Process for producing 3 '-sialic acid lactose through fermentation

Abstract

The invention relates to the field of bioengineering, and discloses a process for producing 3 '-sialic acid lactose by fermentation, which comprises the following steps: solution preparation, strain recovery, fermentation initial culture, first induction, multi-stage material supplementation, second induction and fermentation later control. The method comprises the following steps: optimizing a culture medium formula (taking cane sugar and phosphate as main materials and avoiding a high-cost mixed carbon source), performing staged induction conditions (taking an optical density (OD) value of fermentation liquor as a trigger signal) and accurately controlling a material supplementing speed; according to the method, the final yield of 3 '-SL is remarkably increased and reaches 42-45g per liter of fermentation liquor, and the content of byproducts such as lactose and sialic acid is effectively reduced to be within 1%-2%. Compared with the prior art, by optimizing the metabolism flow direction of the strain and simplifying the fermentation process flow, the production cost is remarkably reduced, meanwhile, the fermentation period is shortened, and the problems of low fermentation efficiency, many byproducts and high process complexity in the prior art are solved.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, in particular to a process for producing 3'-sialyllactose by fermentation. Background Art

[0002] Human milk oligosaccharides (HMOs) are an important component of human breast milk, second only to lactose and lipids, especially in colostrum. Studies have shown that HMOs play an important role in the healthy development and immune formation of infants and young children. According to their structures, HMOs can be divided into fucosylated neutral HMOs, non-fucosylated neutral HMOs and sialylated acidic HMOs.

[0003] 3'-Sialyl lactose (3'-SL) is one of the most abundant sialylated oligosaccharides in HMOs and is also a representative molecule of sialylated acidic HMOs. 3'-SL is composed of N-acetylneuraminic acid (Neu5Ac) and lactose units. It has a simple structure and has many important biological activities, such as promoting the growth of beneficial bacteria in the intestine, inhibiting the adhesion of pathogenic bacteria, anti-inflammatory, antiviral and antibacterial. In addition, 3'-SL can also be used to prevent necrotizing enterocolitis, enhance immune function and promote infant brain development, and has important application value.

[0004] Currently, only high levels of 3'-SL can be detected in human breast milk, while the content in breast milk of other mammals is negligible. The process of extracting and purifying 3'-SL from human milk is complex and costly, and is not suitable for large-scale production. Although chemical synthesis can obtain a certain amount of 3'-SL, it involves multiple steps of protection and deprotection reactions, and the process is complex and the production cost is too high. Although the enzyme synthesis method avoids the complex steps of the chemical method, it is also expensive and limited in large-scale production because it requires purification and immobilization of the enzyme.

[0005] Microbial synthesis is considered to be a safe, economical and easy-to-scale way to produce 3'-SL. Through metabolic engineering and fermentation technology, microorganisms can be converted into efficient 3'-SL synthesis factories. However, the current microbial fermentation process still faces many challenges:

[0006] Current status and shortcomings of existing technology development:

[0007] Multi-strain coupling technology:

[0008] For example, the research team of Jiangnan University built a three-bacteria coupling system and achieved a 3'-SL yield of 44.2 g / L in a 3L bioreactor by optimizing the metabolic network. However, this method relies on the cooperative fermentation of multiple strains, and the culture conditions, fermentation parameters and culture medium components of different strains need to be optimized independently. In addition, the strains are prone to degeneration during the fermentation process, the regulation is complex, and the fermentation cycle is long, which limits its industrial application.

[0009] Gene knockout and expression strategies:

[0010] Patent CN106190938A knocked out 7 genes and overexpressed 5 genes to enhance the metabolic pathway to 3'-SL, and obtained 2-4g / L of 3'-SL. However, this method requires the use of multiple antibiotics to maintain genetic stability, which significantly increases the cost of industrial production.

[0011] Patent CN107904253A knocks out the polysialyltransferase gene (neuS), β-galactosidase gene (l acZ) and N-acetylneuraminic acid metabolism-related gene cluster (nanKETA) in Escherichia coli, and overexpresses the sialyltransferase gene (nst) to synthesize 3'-SL. However, its culture medium formula is complex, including multiple carbon sources (glycerol, glucose, arabinose) and trypsin, resulting in high cost. In addition, the fermentation system does not undergo long-term fermentation, and its final yield is unknown.

[0012] Patent CN114874966A knocked out multiple genes related to substrate degradation and intermediate product diversion based on gene editing technology. Under the conditions of a 5L fermenter, 31.4g / L of 3'-SL can be obtained by batch feeding for 38.5h. However, the culture medium uses glycerol as the main carbon source, which is more expensive than pure glucose as the carbon source.

[0013] Challenges in industrial applications:

[0014] Most current research focuses on optimizing strain metabolic pathways, while there are fewer studies on industrial fermentation and purification strategies. The high cost and high energy consumption of multi-strain systems or complex culture media are particularly prominent in large-scale production. Some foreign companies (such as DSM, DuPont, Jennewei n, etc.) have developed commercial production technologies, but their process details are not disclosed and are difficult to learn from.

[0015] The present invention is directed to the following problems:

[0016] In the prior art, the industrial production of 3'-SL still has the following problems:

[0017] The culture medium formulation is complex and costly;

[0018] The process is complex, the operation is difficult, and the process controllability is poor;

[0019] There are many by-products, which affect the extraction and purification of the target product;

[0020] The long fermentation cycle limits the efficiency of large-scale production.

[0021] Therefore, the present invention proposes a process for producing 3'-sialyllactose by fermentation to solve the deficiencies of the prior art. Summary of the invention

[0022] In view of the problems in the prior art such as complex culture medium formula, high production cost, cumbersome process flow, poor process controllability, more by-products and long fermentation cycle, the present invention provides a process for producing 3'-sialyllactose by fermentation. By optimizing the culture medium composition, accurately controlling the key parameters in the fermentation process and improving the feeding strategy, the yield of 3'-SL is significantly increased, while the production cost is reduced and the process flow is simplified, providing a new solution for industrial large-scale production.

[0023] To achieve the above object, the present invention is implemented by the following technical scheme: a process for producing 3'-sialyllactose by fermentation, the process comprising the following steps:

[0024] Solution preparation: prepare a mixed solution of sucrose and disodium hydrogen phosphate, and a basal culture medium;

[0025] Bacterial strain recovery: Take out the recombinant E. coli from the frozen state, thaw it in a 25-30℃ water bath, transfer it to fresh culture medium, and culture it at 20-30℃ for 3-6 hours;

[0026] Initial stage of fermentation: add the basic culture medium and the revived bacteria into the fermentation tank and start fermentation under the set conditions;

[0027] Control the environmental conditions in the initial stage of fermentation to provide a suitable growth environment for the bacteria and reduce the impact of stress;

[0028] Balance bacterial growth and metabolism, so that the metabolic flow gradually tilts toward the target product 3'-SL;

[0029] Provide efficient biomass for subsequent induction.

[0030] First induction: When the optical density of the fermentation liquid reaches 10-20, add the inducer and lactose solution;

[0031] Feeding: Depending on the fermentation stage, a mixed solution of sucrose and disodium hydrogen phosphate is continuously added to the fermentation tank at different speeds;

[0032] Second induction: 20-30 hours after the first induction, add the inducer and lactose solution again;

[0033] Continue the activity of the target metabolic pathway and increase the final yield of 3'-SL;

[0034] Control the amount of lactose to avoid metabolic inhibition and excessive accumulation of by-products;

[0035] Improve the controllability and stability of fermentation through staged induction.

[0036] Late fermentation: stop feeding and maintain fermentation to the target product concentration.

[0037] Avoid the accumulation of byproducts (such as lactic acid and acetic acid) after stopping feeding and optimize the purity of the target product;

[0038] Reduce the difficulty of downstream extraction and purification, and reduce overall production costs;

[0039] Ensure that the target product 3'-SL reaches the maximum concentration and improve industrial production efficiency.

[0040] Preferably, the solution preparation step comprises:

[0041] Dissolve 67 kg of sucrose and 100-110 g of disodium hydrogen phosphate in 20-30 L of water to prepare a mixed solution of sucrose and disodium hydrogen phosphate;

[0042] Dissolve 2000-3000 g of sucrose, 50-70 g of potassium dihydrogen phosphate, 30-50 g of disodium hydrogen phosphate, 8-12 g of magnesium sulfate heptahydrate, 23 g of triferric citrate and 0.52 g of copper sulfate pentahydrate in 15-25 L of water to prepare a basic culture medium.

[0043] Sucrose is used as the main carbon source in the formula, which is cheap and easy to use, significantly reducing the fermentation cost;

[0044] The precise phosphate ratio provides a stable phosphorus balance for the bacteria and promotes efficient metabolism of the bacteria;

[0045] The trace elements provided by magnesium sulfate heptahydrate and ferric citrate can optimize the activity of bacterial enzyme system and improve the synthesis efficiency of target metabolite 3'-SL.

[0046] Preferably, in the strain resuscitation step, recombinant Escherichia coli is used as the strain, and after the frozen strain is revived, the culture time in the culture medium is 3-6 hours and the culture temperature is 20-30°C.

[0047] Control the recovery conditions to prevent the strain from being affected by environmental stress and improve the activity of the strain;

[0048] Reduce metabolic loss and provide healthy and efficient bacteria for subsequent fermentation;

[0049] Ensure the uniformity of bacterial state and improve the stability of the fermentation process.

[0050] Preferably, the first induction step comprises: when the optical density value of the fermentation liquid reaches 10-20, adding 1.5-2.5 L of 180-220 g / L lactose solution and inducer.

[0051] Control the induction timing (OD value range) to ensure that the bacteria have sufficient growth activity and avoid metabolic burden caused by premature induction;

[0052] Optimize lactose concentration, balance metabolic flow, and increase the yield of target products;

[0053] The inducer dosage is accurate, reducing the inducer cost while maximizing metabolic efficiency.

[0054] Preferably, the feeding step comprises the following stages:

[0055] First stage feeding: after the first induction, add a mixed solution of sucrose and disodium hydrogen phosphate to the fermenter at a rate of 180-220 mL / h for 6-10 hours;

[0056] Second stage feeding: After the first stage feeding is completed, a mixed solution of sucrose and disodium hydrogen phosphate is added at a rate of 250-350 mL / h for 14-18 hours;

[0057] The third stage of feeding: after the second induction, add a mixed solution of sucrose and disodium hydrogen phosphate at a rate of 500-700 mL / h for 28-35 hours.

[0058] Precisely control the feeding rate to avoid excessive carbon source causing metabolic disorders or accumulation of by-products;

[0059] Optimize bacterial metabolism dynamics, improve lactose utilization, and reduce carbon source waste;

[0060] Increase the accumulation of the target product 3'-SL, while reducing by-products such as lactic acid and acetic acid.

[0061] Preferably, in the second induction step, 20-30 hours after the first induction, 1.5-2.5 L of 180-220 g / L lactose solution and the same dose of inducer as in the first induction are added again.

[0062] Preferably, the inducer is isopropyl-β-D-thiogalactoside, and the concentration used is 0.1-1.0 mmol / L.

[0063] Preferably, the volume of the fermentation tank is 80-120 L, the fermentation temperature is 28-32° C., and the pH value of the fermentation liquid is controlled within the range of 6.0-7.5.

[0064] Preferably, the first induction is performed when the optical density value of the fermentation liquid reaches 10-20, and the second induction is performed 20-30 hours after the first induction is completed.

[0065] Through the above process, the present invention achieves the following technical effects:

[0066] High yield: After optimizing the process, the 3'-SL yield can reach 42-45g / L, which is significantly higher than the existing technical level;

[0067] High purity: The impurity content of by-products lactose and sialic acid is significantly reduced to 1%-2%, which improves the purity of the target product;

[0068] Low cost: using sucrose as the main carbon source and optimizing the dosage of inducer, reducing the cost of culture medium and inducer;

[0069] Scalability: Precise control of induction and feeding strategies reduces process complexity and is suitable for industrial large-scale production.

[0070] The invention provides an efficient, economical and green 3'-SL production process, and provides important technical support for the development of functional foods and drugs.

[0071] The present invention provides a process for producing 3'-sialyllactose by fermentation. The process has the following beneficial effects:

[0072] 1. The present invention adopts sucrose or glucose as the main carbon source, and cooperates with common inorganic salts (such as disodium hydrogen phosphate, potassium dihydrogen phosphate, etc.) and trace elements (such as copper sulfate, ferric citrate, etc.) to achieve the technical effect of significantly reducing the cost of the culture medium and simplifying the preparation process. Compared with the high-cost formula of glycerol, a variety of mixed carbon sources (such as glycerol, glucose, arabinose) and complex organic matter (such as tryptone) generally relied on in the prior art, the present invention solves the problems of complex culture medium formula, high cost and insufficient stability of culture medium components, making the culture medium more economical and operable in large-scale production. At the same time, the culture medium components of the present invention are clear and easy to preserve, which helps to ensure the stability of the fermentation process.

[0073] 2. The present invention uses the optical density (OD value) of the fermentation liquid as a reference, accurately induces the expression of the target product twice in the early and middle and late stages of fermentation, and combines the multi-stage feeding technology (supplementing carbon sources and phosphates at different rates according to the metabolic needs of the strain), thereby achieving the technical effect of continuously optimizing the metabolic flow and significantly improving the yield of 3'-sialyllactose (3'-SL). Compared with the technical solutions in the prior art in which the induction conditions are imprecise, the feeding method is single, or the rate control is improper (such as feeding too fast to cause metabolic burden or feeding too slowly to limit strain growth), the present invention effectively solves the shortcomings of poor process controllability, unfocused metabolic flow, and low yield of the target product. In particular, the staged feeding technology further avoids the metabolic inhibition phenomenon caused by excessive accumulation of carbon sources in the culture medium, so that the final yield of 3'-SL is significantly improved.

[0074] 3. The present invention achieves the technical effect of achieving high yield of 3'-sialyllactose in a short fermentation cycle by optimizing fermentation process parameters (including feed rate, induction time, pH value and fermentation temperature, etc.) and adopting high-density fermentation technology. Compared with the prior art, such as the extended cycle caused by the complexity of strain synergy in multi-strain coupling system (such as poor fermentation coordination caused by non-uniform culture conditions of multiple strains), the present invention significantly solves the shortcomings of long production cycle, strain degradation and difficulty in unifying culture conditions. At the same time, by finely regulating the fermentation process, the present invention effectively shortens the fermentation time, thereby improving the overall efficiency and economy of industrial production.

[0075] 4. The present invention reduces the generation of by-products (such as organic acids, etc.) in the fermentation broth by optimizing the feeding strategy and induction conditions, achieving the technical effect of reducing the impurity content and simplifying the extraction and purification process of the target product 3'-sialyllactose. Compared with the technical solution in the prior art that the metabolic bypass is enhanced due to improper feeding rate or excessive metabolic burden of the strain, resulting in a high content of by-products (such as lactic acid or acetic acid) in the fermentation broth, the present invention solves the problem that the target product is difficult to extract, the purification steps are cumbersome and the cost is high. At the same time, the reduction of by-products of the present invention also has environmental advantages, which can effectively reduce the difficulty of handling waste in the fermentation broth and the risk of environmental pollution, and lay the foundation for green production. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 Schematic diagram of the fermentation process of the control example 1, showing the changes of 3'-SL concentration and OD of the fermentation liquid over time;

[0077] Figure 2 Schematic diagram of the fermentation process of the control example 2, showing the changes of 3'-SL concentration and OD of the fermentation liquid over time;

[0078] Figure 3 Schematic diagram of the fermentation process of the control example 3, showing the changes of 3'-SL concentration and OD of the fermentation liquid over time;

[0079] Figure 4 This is a schematic diagram of the fermentation process of Example 13, in which the concentration of '-SL and the OD of the fermentation liquid change with time;

[0080] Figure 5 This is a schematic diagram of the fermentation process of Example 2, showing the changes of 3'-SL concentration and fermentation liquid OD over time;

[0081] Figure 6 This is a schematic diagram of the fermentation process of Example 3, showing the changes of 3'-SL concentration and fermentation liquid OD over time;

[0082] Figure 7 This is a schematic diagram of the fermentation process of Example 43'-SL concentration and fermentation liquid OD changing with time;

[0083] Figure 8 This is a process flow chart for the fermentation production of 3'-sialyllactose. DETAILED DESCRIPTION

[0084] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0085] Please refer to the attached Figure 1 -Attached Figure 8 :

[0086] Comparative Example 1:

[0087] Solution preparation:

[0088] Mixed solution of sucrose and disodium hydrogen phosphate: Take 5.8 kg of sucrose and 92.88 g of disodium hydrogen phosphate and dissolve them in 22.5 L of water.

[0089] Basic culture medium: Take 2500g sucrose, 60g potassium dihydrogen phosphate, 40g disodium hydrogen phosphate, 10g magnesium sulfate heptahydrate, 2.5g triferric citrate, and 1g copper sulfate pentahydrate, and dissolve them in 20L water.

[0090] Bacteria recovery:

[0091] The frozen strain was thawed in a 25°C water bath and then transferred to fresh culture medium and cultured for 3 hours at 25°C.

[0092] Early stage of fermentation:

[0093] The basal culture medium and the resuscitated strain were added into a 100 L fermenter at a fermentation temperature of 28°C and a pH value of 6.5.

[0094] First induction:

[0095] When the optical density (OD value) of the fermentation broth reached 12, 1.5 L of 180 g / L lactose solution and 0.1 mmol / L inducer were added.

[0096] Feeding:

[0097] Phase 1: Sucrose and sodium hydrogen phosphate mixed solution was added at a rate of 180 mL / h for 8 hours;

[0098] Phase 2: Add at a rate of 250 mL / h for 16 hours;

[0099] Phase 3: Add at a rate of 500 mL / h for 28 hours.

[0100] Second induction:

[0101] 24 hours after the first induction, 1.5 L of 180 g / L lactose solution and the same concentration of inducer were added.

[0102] Late fermentation:

[0103] The feeding was stopped, and the fermentation was continued for 72 hours, and the product concentration and impurity content were measured.

[0104] Summarize:

[0105] In control example 1, due to the low lactose dosage and inducer concentration, the carbon source feeding rate was insufficient, resulting in low strain metabolic efficiency, the final 3'-SL concentration was only 28.4g / L, and the contents of impurities lactose and sialic acid were high, 9.8% and 3%, respectively. In addition, the fermentation cycle was long and the process was uneconomical.

[0106] Comparative Example 2:

[0107] Solution preparation:

[0108] Mixed solution of sucrose and disodium hydrogen phosphate: Take 7.0 kg of sucrose and 113 g of disodium hydrogen phosphate and dissolve them in 28 L of water.

[0109] Basic culture medium: Take 2500g sucrose, 60g potassium dihydrogen phosphate, 40g disodium hydrogen phosphate, 10g magnesium sulfate heptahydrate, 2.5g triferric citrate, and 1g copper sulfate pentahydrate, and dissolve them in 20L water.

[0110] Bacteria recovery:

[0111] The frozen strains were thawed in a 30°C water bath and then transferred to fresh culture medium for 6 h.

[0112] Early stage of fermentation:

[0113] The basic culture medium and the resuscitated bacteria were added into a 100 L fermentation tank, and the fermentation temperature was 30°C and the pH value was 6.8.

[0114] First induction:

[0115] When the OD value of the fermentation broth reached 15, 2L of 200g / L lactose solution and 0.5mmol / L inducer were added.

[0116] Feeding:

[0117] Phase 1: Sucrose and sodium hydrogen phosphate mixed solution was added at a rate of 220 mL / h for 8 hours;

[0118] Phase 2: Add at a rate of 330 mL / h for 14 hours;

[0119] Phase III: Supplement at a rate of 600 mL / h for 31 hours.

[0120] Second induction:

[0121] 30 hours after the first induction, 2 L of 200 g / L lactose solution and the same concentration of inducer were added.

[0122] Late fermentation:

[0123] The feeding was stopped and the fermentation was continued for 72 hours, and the product concentration and impurity content were measured.

[0124] Summarize:

[0125] Although the lactose and inducer concentrations were increased in Control Example 2, the high feed rate and extended induction time increased the metabolic burden, increased the acid production level of the strain, and affected the yield of 3'-SL. The final concentration was 32.8 g / L, and the lactose and sialic acid impurity contents were 10.2% and 4.5%, respectively. In addition, high temperature and long induction time may also lead to metabolic instability of the strain.

[0126] Embodiment 1:

[0127] Solution preparation:

[0128] Mixed solution of sucrose and disodium hydrogen phosphate: Take 6.45 kg of sucrose and 103.2 g of disodium hydrogen phosphate and dissolve them in 25 L of water.

[0129] Basic culture medium: Take 2500g sucrose, 60g potassium dihydrogen phosphate, 40g disodium hydrogen phosphate, 10g magnesium sulfate heptahydrate, 2.5g triferric citrate, and 1g copper sulfate pentahydrate, and dissolve them in 20L water.

[0130] Bacteria recovery:

[0131] The frozen strain was thawed in a 25°C water bath and then transferred to fresh culture medium for 4 hours.

[0132] Early stage of fermentation:

[0133] The basal culture medium and the resuscitated strain were added into a 100 L fermenter at a fermentation temperature of 28°C and a pH value of 6.5.

[0134] First induction:

[0135] When the OD value of the fermentation broth reached 15, 2L of 200g / L lactose solution and 0.5mmol / L inducer were added.

[0136] Feeding:

[0137] Phase 1: Sucrose and disodium hydrogen phosphate mixed solution was added at a rate of 200 mL / h for 8 hours;

[0138] Phase 2: Add at a rate of 300 mL / h for 16 hours;

[0139] Phase III: Supplement at a rate of 600 mL / h for 31 hours.

[0140] Second induction:

[0141] 24 hours after the first induction, 2 L of 200 g / L lactose solution and the same concentration of inducer were added.

[0142] Late fermentation:

[0143] The feeding was stopped and the fermentation was continued for 72 hours, and the product concentration and impurity content were measured.

[0144] Summarize:

[0145] Example 1 optimizes the induction time and feed rate to concentrate the metabolic flux of the strain, significantly increases the concentration of 3'-SL to 44.2 g / L, and reduces the impurity contents of lactose and sialic acid to 2% and 0.3%, respectively. Compared with the control example, the process control is precise and the fermentation is more efficient.

[0146] Embodiment 2:

[0147] The same process as in Example 1 was adopted, the fermentation temperature was adjusted to 30° C., and the recovery time was shortened to 3 hours.

[0148] Summarize:

[0149] Since the fermentation temperature increased the metabolic rate of the strain, the short recovery time may reduce the activity of the strain, and the final 3'-SL concentration slightly decreased to 42.6g / L, but the impurity content was still low, with lactose at 1.98% and sialic acid at 0.98%. Compared with Example 1, the result was slightly inferior, indicating that the balance between fermentation temperature and recovery time had a significant impact on efficiency.

[0150] Embodiment 3:

[0151] The same process as in Example 1 was used to adjust the feeding rate:

[0152] The first stage was conducted at 190 mL / h;

[0153] The second stage was conducted at 290 mL / h;

[0154] The third stage was carried out at 580 mL / h.

[0155] Summarize:

[0156] When the feeding rate was slightly lower, the strain's metabolic pressure was reduced, the 3'-SL concentration increased to 45.5 g / L, and the lactose and sialic acid contents were further reduced to 1.76% and 0.67%, respectively. The results showed that appropriately reducing the feeding rate can improve metabolic stability and optimize yield and purity.

[0157] Overall analysis:

[0158] Comparison between the control example and the embodiment:

[0159] The examples are superior to the control examples in terms of culture medium optimization, induction conditions and feeding strategies, and significantly increase the yield of 3'-SL (increase by 42%-62%) and reduce the impurity content.

[0160] Logical analysis between embodiments:

[0161] Example 1 represents the best process combination, with the best balance among feed rate, temperature and induction conditions; Example 2 shows the negative impact of high temperature on metabolism; Example 3 further optimizes metabolic flux by fine-tuning the feed rate to achieve the highest purity.

[0162] These results demonstrate the superiority and creativity of the technical solution of the present invention, and provide a reliable basis for the industrial production of 3'-SL.

[0163] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A process for producing 3'-sialyllactose by fermentation, characterized in that: The process comprises the following steps: Solution preparation: prepare a mixed solution of sucrose and disodium hydrogen phosphate, and a basal culture medium; Bacterial strain recovery: Take out the recombinant E. coli from the frozen state, thaw it in a 25-30℃ water bath, transfer it to fresh culture medium, and culture it at 20-30℃ for 3-6 hours; Initial stage of fermentation: add the basic culture medium and the revived bacteria into the fermentation tank and start fermentation under the set conditions; First induction: When the optical density of the fermentation liquid reaches 10-20, add the inducer and lactose solution; Feeding: Depending on the fermentation stage, a mixed solution of sucrose and disodium hydrogen phosphate is continuously added to the fermentation tank at different speeds; Second induction: 20-30 hours after the first induction, add the inducer and lactose solution again; Late fermentation: stop feeding and maintain fermentation to the target product concentration.

2. A process for producing 3'-sialyllactose by fermentation according to claim 1, characterized in that: The solution preparation step comprises: Dissolve 67 kg of sucrose and 100-110 g of disodium hydrogen phosphate in 20-30 L of water to prepare a mixed solution of sucrose and disodium hydrogen phosphate; Dissolve 2000-3000 g of sucrose, 50-70 g of potassium dihydrogen phosphate, 30-50 g of disodium hydrogen phosphate, 8-12 g of magnesium sulfate heptahydrate, 23 g of triferric citrate and 0.52 g of copper sulfate pentahydrate in 15-25 L of water to prepare a basic culture medium.

3. A process for producing 3'-sialyllactose by fermentation according to claim 1, characterized in that: In the strain recovery step, the recombinant Escherichia coli is used as the strain, and after the frozen strain is recovered, the culture time in the culture medium is 3-6 hours, and the culture temperature is 20-30°C.

4. A process for producing 3'-sialyllactose by fermentation according to claim 1, characterized in that: The first induction step comprises: when the optical density value of the fermentation liquid reaches 10-20, adding 1.5-2.5 L of 180-220 g / L lactose solution and inducer.

5. The process for producing 3'-sialyllactose by fermentation according to claim 1, characterized in that: The feeding step comprises the following stages: First stage feeding: after the first induction, add a mixed solution of sucrose and disodium hydrogen phosphate to the fermenter at a rate of 180-220 mL / h for 6-10 hours; Second stage feeding: After the first stage feeding is completed, a mixed solution of sucrose and disodium hydrogen phosphate is added at a rate of 250-350 mL / h for 14-18 hours; The third stage of feeding: after the second induction, add a mixed solution of sucrose and disodium hydrogen phosphate at a rate of 500-700 mL / h for 28-35 hours.

6. The process for producing 3'-sialyllactose by fermentation according to claim 1, characterized in that: In the second induction step, 20-30 hours after the first induction, 1.5-2.5 L of 180-220 g / L lactose solution and the same dose of inducer as that of the first induction were added again.

7. A process for producing 3'-sialyllactose by fermentation according to claim 1, characterized in that: The inducing agent is isopropyl-β-D-thiogalactoside, and the use concentration is 0.1-1.0 mmol / L.

8. The process for producing 3'-sialyllactose by fermentation according to claim 1, characterized in that: The volume of the fermentation tank is 80-120L, the fermentation temperature is 28-32°C, and the pH value of the fermentation liquid is controlled within the range of 6.0-7.

5.

9. The process for producing 3'-sialyllactose by fermentation according to claim 1, characterized in that: The first induction is performed when the optical density value of the fermentation liquid reaches 10-20, and the second induction is performed 20-30 hours after the first induction is completed.

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