] Preferably, the secondary crystallization adopts a gradient cooling method: cooling at 0.5℃ / h for 40-50℃, cooling at 0.3℃ / h for 30-40℃, and cooling to 25℃ at 0.2℃ / h for 25-30℃.

[0068] Preferably, the secondary crystallization adopts a gradient cooling method: cooling at 0.8℃ / h for 40-50℃, cooling at 0.5℃ / h for 30-40℃, and cooling to 25℃ at 0.3℃ / h for 25-30℃.

[0069] Thirdly, the present invention provides the application of the aforementioned culture medium in the fermentation production of 2'-fucosylated lactose and in the preparation of food, health products, and pharmaceuticals.

[0070] The present invention has at least the following beneficial effects: 1. Compared with the prior art, the present invention has significantly better technical effects in terms of 2'-FL fermentation yield, product purity, crystal quality and industrial applicability.

[0071] According to experimental tests, the yield of 2'-FL in the fermentation broth of this invention is increased from the highest of 30.5 g / L in the prior art (CN113186142A, 3L fermenter) to 180-200 g / L (10000L scale fermentation), which is more than 5-6 times higher, and fully meets the production requirements of industrial production.

[0072] According to experimental tests, the purity of the final product of this invention is increased from the highest level of 95% (CN115073539B) of the prior art to 99.0%-99.5%, an increase of more than 4-4.5 percentage points, which far exceeds the purity requirements of food-grade products.

[0073] According to experimental tests, the angle of repose of the crystal product of this invention is reduced from more than 40° in the prior art to 30°-33°, and the fluidity is improved from "poor" to "excellent"; the hygroscopicity is reduced from more than 0.1% in 8 hours in the prior art to 0.03%-0.07%, and the hygroscopicity is reduced from "strong" to "extremely low", which significantly improves the product's storage stability and ease of use.

[0074] 2. Compared with existing technologies, this invention provides a whole-chain optimization scheme with a different technical concept, achieving a leapfrog improvement in technical performance. The main differences in technical concept between this invention and existing technologies include: Existing technologies mostly adopt a "single carbon source + single precursor" supply mode. This invention innovatively proposes a "glycerol-maltodextrin compound carbon source + lactose-galactomannan compound precursor" system to solve the problems of dissolved oxygen fluctuation and by-product accumulation from the source. Existing technologies employ a "constant concentration" fermentation control strategy, while this invention establishes a "OD-based" fermentation control strategy. 600 The "segmented dynamic control mode of key nodes" enables precise matching between cell growth and product synthesis. Existing technologies focus on improving a single link (strain modification or purification process). This invention constructs a complete chain of technologies, including "fermentation medium design - fermentation process regulation - purification and crystallization optimization", to achieve synergistic improvement in yield, purity and quality.

[0075] 3. Compared with existing technologies, this invention provides a technical solution representing a new technological development trend for the large-scale green production of 2'-FL. The solution incorporates features such as mother liquor recycling (recycling when crystallization amount is ≥15%), low byproduct accumulation, and compatibility with conventional equipment. These features reduce raw material loss and environmental impact, lower the technological threshold for industrialization, and align with the development direction of the bio-manufacturing industry, which emphasizes "high efficiency, energy saving, and low cost." This provides a referable technological paradigm for the large-scale production of similar human milk oligosaccharides.

[0076] Preservation Instructions Preserved strain: Escherichia coli BLCY-019; Category Naming: Escherichia coli BLCY-019; Accession number: CCTCC NO: M 2026303; Preservation period: January 30, 2026; Depository: China Center for Type Culture Collection; Address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. Attached Figure Description

[0077] Figure 1 This is a microscope image (100x) of the crystal obtained after crystallization in Example 1.

[0078] Figure 2 This is a microscope image (100x) of the crystal obtained after crystallization in Example 2.

[0079] Figure 3 This is a microscope image (100x) of the crystal obtained after crystallization in Example 3.

[0080] Figure 4 This is a microscope image (100x) of the crystal obtained after crystallization in Comparative Example 5.

[0081] Figure 5 This is a microscope image (100x) of the crystal obtained after crystallization in Comparative Example 6.

[0082] Figure 6This is a microscope image (100x) of the crystal obtained after crystallization in Comparative Example 8.

[0083] Figure 7 This is a microscope image (100x) of the crystal obtained after crystallization in Comparative Example 9.

[0084] Figure 8 This is a microscope image (100x) of the crystal obtained after crystallization in Comparative Example 10.

[0085] Figure 9 This is a microscope image (100x) of the crystal obtained after crystallization in Comparative Example 11.

[0086] Figure 10 This is a microscope image (100x) of the crystal obtained after crystallization in Comparative Example 15.

[0087] Figure 11 This is a microscope image (100x) of the crystal obtained after crystallization in Comparative Example 16.

[0088] Figure 12 This is a microscope image (100x) of the crystal obtained after crystallization in Comparative Example 17. Detailed Implementation

[0089] Unless otherwise specified, all raw materials and reagents used in this invention were purchased from commercial suppliers, and experiments were conducted in accordance with the operating instructions. Unless otherwise specified, all instruments, equipment, and apparatus used in this invention are conventional instruments, equipment, and apparatus, and experiments were conducted in accordance with the operating instructions and the accompanying reagents.

[0090] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conditions are performed under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention.

[0091] Data analysis and statistical analysis were performed using professional data processing software. One-way ANOVA was used for significance analysis, and P<0.05 was considered to indicate a significant difference.

[0092] This invention uses recombinant Escherichia coli (Escherichia coli BLCY-019) as the production strain, and prepares shake flask seeds and 1000L seed tank seeds by sequentially expanding the seed culture from glycerol tube seeds.

[0093] Shake-flask seed culture medium and culture conditions: peptone 8-10‰ m / v, yeast extract 4-6‰ m / v, sodium chloride 8-10‰ m / v, balance water. Inoculation amount: 0.07% to 0.1%, culture at 37℃ with shaking for 8-12 hours.

[0094] 1000L seed culture medium and culture conditions: peptone 8-10‰ m / v, corn steep liquor powder 5-7‰ m / v (purchased from Jining Tongsheng Biotechnology Development Co., Ltd.), sodium chloride 8-10‰ m / v, balance water. Inoculation amount: 0.08% to 0.1%, cultured at 37℃ for 10-14 hours.

[0095] The initial fermentation culture media involved in the following examples are shown in Table 1, the first feeding culture medium is shown in Table 2, and the second feeding culture medium is: 18-24% m / v lactose-galactomannan mixture, with a mass ratio of lactose to galactomannan of (9-19):1.

[0096] Lactose was purchased from Sinopharm Chemical Reagent Co., Ltd.; galactomannan was purchased from Hubei Haijia Biotechnology Co., Ltd., and is a guar gum hydrolysate with a molecular weight of 5-20 KD.

[0097] Table 1 Initial fermentation culture medium

[0098] The preparation method of the trace metal liquid described in Table 1 is as follows: Na2EDTA: 1.0‰ m / v; Zinc sulfate heptahydrate (ZnSO4) 7H2O): 0.45‰ m / v; Manganese chloride dihydrate (MnCl2·2H2O): 0.5‰ m / v; Cobalt chloride hexahydrate (CoCl2) 6H2O): 0.03‰ m / v; Copper sulfate pentahydrate (CuSO4) 5H2O): 0.03‰ m / v; Sodium molybdate dihydrate (Na2MoO4) 2H2O): 0.04‰ m / v; Calcium chloride dihydrate (CaCl2) 2H2O): 0.6‰ m / v; Ferrous sulfate heptahydrate (FeSO4) 7H2O): 0.3‰ m / v; Nickel chloride hexahydrate (NiCl2·6H2O): 0.03‰ m / v; Boric acid (H3BO3): 0.1‰ m / v.

[0099] Na₂EDTA and zinc sulfate heptahydrate were added to water, and the pH was adjusted to 6.0 with 1M sodium hydroxide. Other substances were added, and the pH was adjusted to 4.0 with 1M hydrochloric acid. Water was then added to bring the volume to a final depth. The mixture was sterilely filtered through a 0.22 μm filter membrane into a sterile container and stored at 4°C.

[0100] Table 2 Feeding Culture Medium 1

[0101] Example 1: Preparation of high-purity 2'-fucosylated lactose A method for preparing high-purity 2'-fucosylated lactose is as follows: (1) Seed culture The preserved recombinant Escherichia coli (Escherichia coli BLCY-019) glycerol tube seed was inoculated into shake flask seed culture medium at an inoculation rate of 0.8‰ v / v and cultured with shaking at 37℃ for 10 h to obtain shake flask seed solution.

[0102] The shake flask seed culture medium is prepared as follows: peptone 9‰ m / v, yeast extract 5‰ m / v, sodium chloride 9‰ m / v, with the remainder being water.

[0103] The above shake flask seed solution was inoculated into a 1000 L seed tank at an inoculation rate of 0.9‰ v / v and cultured at 37℃ for 12 h to obtain the seed tank seed solution.

[0104] The seed culture medium in the seed tank is prepared as follows: 9‰ peptone m / v, 6‰ corn steep liquor powder m / v, 9‰ sodium chloride m / v, with the remainder being water.

[0105] (2) Fermentation production The seed culture from the seed tank described in (1) was inoculated into a 10000L fermenter for fermentation. The initial culture medium volume was 3000 L, and the inoculation amount was 8% v / v. After inoculation, the fermentation temperature was controlled at 37℃, pH 7.0, aeration ratio of 0.9 vvm, and stirring speed of 120 rpm.

[0106] The initial fermentation medium was prepared as follows: potassium dihydrogen phosphate 6.5‰ m / v, magnesium sulfate heptahydrate 1.5‰ m / v, diammonium hydrogen phosphate 2.5‰ m / v, citric acid 1.5‰ m / v, industrial yeast powder 5.5‰ m / v, porcine bone peptone 2.5‰ m / v, a compound carbon source 3.5% m / v, trace metal solution 9‰ m / v, with the remainder being water, adjusted to pH 7.0. The compound carbon source contained glycerol to maltodextrin in a mass ratio of 18:1, and the maltodextrin had a DE value of 8.36.

[0107] When fermentation broth OD 600When the concentration reaches 13, adjust the fermentation temperature to 23°C. Add IPTG to the fermentation broth to a final concentration of 0.15 mM. Simultaneously, begin feeding fed medium 1 and medium 2. By controlling the flow rate, maintain the glycerol concentration in the fermentation broth at 17.5 g / L and the lactose concentration at 4.5 g / L. Increase the aeration ratio to 1.1 vvm and the stirring speed to 180 rpm.

[0108] The first supplemental culture medium, by mass percentage, contains 22% magnesium sulfate heptahydrate, 6% industrial yeast extract, 47% glycerol, and 3% porcine bone peptone. The second supplemental culture medium is a 21% m / v lactose-galactomannan mixture, with a lactose to galactomannan mass ratio of 14:1.

[0109] When fermentation broth OD 600 When the concentration reaches 55, add IPTG to the fermentation broth to a final concentration of 0.09 mM. Adjust the feed rate to control the glycerol concentration at 15 g / L and the lactose concentration at 6.5 g / L. Further increase the aeration ratio to 1.3 vvm and the stirring speed to 250 rpm.

[0110] When fermentation broth OD 600 When the concentration reaches 90%, add IPTG to a final concentration of 0.09 mM into the fermentation broth. Adjust the feed rate to control the glycerol concentration at 12.5 g / L and the lactose concentration at 7.5 g / L. Increase the aeration ratio to 1.4 vvm and the stirring speed to 350 rpm.

[0111] When fermentation broth OD 600 When the concentration reaches 140, add IPTG to the fermentation broth to a final concentration of 0.09 mM. Adjust the feed rate to control the glycerol concentration at 8.5 g / L and the lactose concentration at 9 g / L. Maintain an aeration ratio of 1.4 vvm and a stirring speed of 350 rpm.

[0112] Fermentation was terminated when the concentration of 2'-FL in the fermentation broth ceased to increase. Testing showed that under the fermentation conditions of this embodiment, the yield of 2'-FL in the fermentation broth was 200 g / L, and the product purity was 95%.

[0113] (3) Purification and crystallization Sterilization and centrifugation: After fermentation, the fermentation broth described in (2) was sterilized at 85°C for 25 min. Then, a disc centrifuge was used to separate the solids and liquids at 22,000 rpm to remove the cells and collect the supernatant.

[0114] Ceramic membrane filtration: The supernatant was sequentially filtered through a series of 100 nm and 50 nm ceramic membranes, with the operating temperature controlled at 35℃ and the operating pressure at 0.3 MPa. Testing showed that the transmittance of the filtered liquid reached 72%.

[0115] Decolorization: The ceramic membrane filtrate was decolorized by passing it through a combination of an immobilized carbon column and an Ama filter. The feed temperature was controlled at 75℃, the pH at 5.0, and the system flow rate at 10 m³ / s. 3 / h. The amount of diatomaceous earth added to the Ama filter is 0.08% of the dry basis of the liquid. Testing showed that the light transmittance of the liquid increased to 96% after decolorization.

[0116] Desalination: Desalination was performed in two steps. First, electrodialysis was conducted using 25 pairs of homogeneous membranes at a controlled current of 2.5 A, with a 7.5% sodium chloride solution as the aqueous electrode phase. After electrodialysis, the conductivity of the feed solution decreased to 1250 μS / cm. Subsequently, the feed solution was passed sequentially through a cation-anion-cation-anion-cation exchange column for deep desalination, with the feed solution temperature controlled at 40℃ and the flow rate at 3 BV / h. After ion exchange desalination, the conductivity of the feed solution decreased to 30 μS / cm.

[0117] Concentration: The desalted liquid is concentrated using a six-effect evaporator, with the concentration temperature controlled at 70℃ and the vacuum degree at -0.08 MPa, until the sugar concentration is 74%.

[0118] Single-stage evaporation crystallization: The concentrated liquid is transferred to an evaporation crystallization reactor and evaporated and crystallized at 49°C and a vacuum of -0.09 MPa. The feed water rate is controlled at 400 kg / h, and the evaporation rate is adjusted to control the crystallization amount at 20%. Subsequently, the crystals and mother liquor are separated at 1000 rpm to obtain primary crystals with a water content of 1%. The separated mother liquor is returned to the primary crystallization process for recycling. When the crystallization amount drops below 15%, the recycling of this batch of mother liquor is stopped. After single-stage evaporation crystallization, the product crystal purity is 98.5%.

[0119] Secondary cooling crystallization: Dissolve the primary crystals, adjust the sugar concentration to 68%, and adjust the temperature to 49℃. Add 2‰ (dry weight) of 2'-FL (77 μm particle size, 99.5% purity) as seed crystals, and grow crystals at 49℃ for 5 h. After crystallization, perform gradient cooling: cool at a rate of 0.6℃ / h in the 40℃-50℃ range; at a rate of 0.4℃ / h in the 30℃-40℃ range; and at a rate of 0.2℃ / h in the 25℃-30℃ range, until reaching 25℃. After crystallization, centrifuge at 1000 rpm to separate the crystals from the mother liquor, obtaining secondary crystals with a water content of 1%.

[0120] Drying: The secondary crystals were placed in a fluidized bed drying chamber and dried by controlling the hot air temperature at 60℃ and the air velocity at 11m / s to obtain 2'-FL crystals.

[0121] (4) Quality analysis of 2'-FL crystals: After crystallization, the microscope image (100x) of the obtained crystals is shown below. Figure 1 As shown in Table 3, 2'-FL exhibits a distinct crystalline morphology, with crystal size distribution. The crystals are mainly concentrated in the 40-120 mesh range, accounting for 90.5% of the total, achieving a purity of 99.5%. The moisture content is 0.3%, the angle of repose is 30°, and the product has good flowability. After being placed in an environment at room temperature and 40% RH for 8 hours, the weight gain was 0.05%, demonstrating extremely low hygroscopicity.

[0122] Table 3. Crystal size distribution after crystallization.

[0123] Example 2: Preparation of high-purity 2'-fucosylated lactose A method for preparing high-purity 2'-fucosylated lactose is as follows: (1) Seed culture The preserved recombinant Escherichia coli (Escherichia coli BLCY-019) glycerol tube seed was inoculated into shake flask seed culture medium at an inoculation rate of 0.7‰ v / v and cultured at 37℃ with shaking for 8 h to obtain shake flask seed solution.

[0124] The above shake flask seed solution was inoculated into a 1000L seed tank at an inoculation rate of 0.8‰ v / v and cultured at 37℃ for 10h to obtain the seed tank seed solution.

[0125] The shake flask seed culture medium is prepared as follows: 8‰ peptone m / v, 4‰ yeast powder m / v, 8‰ sodium chloride m / v, and the remainder is water.

[0126] The seed culture medium in the seed tank is prepared as follows: 8‰ peptone m / v, 5‰ corn steep liquor powder m / v, 8‰ sodium chloride m / v, and the remainder is water.

[0127] (2) Fermentation production The seed culture from the seed tank described in (1) was inoculated into a 10000L fermenter at an inoculation rate of 5% v / v. After inoculation, the fermentation temperature was controlled at 37℃, pH 6.8, aeration ratio of 0.8 vvm, and stirring speed of 100 rpm for cultivation.

[0128] The initial fermentation medium was prepared as follows: potassium dihydrogen phosphate 5‰ m / v, magnesium sulfate heptahydrate 1‰ m / v, diammonium hydrogen phosphate 2‰ m / v, citric acid 1‰ m / v, industrial yeast powder 5‰ m / v, porcine bone peptone 2‰ m / v, compound carbon source 3% m / v, trace metal solution 8‰ m / v, with the remainder being water, adjusted to pH 6.8. The compound carbon source contained glycerol to maltodextrin in a mass ratio of 15:1, and the maltodextrin had a DE value of 6.71.

[0129] When fermentation broth OD 600 When the concentration reaches 10, adjust the fermentation temperature to 22°C. Add IPTG to the fermentation broth to a final concentration of 0.1 mM. Simultaneously begin feeding fed medium 1 and fed medium 2. By controlling the flow rate, maintain the glycerol concentration in the fermentation broth at 15 g / L and the lactose concentration at 3 g / L. Increase the aeration ratio to 1.0 vvm and the stirring speed to 150 rpm.

[0130] The first supplemental culture medium, by mass percentage, contains 20% magnesium sulfate heptahydrate, 5% industrial yeast extract, 40% glycerol, and 2.5% porcine bone peptone. The second supplemental culture medium is an 18% m / v lactose-galactomannan mixture, with a lactose to galactomannan mass ratio of 9:1.

[0131] When fermentation broth OD 600 When the concentration reaches 50, add IPTG to the fermentation broth to a final concentration of 0.08 mM. Adjust the feed rate to control the glycerol concentration at 12 g / L and the lactose concentration at 5 g / L. Further increase the aeration ratio to 1.2 vvm and the stirring speed to 200 rpm.

[0132] When fermentation broth OD 600 When the concentration reaches 80%, add IPTG to a final concentration of 0.08 mM into the fermentation broth. Adjust the feed rate to control the glycerol concentration at 10 g / L and the lactose concentration at 6 g / L. Increase the aeration ratio to 1.3 vvm and the stirring speed to 300 rpm.

[0133] When fermentation broth OD 600 When the concentration reaches 130, add IPTG to the fermentation broth to a final concentration of 0.08 mM. Adjust the feed rate to control the glycerol concentration at 7 g / L and the lactose concentration at 8 g / L. Maintain an aeration ratio of 1.3 vvm and a stirring speed of 300 rpm.

[0134] Fermentation was terminated when the concentration of 2'-FL in the fermentation broth ceased to increase. Testing showed that under the fermentation conditions of this embodiment, the yield of 2'-FL in the fermentation broth was 185 g / L, and the product purity was 93%.

[0135] (3) Purification and crystallization Sterilization and centrifugation: After fermentation, the fermentation broth described in (2) was sterilized at 85°C for 20 min. Then, a disc centrifuge was used to separate the solids and liquids at 18,000 rpm to remove the cells and collect the supernatant.

[0136] Ceramic membrane filtration: The supernatant was sequentially filtered through a series of 100 nm and 50 nm ceramic membranes, with the operating temperature controlled at 30℃ and the operating pressure at 0.2 MPa. Testing showed that the transmittance of the filtered liquid reached 70%.

[0137] Decolorization: The ceramic membrane filtrate was decolorized by passing it through a combination of an immobilized carbon column and an Ama filter. The feed temperature was controlled at 70℃, the pH at 4.5, and the system flow rate at 8 m³ / s. 3 / h. The amount of diatomaceous earth added to the Ama filter is 0.05% of the dry basis of the liquid. Testing showed that the light transmittance of the liquid increased to 95% after decolorization.

[0138] Desalination: Desalination is performed in two steps. First, electrodialysis desalination is carried out using 20 pairs of homogeneous membranes at a controlled current of 2.0 A, with a 5% sodium chloride solution as the aqueous electrode phase. After electrodialysis, the conductivity of the feed solution decreased to 1500 μS / cm. Subsequently, the feed solution is passed sequentially through a "cation-anion-cation-anion-cation" ion exchange column (preferably D001 cation resin and D354 anion resin) for deep desalination, with the feed solution temperature controlled at 35℃ and the flow rate at 2 BV / h. After ion exchange desalination, the conductivity of the feed solution decreased to 50 μS / cm.

[0139] Concentration: The desalted liquid is concentrated using a six-effect evaporator, with the concentration temperature controlled at 65℃ and the vacuum degree at -0.06 MPa, until the sugar concentration is 72%.

[0140] Single-stage evaporation crystallization: The concentrated liquid is transferred to an evaporation crystallization reactor and evaporated and crystallized at 48℃ and a vacuum of -0.08 MPa. The feed water rate is controlled at 300 kg / h, and the evaporation rate is adjusted to control the crystallization amount at 18%. Subsequently, the crystals and mother liquor are separated at 800 rpm to obtain primary crystals with a water content of 2%. The separated mother liquor is returned to the primary crystallization process for recycling. When the crystallization amount drops below 15%, the recycling of this batch of mother liquor is stopped. After single-stage evaporation crystallization, the product crystal purity is 98%.

[0141] Secondary cooling crystallization: Dissolve the primary crystals, adjust the sugar concentration to 67%, and adjust the temperature to 48℃. Add 1‰ (dry weight) of 2'-FL (75 μm particle size, 99% purity) as seed crystals, and grow crystals at 48℃ for 4 h. After crystallization, perform gradient cooling: cool at a rate of 0.5℃ / h in the 40℃-50℃ range; at a rate of 0.3℃ / h in the 30℃-40℃ range; and at a rate of 0.2℃ / h in the 25℃-30℃ range, until reaching 25℃. After crystallization, centrifuge at 800 rpm to separate the crystals from the mother liquor, obtaining secondary crystals with a moisture content of 2%.

[0142] Drying: The secondary crystals were placed in a fluidized bed drying chamber and dried by controlling the hot air temperature at 50℃ and the air velocity at 8 m / s to obtain 2'-FL crystals.

[0143] (4) Quality analysis of 2'-FL crystals: After crystallization, the microscope image (100x) of the obtained crystals is shown below. Figure 2 As shown in Table 4, 2'-FL exhibits a distinct crystalline morphology, with crystal size distribution. The crystals are mainly concentrated in the 40-120 mesh range, accounting for 88.5%, with a purity of 99.2%. The moisture content is 0.7%, the angle of repose is 33°, and the product has good flowability. After being placed in an environment at room temperature and 35% RH for 8 hours, the weight gain was 0.07%, indicating low hygroscopicity.

[0144] Table 4. Crystal size distribution after crystallization is complete.

[0145] Example 3: Preparation of high-purity 2'-fucosylated lactose A method for preparing high-purity 2'-fucosylated lactose is as follows: (1) Seed culture The preserved recombinant Escherichia coli (Escherichia coli BLCY-019) glycerol tube seed was inoculated into shake flask seed culture medium at an inoculation rate of 1‰ v / v and cultured at 37℃ with shaking for 12 h to obtain shake flask seed solution.

[0146] The shake flask seed culture medium is prepared as follows: 10‰ peptone m / v, 6‰ yeast powder m / v, 10‰ sodium chloride m / v, and the remainder is water.

[0147] The above shake flask seed solution was inoculated into a 1000 L seed tank at an inoculation rate of 1‰ v / v and cultured at 37℃ for 14 h to obtain the seed tank seed solution.

[0148] The seed culture medium in the seed tank is prepared as follows: 10‰ peptone m / v, 7‰ corn steep liquor powder m / v, 10‰ sodium chloride m / v, and the remainder is water.

[0149] (2) Fermentation production The seed culture from the seed tank described in (1) was inoculated into a fermenter for fermentation. The initial fermentation medium was 10,000 L, and the inoculation amount was 10% v / v. After inoculation, the fermentation temperature was controlled at 37℃, pH 7.2, aeration ratio of 1.0 vvm, and stirring speed of 150 rpm.

[0150] The 10,000 L initial fermentation medium was prepared as follows: potassium dihydrogen phosphate 8‰ m / v, magnesium sulfate heptahydrate 2‰ m / v, diammonium hydrogen phosphate 3‰ m / v, citric acid 2‰ m / v, industrial yeast powder 6‰ m / v, porcine bone peptone 3‰ m / v, compound carbon source 4% m / v, trace metal solution 1% m / v, and water was added to bring the volume to 10,000 L, and the pH was adjusted to 7.2. The compound carbon source contained glycerol to maltodextrin in a mass ratio of 20:1, and the maltodextrin had a DE value of 9.84.

[0151] When fermentation broth OD 600 When the temperature reaches 15°C, adjust the fermentation temperature to 25°C. Add IPTG to the fermentation broth to a final concentration of 0.2 mM. Simultaneously begin feeding fed medium 1 and fed medium 2. By controlling the flow rate, maintain the glycerol concentration in the fermentation broth at 20 g / L and the lactose concentration at 6 g / L. Increase the aeration ratio to 1.2 vvm and the stirring speed to 200 rpm.

[0152] The first supplemental culture medium, by mass percentage, contains 24% magnesium sulfate heptahydrate, 7% industrial yeast extract, 55% glycerol, and 3.5% porcine bone peptone. The second supplemental culture medium is a 24% m / v lactose-galactomannan mixture, with a lactose to galactomannan mass ratio of 19:1.

[0153] When fermentation broth OD 600 When the concentration reaches 60%, add IPTG to a final concentration of 0.1 mM into the fermentation broth. Adjust the feed rate to control the glycerol concentration at 17 g / L and the lactose concentration at 8 g / L. Further increase the aeration ratio to 1.5 vvm and the stirring speed to 300 rpm.

[0154] When fermentation broth OD 600 When the concentration reaches 100, add IPTG to the fermentation broth to a final concentration of 0.1 mM. Adjust the feed rate to control the glycerol concentration at 15 g / L and the lactose concentration at 9 g / L. Maintain an aeration ratio of 1.5 vvm and increase the stirring speed to 400 rpm.

[0155] When fermentation broth OD 600 When the concentration reaches 150, add IPTG to the fermentation broth to a final concentration of 0.1 mM. Adjust the feed rate to control the glycerol concentration at 10 g / L and the lactose concentration at 10 g / L. Maintain an aeration ratio of 1.5 vvm and a stirring speed of 400 rpm.

[0156] Fermentation was terminated when the concentration of 2'-FL in the fermentation broth ceased to increase. Testing showed that under the fermentation conditions of this embodiment, the yield of 2'-FL in the fermentation broth was 190 g / L, and the product purity was 93%.

[0157] (3) Purification and crystallization Sterilization and centrifugation: After fermentation, the fermentation broth described in (2) was sterilized at 85°C for 30 min. Then, a disc centrifuge was used to separate the solids and liquids at 25,000 rpm to remove the cells and collect the supernatant.

[0158] Ceramic membrane filtration: The supernatant was sequentially filtered through a series of 100 nm and 50 nm ceramic membranes, with the operating temperature controlled at 40℃ and the operating pressure at 0.4 MPa. Testing showed that the transmittance of the filtered liquid reached 73%.

[0159] Decolorization: The ceramic membrane filtrate was decolorized by passing it through an immobilized carbon column in combination with an Ama filter. The feed temperature was controlled at 80℃, the pH at 5.5, and the system flow rate at 12 m³ / h. The amount of diatomaceous earth added to the Ama filter was 0.1% of the dry basis of the feed. Testing showed that the transmittance of the decolorized feed increased to 97%.

[0160] Desalination: Desalination was performed in two steps. First, electrodialysis was conducted using 30 pairs of homogeneous membranes at a controlled current of 3.0 A, with a 10% sodium chloride solution as the aqueous electrode phase. After electrodialysis, the conductivity of the feed solution decreased to 1000 μS / cm. Subsequently, the feed solution was passed sequentially through a cation-anion-cation-anion-cation exchange column for deep desalination, with the feed solution temperature controlled at 45℃ and the flow rate at 4 BV / h. After ion exchange desalination, the conductivity of the feed solution decreased to 10 μS / cm.

[0161] Concentration: The desalted liquid is concentrated using a six-effect evaporator, with the concentration temperature controlled at 75℃ and the vacuum degree at -0.1 MPa, until the sugar concentration is 75%.

[0162] Single-stage evaporation crystallization: The concentrated liquid is transferred to an evaporation crystallization reactor and evaporated and crystallized at 50°C and a vacuum of -0.1 MPa. The feed water rate is controlled at 500 kg / h, and the evaporation rate is adjusted to control the crystallization amount at 22%. Subsequently, the crystals and mother liquor are separated at a rotation speed of 1300 rpm to obtain the first crystal, with a water content of 0.8%. The separated mother liquor is returned to the first crystallization process for recycling. When the crystallization amount drops below 15%, the recycling of that batch of mother liquor is stopped. After single-stage evaporation crystallization, the product crystal purity is 98.5%.

[0163] Secondary cooling crystallization: Dissolve the primary crystals, adjust the sugar concentration to 70%, and adjust the temperature to 50℃. Add 3‰ (dry weight) of 2'-FL (80 μm particle size, 100% purity) as seed crystals, and grow crystals at 50℃ for 6 h. After crystallization, perform gradient cooling: cool at a rate of 0.8℃ / h in the 40℃-50℃ range; at a rate of 0.5℃ / h in the 30℃-40℃ range; and at a rate of 0.3℃ / h in the 25℃-30℃ range, until reaching 25℃. After crystallization, centrifuge at 1300 rpm to separate the crystals from the mother liquor, obtaining secondary crystals with a moisture content of 0.8%.

[0164] Drying: The secondary crystals were placed in a fluidized bed drying chamber and dried by controlling the hot air temperature at 70℃ and the air velocity at 15 m / s to obtain 2'-FL crystals.

[0165] (4) Quality analysis of 2'-FL crystals: After crystallization, the microscope image (100x) of the obtained crystals is shown below. Figure 3 As shown in Table 5, 2'-FL exhibits a distinct crystalline morphology, with crystal size distribution primarily concentrated in the 40-120 mesh range, accounting for 87.4% of the total. The purity reaches 99.0%, with a moisture content of 0.5% and an angle of repose of 32°, indicating excellent product flowability. After being placed at room temperature and 45% RH for 8 hours, the product showed a weight gain of only 0.03%, demonstrating extremely low hygroscopicity.

[0166] Table 5. Crystal size distribution after crystallization.

[0167] Comparative Example A comparative example was set up with reference to Example 1. The difference between the comparative example and Example 1 is shown in Table 6: Settings in Table 6

[0168] Comparative Example 1 Comparative Example 1 was similar to Example 1, except that only 3.5% m / v glycerol was used as the sole carbon source in the initial fermentation medium. Under the fermentation conditions of Comparative Example 1, the yield of 2'-FL in the fermentation broth was 145 g / L, and the product purity was 86%. No crystalline product was obtained during subsequent crystallization.

[0169] Comparative Example 1 illustrates that replacing the compound carbon source with glycerol alone led to a significant decrease in the yield and purity of 2'-FL. The reasons are as follows: Glycerol, as a rapidly utilized carbon source, relies primarily on aerobic respiration for its metabolism. This process involves extremely high oxygen consumption, a sharp drop in dissolved oxygen, and a high redox potential difference, triggering anaerobic metabolism and producing organic acid byproducts such as acetic acid. In contrast, Example 1 used glycerol and maltodextrin as a compound carbon source. Maltodextrin exhibits a slower degradation rate, lower oxygen consumption, and a lower oxygen-reduction potential difference, providing a stable carbon source without causing drastic oxygen fluctuations. This effectively prevented the accumulation of byproducts such as acetic acid, maintained the metabolic activity of the cells, and thus ensured the efficient synthesis of 2'-FL in the later stages. In the Comparative Example 1 system, the increased impurity content in the mother liquor made it difficult to establish effective supersaturation during crystallization, hindering crystal nucleus formation and ultimately preventing the acquisition of crystalline products.

[0170] Comparative Example 2 Comparative Example 2 was similar to Example 1, except that glycerol and glucose were used as a combined carbon source in the initial fermentation medium (glycerol to glucose mass ratio of 18:1, total addition amount still 3.5% m / v). Under the fermentation conditions of Comparative Example 2, the yield of 2'-FL in the fermentation broth was 138 g / L, and the product purity was 82%. No crystalline product was obtained during the subsequent crystallization process.

[0171] Comparative Example 2 illustrates that replacing the mixed carbon source in the initial fermentation medium with a glucose-glycerol mixture significantly reduced the yield and purity of 2'-FL. This is because both glucose and glycerol are rapidly utilized carbon sources, causing a surge in oxygen consumption during the initial fermentation stage, leading to decreased dissolved oxygen and a high redox potential. This results in the formation of byproducts such as acetic acid, reducing product purity. Furthermore, glucose's preferential use as a carbon source triggers carbon decomposition metabolite repression, causing fluctuations in the cell's metabolic state during carbon source utilization, thus reducing 2'-FL yield. Increased impurity content in the mother liquor makes it difficult to establish effective supersaturation during crystallization, hindering crystal nucleus formation and ultimately preventing the acquisition of crystalline products.

[0172] Comparative Example 3 Comparative Example 3 was similar to Example 1, except that maltodextrin was replaced with a compound carbon source of 30.37 in the initial fermentation medium. Under the fermentation conditions of Comparative Example 3, the yield of 2'-FL in the fermentation broth was 130 g / L, and the product purity was 82%. No crystalline product was obtained during the subsequent crystallization process.

[0173] Comparative Example 3 shows that the high DE value and high reducing sugar content of maltodextrin caused carbon metabolism disorders during the fermentation process, resulting in a decrease in 2'-FL yield. Increased impurity content in the mother liquor made it difficult to establish effective supersaturation during crystallization, hindering crystal nucleus formation and ultimately preventing the acquisition of crystalline products.

[0174] Comparative Example 4 Comparative Example 4 was similar to Example 1, except that only 3.5% m / v glucose was used as the sole carbon source in the initial fermentation medium. Under the fermentation conditions of Comparative Example 4, the yield of 2'-FL in the fermentation broth was 128 g / L, and the product purity was 80%. No crystalline product was obtained during subsequent crystallization.

[0175] Comparative Example 4 illustrates that replacing the compound carbon source with glucose alone led to a significant decrease in the yield and purity of 2'-FL. The reasons are as follows: Glucose, as a rapidly utilized carbon source, primarily relies on aerobic respiration for its metabolism. This process involves extremely high oxygen consumption, a sharp drop in dissolved oxygen, and a high redox potential difference, triggering anaerobic metabolism and producing organic acid byproducts such as acetic acid. In the Comparative Example 4 system, the increased impurity content in the mother liquor made it difficult to establish effective supersaturation during crystallization, hindering crystal nucleus formation and ultimately preventing the acquisition of crystalline products.

[0176] Comparative Example 5 Comparative Example 5 was similar to Example 1, except that the mass ratio of glycerol to maltodextrin in the initial carbon source of the fermentation medium was 14:1. Under the fermentation conditions of Comparative Example 5, the yield of 2'-FL in the fermentation broth was 162 g / L, and the product purity was 89%. After crystallization, the microscope image (100x) of the obtained crystals is shown below. Figure 4 As shown in Table 7, the crystal size distribution is as follows: 40-80 mesh accounts for 28.6%, 80-120 mesh accounts for 24.5%, 120-160 mesh accounts for 21.3%, and below 160 mesh accounts for 25.6%. The crystal size distribution is too wide, the proportion of fine crystals is increased, the purity is 97.9%, the moisture content is 1.5%, the angle of repose is 43°, and the product's flowability is poor. After placing the product in an environment at room temperature and 35% RH for 8 hours, its weight gain was 0.12%.

[0177] Comparative Example 5 illustrates that when the ratio of glycerol to maltodextrin is below the preferred range of this invention, both the yield and purity of 2'-FL decrease. This is because, while maltodextrin helps maintain a low redox potential and reduces oxygen consumption, insufficient glycerol leads to a slower initial cell growth rate. Maltodextrin is not preferentially utilized as a rapid carbon source, and its slow degradation characteristics cannot meet the conditions required for rapid cell proliferation, preventing the cells from reaching high density at the optimal time, thus affecting the product synthesis efficiency in the subsequent induction phase. The blocked 2'-FL synthesis pathway easily generates organic acid byproducts such as acetic acid. Only by controlling the ratio of glycerol to maltodextrin within a suitable range can a balance between rapid expansion and low-oxygen metabolism be achieved. During crystallization of the yeast broth in Comparative Example 5, crystal purity decreased, particle size distribution became too wide, moisture content increased, fluidity worsened, and hygroscopicity increased.

[0178] Table 7. Crystal size distribution after crystallization.

[0179] Comparative Example 6 Comparative Example 6 was similar to Example 1, except that the mass ratio of glycerol to maltodextrin in the initial fermentation medium was 21:1. Under the fermentation conditions of Comparative Example 6, the yield of 2'-FL in the fermentation broth was 158 g / L, and the product purity was 88%. After crystallization, the microscope image (100x) of the obtained crystals is shown below. Figure 5 As shown in Table 8, the crystal size distribution is as follows: 40-80 mesh accounts for 30.0%, 80-120 mesh accounts for 27.1%, 120-160 mesh accounts for 24.9%, and below 160 mesh accounts for 18.0%. The crystal size distribution is too wide, the proportion of fine crystals is increased, the purity is 98.1%, the moisture content is 1.8%, the angle of repose is 39°, and the product's flowability is poor. After placing the product in an environment at room temperature and 35% RH for 8 hours, its weight gain was 0.15%.

[0180] Comparative Example 6 illustrates that when the ratio of glycerol to maltodextrin exceeds the preferred range of this invention, the yield and purity of 2'-FL also decrease. This is because a high proportion of glycerol leads to excessively high oxygen consumption in the early stages of fermentation, increasing the redox potential and causing metabolic flux to shift towards byproducts such as acetic acid. The yeast broth in Comparative Example 6 has low purity. During crystallization, crystal purity decreases, particle size distribution becomes too wide, moisture content increases, fluidity deteriorates, and hygroscopicity increases.

[0181] Table 8. Crystal size distribution after crystallization.

[0182] Comparative Example 7 Comparative Example 7 was similar to Example 1, except that the supplemental culture medium was a lactose solution. Under the fermentation conditions of Comparative Example 7, the yield of 2'-FL in the fermentation broth was 152 g / L, and the product purity was 85%. No crystalline product was obtained during the subsequent crystallization process.

[0183] Comparative Example 7 illustrates that replacing the lactose-galactomannan mixture in fed culture medium II with a single lactose solution significantly reduced the yield and purity of 2'-FL. This is because, in the later stages of fermentation, with high cell density and insufficient dissolved oxygen, lactose easily enters the anaerobic metabolic pathway and is converted into byproducts such as lactic acid and acetic acid, resulting not only in lactose waste but also a deterioration of the fermentation environment. In contrast, the galactomannan in Example 1 does not enter the anaerobic pathway and does not produce byproducts such as lactic acid and acetic acid. Furthermore, galactomannan does not act as a rapid carbon source and can continuously provide galactosyl groups for 2'-FL synthesis. The yeast broth in Comparative Example 7 had excessively low purity; under these conditions, supersaturation was difficult to establish, crystal nucleus formation was inhibited, and crystalline products could not be obtained.

[0184] Comparative Example 8 Comparative Example 8 was similar to Example 1, except that the mass ratio of the lactose-galactomannan mixture in the fed culture medium II was 8:1. Under the fermentation conditions of Comparative Example 8, the yield of 2'-FL in the fermentation broth was 165 g / L, and the product purity was 88%. After crystallization, the microscope image (100x) of the obtained crystals is shown below. Figure 6 As shown in Table 9, the crystal size distribution is as follows: 40-80 mesh accounts for 35.8%, 80-120 mesh accounts for 24.5%, 120-160 mesh accounts for 23.7%, and below 160 mesh accounts for 16.0%. The crystal size distribution is too wide, the proportion of fine crystals is increased, the purity is 97.6%, the moisture content is 1.7%, the angle of repose is 38°, and the product's flowability is poor. After placing the product in an environment at room temperature and 35% RH for 8 hours, its weight gain was 0.14%.

[0185] Comparative Example 8 illustrates that when the ratio of lactose to galactomannan is lower than the preferred range of this invention, both the yield and purity of 2'-FL decrease significantly. This is because galactomannan is not utilized as a rapid carbon source; when the galactomannan ratio is too high, the supply of lactose, as a direct precursor, is relatively insufficient, thus limiting the rate of product synthesis. The rate at which galactomannan releases galactose is gradual and continuous, but it cannot meet the large demand for precursors in the later stages of high-density fermentation. The 2'-FL synthesis pathway is blocked, easily generating organic acid byproducts such as acetic acid. When the yeast broth of Comparative Example 8 was crystallized, the crystal purity decreased, the particle size distribution became too wide, the moisture content increased, the flowability worsened, and the hygroscopicity increased.

[0186] Table 9. Crystal size distribution after crystallization is complete.

[0187] Comparative Example 9 Comparative Example 9 was similar to Example 1, except that the mass ratio of the lactose-galactomannan mixture in the fed culture medium II was 20:1. Under the fermentation conditions of Comparative Example 9, the yield of 2'-FL in the fermentation broth was 161 g / L, and the product purity was 87%. After crystallization, the microscope image (100x) of the obtained crystals is shown below. Figure 7 As shown in Table 10, the crystal size distribution is as follows: 40-80 mesh accounts for 29.1%, 80-120 mesh accounts for 26.2%, 120-160 mesh accounts for 24.3%, and below 160 mesh accounts for 20.4%. The crystal size distribution is too wide, the proportion of fine crystals is increased, the purity is 97.6%, the moisture content is 2.0%, the angle of repose is 41°, and the product's flowability is poor. After placing the product in an environment at room temperature and 35% RH for 8 hours, its weight gain was 0.19%.

[0188] Comparative Example 9 illustrates that when the ratio of lactose to galactomannan exceeds the preferred range of this invention, both the yield and purity of 2'-FL decrease significantly. This is because when the galactomannan ratio is too low, lactose dominates. Its rapid metabolic characteristics inevitably lead to some lactose entering the anaerobic pathway, generating byproducts such as lactic acid and acetic acid, resulting in reduced product purity. Simultaneously, the lack of a continuous supply of galactose from galactomannan further reduces the yield of 2'-FL. When the yeast broth in Comparative Example 9 was crystallized, the crystal purity decreased, the particle size distribution became too wide, the moisture content increased, the flowability worsened, and the hygroscopicity increased.

[0189] Table 10 Crystal size distribution after crystallization is complete

[0190] Comparative Example 10 Comparative Example 10 was similar to Example 1, except that the concentration of MnCl2·2H2O in the initial fermentation medium was replaced with 0.6‰ m / v. Under the fermentation conditions of Comparative Example 8, the yield of 2'-FL in the fermentation broth was 165 g / L, and the product purity was 88%. After crystallization, the microscope image (100x) of the obtained crystals is shown below. Figure 8 As shown in Table 11, the crystal size distribution is as follows: 40-80 mesh accounts for 32.5%, 80-120 mesh accounts for 25.6%, 120-160 mesh accounts for 24.8%, and below 160 mesh accounts for 17.1%. The crystal size distribution is too wide, the proportion of fine crystals is increased, the purity is 97.8%, the moisture content is 1.7%, the angle of repose is 39°, and the product's flowability is poor. After placing the product in an environment at room temperature and 35% RH for 8 hours, its weight gain was 0.15%.

[0191] Comparative Example 10 illustrates that manganese ions can affect the stability and activity of related enzymes in the synthetic pathway. Insufficient concentration leads to reduced utilization of galactomannan, resulting in decreased 2'-FL yield. The cells utilize more lactose, which, due to its rapid metabolism, inevitably enters the anaerobic pathway, generating byproducts such as lactic acid and acetic acid, thus reducing product purity. During crystallization of the yeast broth from Comparative Example 10, crystal purity decreased, particle size distribution became too wide, moisture content increased, flowability worsened, and hygroscopicity increased.

[0192] Table 11 Crystal size distribution after crystallization is complete

[0193] Comparative Example 11 Comparative Example 11 was similar to Example 1, except that the concentration of CaCl2·2H2O in the initial fermentation medium was replaced with 0.3‰ m / v. Under the fermentation conditions of Comparative Example 11, the yield of 2'-FL in the fermentation broth was 172 g / L, and the product purity was 90%. After crystallization, the microscope image (100x) of the obtained crystals is shown below. Figure 9 As shown in Table 12, the crystal size distribution is as follows: 40-80 mesh accounts for 31.2%, 80-120 mesh accounts for 28.5%, 120-160 mesh accounts for 23.5%, and below 160 mesh accounts for 16.8%. The crystal size distribution is too wide, the proportion of fine crystals is increased, the purity is 97.5%, the moisture content is 1.6%, the angle of repose is 38°, and the product's flowability is poor. After placing the product in an environment at room temperature and 35% RH for 8 hours, its weight gain was 0.15%.

[0194] Comparative Example 11 shows that calcium ions can affect the stability and activity of related enzymes in the synthesis pathway. Insufficient concentration will lead to a decrease in the utilization rate of galactomannan, resulting in a decrease in the yield of 2'-FL. The cells will utilize more lactose. Due to the rapid metabolism of lactose, it will inevitably enter the anaerobic pathway, generating byproducts such as lactic acid and acetic acid, which will reduce the purity of the yeast culture. At the same time, the unused galactomannan in the system will further increase the viscosity of the mother liquor. Under these conditions, crystallization will result in a decrease in crystal purity, an increase in the proportion of fine crystals, an increase in water content, a decrease in fluidity, and an increase in hygroscopicity.

[0195] Table 12 Crystal size distribution after crystallization is complete

[0196] Comparative Example 12 Comparative Example 12 was similar to Example 1, except that NiCl2·6H2O was not added to the initial fermentation medium containing trace amounts of metal liquid. Under the fermentation conditions of Comparative Example 12, the yield of 2'-FL in the fermentation broth was 158 g / L, and the product purity was 85%. No crystalline product was obtained during the subsequent crystallization process.

[0197] Comparative Example 12 illustrates that nickel ions affect the stability and activity of related enzymes in the synthetic pathway. The absence of nickel ions leads to extremely low utilization of galactomannan by the strain, with the cells utilizing more lactose. Due to lactose's rapid metabolic characteristics, it enters the anaerobic pathway, generating byproducts such as lactic acid and acetic acid, thus reducing the purity and yield of 2'-FL. The low purity of the yeast culture, coupled with the residual unused galactomannan in the system, further increases the viscosity of the mother liquor. Under these conditions, supersaturation is difficult to establish during crystallization, inhibiting crystal nucleus formation and preventing the acquisition of crystalline products.

[0198] Comparative Example 13 Comparative Example 13 is similar to Example 1, except that during the fermentation process, when the OD of the fermentation broth... 600 After induction began at level 13, the glycerol concentration was maintained at 17.5 g / L through feeding. Fermentation was terminated when the 2'-FL concentration in the fermentation broth stopped increasing. Under the fermentation conditions of Comparative Example 13, the yield of 2'-FL in the fermentation broth was 150 g / L, and the product purity was 85%. No crystalline product was obtained during subsequent crystallization.

[0199] Comparative Example 13 illustrates that maintaining a high glycerol concentration during product synthesis significantly reduces the yield and purity of 2'-FL. This is because, in the later stages of induction, the cells should transition from growth to production. However, consistently high glycerol concentrations cause the cells to allocate excessive carbon and energy for proliferation and metabolism, rather than for 2'-FL synthesis. The cells have limited capacity to metabolize glycerol; excess glycerol is converted into organic acid byproducts such as acetic acid, thereby inhibiting metabolic activity, accelerating cell senescence, and reducing the purity and yield of 2'-FL. Excessive accumulation of impurities in the mother liquor makes it difficult to establish supersaturation during crystallization, hindering crystal nucleus formation and ultimately preventing the acquisition of crystalline products.

[0200] Comparative Example 14 Comparative Example 14 is similar to Example 1, except that during the fermentation process, when the OD of the fermentation broth... 600 After induction began at level 13, the lactose concentration was maintained at 4.5 g / L through feeding. Fermentation was terminated when the 2'-FL concentration in the fermentation broth stopped increasing. Under the fermentation conditions of Comparative Example 14, the yield of 2'-FL in the fermentation broth was 120 g / L, and the product purity was 78%. No crystalline product was obtained during subsequent crystallization.

[0201] Comparative Example 14 shows that maintaining a constant lactose concentration at the initial level during product synthesis leads to a significant decrease in the yield and purity of 2'-FL. The reason is as follows: when the OD of the fermentation broth... 600 At a concentration of 13, the cell density is relatively low, and the 4.5 g / L lactose content is sufficient for initial synthesis. However, as fermentation progresses, the cell density increases rapidly, and the synthesis reaction slows down due to lactose shortage, resulting in a decrease in 2'-FL yield. Furthermore, with sufficient carbon source but insufficient lactose, the metabolic flow shifts towards byproduct formation, thus reducing product purity. Low mother liquor purity makes it difficult to establish supersaturation during crystallization, hindering crystal nucleus formation and ultimately preventing the acquisition of crystalline products.

[0202] Comparative Example 15 Comparative Example 15 is similar to Example 1, except that after one evaporation crystallization and separation to obtain the primary crystals, a second cooling crystallization is not performed; instead, the primary crystals are directly dried to obtain 2'-FL crystals. Quality analysis of the 2'-FL crystals: After crystallization, the microscope image (100x) of the obtained crystals is shown below. Figure 10 As shown in Table 13, the crystal size distribution is as follows: 40-80 mesh crystals account for 30.4%, 80-120 mesh for 25.7%, 120-160 mesh for 29.4%, and below 160 mesh for 14.5%. The crystal size distribution is too wide, with an increase in small crystals. The purity is 98%, the moisture content is 2%, the angle of repose is 37°, and the product's flowability is poor. After placing the product in an environment at room temperature and 35% RH for 8 hours, its weight gain was 0.15%.

[0203] Comparative Example 15 shows that after only one evaporation crystallization, the purity of 2'-FL crystals decreased, the crystal size distribution became uneven, the moisture content increased, the fluidity worsened, and the hygroscopicity increased. The reasons are as follows: Single evaporation crystallization takes place in a concentrated solution with a complex composition, resulting in strong crystallization driving force and rapid growth. This easily leads to impurities with similar structures (such as glucose, galactose, DFL, etc.) and trace amounts of mother liquor being embedded or encapsulated within the crystals, making them difficult to remove by centrifugation and thus reducing crystal purity. Simultaneously, the resulting crystals have uneven particle size distribution and irregular morphology, making it easy for moisture to remain in the crystal gaps and interlayers during centrifugation, resulting in high moisture content, poor fluidity, and increased hygroscopicity.

[0204] Table 13 Crystal size distribution after crystallization is complete

[0205] Comparative Example 16 Comparative Example 16 is similar to Example 1, except that the secondary cooling crystallization did not involve segmented crystallization; instead, the temperature was lowered at a constant rate of 0.5°C / h within the range of 25°C-50°C. Quality analysis of the 2'-FL crystals was performed: Microscopic images (100x) of the obtained crystals after crystallization are shown below. Figure 11 As shown in Table 14, the crystal size distribution is as follows: 40-80 mesh crystals account for 24.7%, 80-120 mesh for 32.9%, 120-160 mesh for 28.8%, and below 160 mesh for 13.6%. The crystal size distribution is too wide, with an increase in small crystals. The purity is 98.5%, the moisture content is 1%, the angle of repose is 37°, and the product's flowability is poor. After placing the product in an environment at room temperature and 35% RH for 8 hours, its weight gain was 0.12%.

[0206] Comparative Example 16 illustrates that without segmented gradient cooling during secondary cooling crystallization, the purity of 2'-FL crystals decreases, crystal size distribution becomes uneven and irregular in shape, moisture content increases, fluidity deteriorates, and hygroscopicity increases. The reasons are as follows: Constant rapid cooling causes the system to instantly enter a highly supersaturated state, inducing explosive nucleation and forming numerous fine crystal nuclei. This results in uneven crystal size and irregular morphology, leading to poor fluidity during crystal accumulation and more residual moisture during subsequent separation. It also makes the crystals more susceptible to absorbing moisture from the environment, resulting in higher moisture content and increased hygroscopicity. Furthermore, it hinders the removal of impurity molecules during crystallization, causing impurity encapsulation and reducing product purity.

[0207] Table 14 Crystal size distribution after crystallization is complete

[0208] Comparative Example 17 Comparative Example 17 is similar to Example 1, except that in one evaporation crystallization step, when the amount of crystals decreased to below 15%, the batch of mother liquor was still recycled. After crystallization, the resulting crystals were visualized under a microscope (100x magnification) as shown below. Figure 12 As shown in Table 15, the crystal size distribution is as follows: 40-80 mesh crystals account for 28.3%, 80-120 mesh for 27.9%, 120-160 mesh for 22.8%, and below 160 mesh for 21.0%. The crystal size distribution is too wide, with an increase in small crystals. Testing revealed that the final 2'-FL product obtained in Comparative Example 5 had a purity of only 95%, a moisture content of 2.2%, and an angle of repose of 41°. The product exhibited poor flowability and was prone to clumping. After being placed in an environment at room temperature and 35% RH for 8 hours, the product showed a weight gain of 0.4%, indicating a significant increase in hygroscopicity.

[0209] Comparative Example 17 shows that unlimited recycling of mother liquor will cause impurities to continuously accumulate in the system, interfering with the crystallization process, resulting in decreased crystal purity, irregular crystal shape, uneven size distribution, and consequently, increased product moisture content, decreased fluidity, and increased hygroscopicity.

[0210] Table 15 Crystal size distribution after crystallization is complete

[0211] Example 1: Particle Flowability Test The angle of repose is an important indicator of flowability; when the angle of repose is <30°, the flowability is excellent. The angle of repose of 2'-FL prepared in Examples 1-3 and Comparative Examples 5-6, 8-11, and 15-17 was tested, and the results are shown in Table 16.

[0212] Method for determining the angle of repose: The fixed funnel method is used to determine the angle of repose of the particles. A dry and clean funnel is fixed on an iron stand, and the opening of the lowest funnel is 2.5 cm (H) away from the horizontally placed coordinate paper. 2'-FL is poured into the funnel along the funnel wall until the tip of the particle cone formed by the last funnel touches the opening of the funnel. The diameter of the cone (2R) is measured from the coordinate paper. Then tanθ=H / R, and the angle θ is the angle of repose.

[0213] Table 16 Particle Flowability Test

[0214] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A culture medium for fermentation of 2'-fucosylated lactose, characterized in that, Includes seed culture medium, initial culture medium, and supplemental culture medium; The initial culture medium, using water as a solvent, comprises: potassium dihydrogen phosphate 5‰-8‰ m / v, magnesium sulfate heptahydrate 1‰-2‰ m / v, diammonium hydrogen phosphate 2‰-3‰ m / v, citric acid 1‰-2‰ m / v, industrial yeast powder 5‰-6‰ m / v, porcine bone peptone 2‰-3‰ m / v, a compound carbon source 3%-4% m / v, and trace metal solution 8‰-1% m / v; the compound carbon source is composed of glycerol and maltodextrin in a mass ratio of 15-20:1, and the DE value of the maltodextrin is 6-10; The composition of the trace metal liquid is as follows: Na2EDTA 0.8-1.2‰ m / v, zinc sulfate heptahydrate 0.3-0.5‰ m / v, manganese chloride dihydrate 0.3-0.5‰ m / v, cobalt chloride hexahydrate 0.01-0.03‰ m / v, copper sulfate pentahydrate 0.01-0.03‰ m / v, sodium molybdate dihydrate 0.03-0.05‰ m / v, calcium chloride dihydrate 0.4-0.6‰ m / v, ferrous sulfate heptahydrate 0.1-0.3‰ m / v, nickel chloride hexahydrate 0.01-0.03‰ m / v, and boric acid 0.05-0.1‰ m / v; The feed medium includes feed medium one and feed medium two. Feed medium two is a lactose-galactomannan mixture with a mass concentration of 18%-24% m / v, and the mass ratio of lactose to galactomannan is 9-19:

1.

2. The culture medium according to claim 1, characterized in that, The supplemental culture medium comprises, by weight percentage: 20%-24% magnesium sulfate heptahydrate, 5%-7% industrial yeast powder, 40%-55% glycerol, and 2.5%-3.5% porcine bone peptone.

3. The culture medium according to claim 1, characterized in that, The seed culture medium includes shake flask seed culture medium and seed tank culture medium; the shake flask seed culture medium contains 8‰-10‰ m / v peptone, 4‰-6‰ m / v yeast extract, and 8‰-10‰ m / v sodium chloride; the seed tank culture medium contains 8‰-10‰ m / v peptone, 5‰-7‰ m / v corn steep liquor, and 8‰-10‰ m / v sodium chloride, with water as the solvent.

4. A method for producing 2'-FL by fermentation, characterized in that, Using the culture medium described in any one of claims 1-3, with *Escherichia coli* BLCY-019 (accession number CCTCC NO: M 2026303) as the strain, and according to OD... 600 Node segmented control: OD 600 When the concentration is 10-15, adjust the temperature to 22-25℃, the glycerol concentration to 15-20 g / L, and the lactose concentration to 3-6 g / L; OD 600 When OD600 = 50-60, the glycerol concentration is 12-17 g / L, and the lactose concentration is 5-8 g / L; when OD600 = 80-100, the glycerol concentration is 10-15 g / L, and the lactose concentration is 6-9 g / L; 600 When the concentration is 130-150, the glycerol concentration is 7-10 g / L and the lactose concentration is 8-10 g / L.

5. The method according to claim 4, characterized in that, IPTG adds OD in stages. 600 =10-15, add to a final concentration of 0.1-0.2 mM; OD 600 When the concentration is 50-60, 80-100, or 130-150, add more water to bring the final concentration to 0.08-0.1 mM.

6. The method according to claim 4, characterized in that, Segmented control of airflow ratio and stirring speed: OD 600 When the aeration ratio is 10-15, the ventilation ratio is 1.0-1.2 vvm, and the stirring speed is 150-200 rpm; OD 600 When the concentration is 50-150, the aeration ratio is 1.0-1.5 vvm, and the stirring speed is 150-400 rpm.

7. The method according to claim 4, characterized in that, The fermentation broth is sterilized, centrifuged to remove bacteria, finely filtered through a ceramic membrane, decolorized, desalted by electrodialysis and ion exchange, concentrated to a sugar concentration of 72-75%, crystallized by primary evaporation, crystallized by secondary gradient cooling, and dried to obtain the 2'-FL product. The secondary crystallization adopts a gradient cooling method: cooling at 0.5-0.8℃ / h at 40-50℃, cooling at 0.3-0.5℃ / h at 30-40℃, and cooling to 25℃ at 0.2-0.3℃ / h at 25-30℃.

8. The use of the culture medium according to any one of claims 1-3 in the fermentation production of 2'-fucosylated lactose.

9. The use of 2'-fucosylated lactose obtained by fermentation in any of the culture media described in claims 1-3 in the preparation of food, health products, and pharmaceuticals.

Images