Method for improving purity of 6-SL

Abstract

The invention relates to the field of biology, and discloses a method for improving the purity of 6-SL, which comprises the following steps: inactivating fermentation liquor, performing microfiltration to remove thalli, performing enzymolysis and color removal by using lactase, and simultaneously adding divalent metal salt to precipitate impurities and active carbon to remove pigments; separating impurities and concentrating a target product by adopting ultrafiltration and nanofiltration in sequence; further purifying through electrodialysis desalination and ion exchange; and finally, carrying out evaporation concentration, secondary decoloration and spray drying to obtain a high-purity and high-whiteness 6-SL finished product. Through key steps of optimizing electrodialysis pH, ion exchange resin types, regeneration frequency, powdered carbon decoloration conditions and the like, impurities and pigments are effectively removed, the purity (greater than or equal to 80%) and whiteness of the product are improved, meanwhile, the loss rate is remarkably reduced, and the average recovery rate of the product reaches 80% or above. The method is simple and efficient in process flow, has the characteristics of low energy consumption and low pollution, and is suitable for large-scale industrial production of 6-SL.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a method for improving the purity of 6-SL. Background Art

[0002] Human milk oligosaccharides (HMOs) are a class of natural bioactive substances with complex structures and rich functions. They are widely present in human breast milk and are an important component of the healthy development of infants and young children. Scientific research shows that HMOs can block pathogens from adhering to the surface of host cells, promote the proliferation of beneficial intestinal bacteria (such as bifidobacteria), enhance host immunity, strengthen the intestinal barrier, and reduce the occurrence of diarrhea, respiratory infections and allergic diseases. In addition, HMOs have also been shown to promote the brain development of infants and young children, which has important health significance. However, due to the extremely low content of HMOs in other natural sources, the possibility of direct extraction from nature is restricted. For this reason, biosynthesis has become the main way to develop HMOs.

[0003] Among the many components of HMOs, 6-sialyllactose (6-SL) has attracted widespread attention due to its unique structure and function. 6-SL is composed of lactose and N-acetylneuraminic acid (NeuAc) connected by glycosidic bonds. As an important sialic acid derivative, 6-SL not only has the common functions of human milk oligosaccharides, such as regulating intestinal flora and enhancing immunity, but also plays a key role in the development of the nervous system. Studies have found that sialic acid can act on nerve cell membranes and synapses, increase the speed of neuronal signal transmission, and thus enhance memory and learning ability. At the same time, sialic acid also shows good effects in antiviral and prevention of pathogen invasion. Therefore, 6-SL has broad application prospects in the fields of infant formula, health foods and pharmaceuticals.

[0004] Production status of sialic acid and 6-SL:

[0005] At present, the production methods of sialic acid and its derivative 6-SL are mainly divided into the following categories:

[0006] Natural product extraction:

[0007] Sialic acid is widely found in animal milk and certain animal and plant sources, such as milk, eggs and plant mucus. However, the sialic acid content in these sources is low, and the extraction process requires complex separation and purification operations, with high production costs, which is difficult to meet the needs of large-scale industrial production.

[0008] Chemical synthesis:

[0009] Synthesizing sialic acid molecules through chemical reaction pathways was one of the important directions of early development. This method prepares the target product through glycosylation, reduction reaction and multi-step chemical reactions, but its application is greatly limited due to the complex steps, many by-products, high environmental pollution and high production costs.

[0010] Microbial fermentation:

[0011] Microbial fermentation is currently one of the main methods for producing sialic acid and 6-SL. Sialic acid or its polymer polysialic acid (PSA) is synthesized by fermentation using recombinant Escherichia coli (such as K1, K92, K235) and other microbial strains. These microbial strains naturally have enzyme systems for synthesizing sialic acid. Further optimization of fermentation conditions and strain genetic background can significantly increase production. At the same time, polysialic acid can also be obtained by enzymatic or chemical hydrolysis to obtain monomeric sialic acid. However, the subsequent separation and purification of fermentation products remains a bottleneck in the current technological development.

[0012] 6-SL purification technology:

[0013] The purification process of 6-SL is an important link in production. Its purity, whiteness and yield directly determine the quality and market competitiveness of the final product. The existing 6-SL purification technologies mainly include the following:

[0014] Chromatographic separation technology:

[0015] Typical technologies such as CN202410524903.3 use chromatography columns for separation and purification. This method uses a chloride-type strongly basic anion exchange resin as a filler, and uses an acidic mobile phase to elute 6-SL and perform freeze-drying. However, this method has the following problems:

[0016] Generates a large amount of acidic wastewater and waste, causing serious environmental pollution;

[0017] The removal effect of pigments and impurities is limited, resulting in a pale yellow or creamy yellow product color;

[0018] The product loss rate is high and the purity is low, usually not exceeding 80%.

[0019] Multi-resin ion exchange technology:

[0020] For example, the technology described in CN102482313 uses a combination of multiple ion exchange resins to purify 6-SL. This technology uses resins to desalt and adsorb 6-SL, and then uses an acidic solution for elution. However, this technology has the following shortcomings:

[0021] The processing technology is complicated, and the use of multiple resins increases the difficulty and cost of operation;

[0022] The purity and yield of the product are both low, and the resin needs a large amount of acid and alkali solution to be regenerated after repeated use, which has a negative impact on the environment;

[0023] It is difficult to meet the requirements of large-scale industrial production.

[0024] Membrane separation technology:

[0025] Membrane separation technology (such as ultrafiltration and nanofiltration) is a new purification method developed in recent years, which can achieve the separation and concentration of sialic acid or 6-SL to a certain extent. However, due to the limitations of membrane selectivity and retention performance, it is difficult to achieve high-purity 6-SL separation using membrane separation technology alone.

[0026] Problems with existing technologies:

[0027] Through the analysis and summary of the above technologies, the current 6-SL production and purification technology mainly has the following bottlenecks:

[0028] Insufficient purity and whiteness:

[0029] Existing technologies are not very effective in removing impurities and pigments. The purity of the final product is generally less than 80%, and the color is yellowish, which makes it difficult to meet the needs of high-end food or pharmaceutical fields.

[0030] The process is complex and the operation is difficult:

[0031] The existing ion exchange and chromatography separation technologies have complicated process flows, involving multiple adsorption, elution or resin replacement, which increases production costs and technical difficulty.

[0032] Serious environmental pollution:

[0033] Many purification technologies require the use of large amounts of acid and alkali solutions to regenerate resins or adjust pH, resulting in large amounts of wastewater and waste, making it difficult to achieve green and environmentally friendly production.

[0034] Limited economic benefits:

[0035] The product loss rate is high and the yield is low, especially in industrial production. These problems lead to a decline in economic benefits and limit the promotion and application of technology.

[0036] Therefore, the present invention proposes a method for improving the purity of 6-SL to solve the shortcomings of the prior art. Summary of the invention

[0037] In view of the problems of insufficient purity, complex process, serious environmental pollution and limited economic benefits in the purification process of 6-sialyllactose (6-SL) in the prior art, the present invention provides an efficient, environmentally friendly and low-cost 6-SL purification method. By optimizing the multi-step purification process, the purity and whiteness of 6-SL are significantly improved, while the product loss rate and the amount of waste generated are reduced, meeting the needs of industrial large-scale production.

[0038] To achieve the above object, the present invention is implemented by the following technical scheme: a method for improving the purity of 6-SL, the method comprising the following steps:

[0039] S1: removing bacteria, inactivating the fermentation broth, and filtering it through a microfiltration membrane with a pore size of 50 nm to 500 nm to obtain a supernatant;

[0040] S2: enzymatic decolorization, adding 2% lactase by volume to the supernatant obtained in step S1 for enzymatic hydrolysis, adding a divalent metal salt to precipitate anions, wherein the divalent metal salt is calcium chloride, magnesium chloride or ferrous chloride, then adding 2% activated carbon by volume, stirring and mixing, heating to 65° C. for inactivation, and then performing plate and frame filtration;

[0041] S3: ultrafiltration, filtering the liquid obtained in step S2 using an ultrafiltration membrane with a molecular weight cutoff of 3000-10000Da;

[0042] S4: Nanofiltration, the liquid filtered in step S3 is concentrated by nanofiltration, and the molecular weight cutoff range of the nanofiltration membrane is 300-1000Da;

[0043] S5: electrodialysis, adjusting the pH of the concentrate in step S4 to 2 to 4, and performing electrodialysis desalination;

[0044] S6: ion exchange, purifying the liquid after desalination in step S5 by ion exchange, wherein the ion exchange uses a strong acid cation exchange resin and a strong base anion exchange resin;

[0045] S7: Evaporation and concentration: the liquid obtained in step S6 is concentrated by rotary evaporation at 65°C to a sugar content of 30-40;

[0046] S8: secondary decolorization, using powdered carbon to make a thin filter cake of the concentrated solution obtained in step S7 and then filtering and decolorizing with activated carbon;

[0047] S9: spray drying, spray drying the liquid filtered in step S8 to obtain a 6-sialyllactose finished product with a purity of ≥80%.

[0048] Preferably, the fermentation liquid in step S1 is inactivated by heating, the inactivation temperature is 80-100° C., and the inactivation time is 10-20 minutes.

[0049] Preferably, in the step S2:

[0050] After adding lactase, the enzymolysis time is 1-3 hours, and the enzymolysis temperature is 30-50°C;

[0051] The mass of the added divalent metal salt is 1-3% of the liquid weight;

[0052] The amount of activated carbon added is 1-3% of the liquid weight;

[0053] The filtration accuracy after plate and frame filtration is 0.5μm to 5μm.

[0054] Preferably, the ultrafiltration in step S3 uses an ultrafiltration membrane with a molecular weight cutoff of 3000-10000Da, the filtration process temperature is 10-30°C, and the filtration pressure is 0.1-0.4MPa.

[0055] Preferably, the molecular weight cutoff of the nanofiltration membrane in step S4 is 300-1000Da, the concentration of the concentrated liquid is 50-100g / L, the nanofiltration process temperature is 10-30°C, and the filtration pressure is 0.2-0.6MPa.

[0056] Preferably, the electrodialysis conditions in step S5 are:

[0057] Adjust pH to 2-4;

[0058] The conductivity is controlled at 0.5-3ms / cm;

[0059] The desalting time is 30-90 minutes.

[0060] Preferably, the ion exchange resin used in step S6 is:

[0061] Strong acid cation exchange resin, resin particle size is 50-300μm;

[0062] Strongly basic anion exchange resin, with a particle size of 50-300 μm;

[0063] The resin was regenerated after every two column volumes using a 4-6% acidic or alkaline solution.

[0064] Preferably, the rotary evaporation concentration temperature in step S7 is 65° C., the vacuum degree is 0.08-0.1 MPa, and the sugar content of the concentrated liquid is 30-40.

[0065] Preferably, the secondary decolorization in step S8 uses powdered carbon to prepare a thin filter cake, the amount of powdered carbon added is 0.5-2% of the weight of the liquid, and the residual pigment and bacteria are removed by filtering through a thin layer of activated carbon.

[0066] Preferably, the spray drying conditions in step S9 are:

[0067] The inlet air temperature is 120-150℃;

[0068] The air outlet temperature is 80-100℃;

[0069] The purity of 6-SL finished product is ≥80%.

[0070] The present invention provides a method for improving the purity of 6-SL. It has the following beneficial effects:

[0071] 1. The present invention adopts a multi-step coordinated optimization purification technology scheme such as microfiltration, enzymatic decolorization, ultrafiltration, nanofiltration, electrodialysis and ion exchange to achieve efficient removal of impurities, pigments and salts in 6-sialyllactose (6-SL), achieving a technical effect of 6-SL purity ≥ 80% and significantly improved product whiteness. Compared with the problem that the single chromatographic separation technology in the prior art (such as CN202410524903.3) relies on chemical reagent elution and is difficult to completely remove impurities, the present invention combines high-efficiency filtration, nanofiltration and electrodialysis in a multi-stage process, which not only improves the purity and visual perception of the product, but also overcomes the technical bottleneck of yellowish product color and low purity caused by the prior art, and better meets the demand for high-quality 6-SL in the food and pharmaceutical industries.

[0072] 2. The present invention optimizes the operating conditions of each step in the purification process, such as using reasonable pH adjustment (pH2-4) and conductivity control (<3ms / cm) in electrodialysis, combined with targeted ion exchange resins, effectively reducing the loss of products in the desalination and impurity removal process, and achieving the technical effect of an average recovery rate of ≥80%. Compared with the problems of high loss rate and low recovery rate of target products caused by multiple adsorption-elution operations in the prior art (such as CN102482313), the present invention significantly reduces the loss rate and improves the overall recovery rate by reducing unnecessary adsorption operations, optimizing material selection and accurately controlling process parameters, thereby overcoming the shortcomings of the prior art that the economic benefits are limited due to excessive loss.

[0073] 3. The present invention adopts a multi-step coordinated and compact purification process. By rationally integrating microfiltration, enzymatic decolorization and membrane separation technology, the repeatability between process steps is significantly reduced, the simplicity of the process is optimized, and the technical effect of efficient and low-cost production of 6-SL is achieved. Compared with the problems of complex process and high frequency of equipment use caused by relying on multiple ion exchange resins or chromatographic separation technology (such as multi-resin adsorption method) in the prior art, the present invention reduces the types of resins and regeneration frequency by combining high-efficiency filtration with electrodialysis, while reducing the amount of acid and alkali solution used, greatly reducing production costs. This technology solves the shortcomings of the existing process flow, high consumables and energy consumption, and provides an economically feasible solution for the large-scale industrial production of 6-SL.

[0074] 4. The present invention achieves effective control of wastewater and waste material discharge by optimizing solvent selection and reducing the use of acid and alkali solutions in the links of enzymatic decolorization, electrodialysis and ion exchange, and achieves the technical effect of green production. Compared with the problem of serious waste liquid and wastewater pollution caused by the large amount of acid and alkali solutions (such as chromatography and ion exchange) required in the resin regeneration link in the prior art, the present invention improves the ion exchange conditions (such as reasonably controlling the resin regeneration frequency and solution concentration), which not only reduces the consumption of chemical reagents, but also greatly reduces the cost and environmental pressure of wastewater treatment. This green and environmentally friendly process route overcomes the shortcomings of the prior art that are poor in environmental protection and do not conform to the concept of sustainable development, and provides important technical support for achieving clean production. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 This is the experimental physical picture of the control example 16-SL;

[0076] Figure 2 The purity of 6-SL was determined by high performance liquid chromatography (HPLC) in Comparative Example 1;

[0077] Figure 3 This is the experimental physical picture of the control example 26-SL;

[0078] Figure 4 The purity of 6-SL was measured by high performance liquid chromatography in comparison example 2;

[0079] Figure 5 This is the experimental physical picture of the control example 36-SL;

[0080] Figure 6 The purity of 6-SL was determined by high performance liquid chromatography in Comparative Example 3;

[0081] Figure 7 This is a physical picture of the experiment of Example 16-SL;

[0082] Figure 8This is a graph showing the purity of 6-SL measured by high performance liquid chromatography in Example 1;

[0083] Fig. 9 This is a physical picture of the experiment of Example 26-SL;

[0084] Fig.10 This is a graph showing the purity of 6-SL measured by high performance liquid chromatography in Example 2;

[0085] Fig.11 This is a physical picture of the experiment of Example 36-SL;

[0086] Fig.12 This is a graph showing the purity of 6-SL measured by high performance liquid chromatography in Example 3;

[0087] Fig.13 This is a physical picture of the experiment of Example 46-SL;

[0088] Fig.14 This is a graph showing the purity of 6-SL measured by high performance liquid chromatography in Example 4;

[0089] Fig.15 Flow chart of the method for improving the purity of 6-SL. DETAILED DESCRIPTION

[0090] 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.

[0091] Please see attached Figure 1 -Attached Fig.15 :

[0092] Comparison example:

[0093] Comparative Example 1:

[0094] Differences from Example 1:

[0095] The molecular weight cut-off of the ultrafiltration membrane is increased to 10,000 Da.

[0096] Results and Analysis:

[0097] Since macromolecular impurities (such as proteins) were not effectively removed, the purity of the finished product was reduced to 81.2%, and the product was slightly yellow. The change in ultrafiltration conditions directly affected the purification effect of the subsequent process, and the final recovery rate was 80.3%.

[0098] Comparative Example 2:

[0099] Differences from Example 1:

[0100] The molecular weight cut-off of the nanofiltration membrane was increased to 1000Da.

[0101] Results and Analysis:

[0102] The impurity removal efficiency decreased, especially the low molecular weight salt and sugar impurities were not completely removed, the purity of the finished product was only 80.5%, and the recovery rate was 81.2%. The inaccurate nanofiltration retention range was the main problem.

[0103] Comparative Example 3:

[0104] Differences from Example 1:

[0105] Electrodialysis was performed directly for desalination without pH adjustment.

[0106] Results and Analysis:

[0107] The deionization efficiency during the electrodialysis process dropped significantly, the residual salt impurities were high, the finished product purity was only 77.3%, and the recovery rate was 80.8%. The lack of pH adjustment had a significant impact on the electrodialysis efficiency, indicating that the optimization of this step is crucial.

[0108] Example:

[0109] Embodiment 1:

[0110] S1: Remove bacteria

[0111] The fermentation broth was heated to 90°C for 15 minutes for inactivation, and then filtered through a ceramic microfiltration membrane with a pore size of 220 nm to remove bacteria and suspended particles to obtain a clear supernatant.

[0112] Heating inactivation effectively destroys the activity of microorganisms, prevents the bacteria in the fermentation broth from continuing to metabolize, and avoids the decomposition or contamination of the target product in the subsequent steps. The microfiltration membrane can efficiently separate bacteria, suspended particles and other insoluble impurities through precise pore size restriction, ensuring the clarity of the supernatant.

[0113] Ensure the stability and purity of the target product and reduce interference from impurities; reduce the burden of subsequent separation and purification steps and improve process efficiency.

[0114] S2: Enzymatic decolorization

[0115] Lactase (final concentration of 2% of the liquid volume) was added to the supernatant obtained from S1, and enzymatic hydrolysis was performed at 40°C for 2 hours to hydrolyze macromolecular lactose; then 2% calcium chloride by weight of the liquid was added to precipitate anionic impurities, and 2% activated carbon by weight of the liquid was added, and the mixture was stirred and mixed, and then continued to be stirred at 40°C for 45 minutes for decolorization. After the mixture was heated to 65°C to inactivate lactase, the precipitate and pigment were removed by plate and frame filtration (filtration accuracy 0.5 μm).

[0116] Lactase: Hydrolyzes macromolecular sugars such as lactose to prevent them from interfering with subsequent purification steps.

[0117] Divalent metal salts: react with anionic impurities to form insoluble precipitates, further reducing impurities.

[0118] Activated carbon: removes pigments and some organic impurities from liquids through adsorption.

[0119] Significantly improve the clarity and purity of the supernatant, laying the foundation for subsequent separation processes; remove pigments and anionic impurities to ensure that the final product color is close to white.

[0120] S3: Ultrafiltration

[0121] The decolorized filtrate was filtered using an ultrafiltration membrane with a molecular weight cutoff of 8000Da to remove macromolecular impurities (such as proteins, etc.). The filtration temperature was 25°C and the pressure was controlled at 0.3MPa.

[0122] Ultrafiltration removes high molecular weight impurities (such as proteins, polymers, etc.) through the screening effect of the molecular weight cutoff membrane, while the target product 6-SL and small molecular substances can enter the dialysate through membrane filtration.

[0123] Efficiently remove high molecular weight impurities and significantly improve liquid purity; retain target products, reduce product loss and improve recovery rate.

[0124] S4: Nanofiltration

[0125] The ultrafiltrate was concentrated using a nanofiltration membrane with a molecular weight cutoff of 500Da to 80g / L. The filtration temperature was controlled at 20°C and the pressure was 0.5MPa, effectively removing low molecular weight impurities and salts.

[0126] Nanofiltration membranes can separate target products from low molecular weight impurities (such as inorganic salts, small molecule impurities, etc.), while concentrating the liquid volume and increasing the relative concentration of the target product.

[0127] Efficiently separate low molecular weight impurities and salts; reduce the processing burden of subsequent electrodialysis and ion exchange, and improve process efficiency.

[0128] S5: Electrodialysis

[0129] The pH of the nanofiltration concentrate was adjusted to 3, the conductivity was controlled not to exceed 2 ms / cm, the desalination time was 60 minutes, and the electrodialysis effectively removed the remaining inorganic salts and soluble impurities.

[0130] Electrodialysis uses an electric field to cause ions to migrate through a selective membrane to an electrode, removing inorganic salts and charged impurities from the liquid. By optimizing pH conditions, the stability of the target product can be enhanced and the efficiency of electrodialysis can be improved.

[0131] Efficiently remove salt and charged impurities to further improve the purity of the liquid; protect the stability of the target product and reduce product loss.

[0132] S6: Ion Exchange

[0133] Strong acid cation exchange resin (particle size 200 μm) and strong basic anion exchange resin (particle size 150 μm) were used for further purification, and the resin was regenerated with 4% acid-base solution after every two column volumes to ensure complete removal of residual ionic impurities.

[0134] Ion exchange resins further remove trace residual ions and charged impurities through specific adsorption, while avoiding adsorption of target products.

[0135] Improve purification accuracy and ensure complete removal of impurities; significantly improve product whiteness and sensory quality.

[0136] S7: Evaporation and concentration

[0137] The liquid was concentrated to 35 % brix by rotary evaporation at 65°C to further increase the concentration of the solution.

[0138] By low-temperature concentration under vacuum conditions, the liquid volume is reduced, the concentration of the target product is increased, and thermal degradation of the target product by high temperature is avoided.

[0139] Reduce the volume of liquid and improve the efficiency of subsequent decolorization and drying; protect the activity of the target product and reduce losses.

[0140] S8: Secondary decolorization

[0141] Add 1% powdered carbon by weight of the liquid to make a thin layer of filter cake, and filter it through activated carbon for deep decolorization to further remove residual pigments and trace bacterial impurities.

[0142] Powdered carbon further removes pigments, odorous substances and a small amount of microorganisms in the liquid through surface adsorption.

[0143] Improve the sensory quality of the finished product and ensure the color is close to pure white; remove bacterial impurities and improve product stability and safety.

[0144] S9: Spray Drying

[0145] The filtrate is spray-dried under the conditions of an inlet air temperature of 140° C. and an outlet air temperature of 90° C. to obtain a high-purity, high-whiteness 6-sialyllactose crystal finished product.

[0146] Spray drying converts the target product into powder or crystals by rapidly evaporating the water in the liquid, thus avoiding thermal degradation of the target product by high temperature.

[0147] The 6-SL finished product with regular crystal form, good particle size and high stability is formed; the high purity (≥80%) and high recovery rate of the target product are ensured.

[0148] Summary and analysis:

[0149] After multiple steps of optimization, the process successfully achieved a 6-SL purity of 82.8% and a recovery rate of 82%. This was mainly due to the synergistic effect of each step: enzymatic hydrolysis significantly reduced lactose interference, ultrafiltration and nanofiltration graded to remove impurities, and electrodialysis and ion exchange further purified, and the final secondary decolorization ensured the whiteness of the finished product. Compared with the control example, the precise control of nanofiltration and electrodialysis was the key factor in improving purity and recovery.

[0150] Embodiment 2:

[0151] Differences from Example 1:

[0152] The enzymatic hydrolysis temperature was lowered to 35°C and the time was extended to 3 hours to reduce the loss of lactase activity;

[0153] The precipitant was replaced with magnesium chloride (2% of the liquid weight) to improve the precipitation efficiency;

[0154] The ultrafiltration molecular weight cut-off is reduced to 6000Da to further optimize impurity removal;

[0155] The concentration of the nanofiltration concentrated liquid was adjusted to 70 g / L to reduce the subsequent processing pressure.

[0156] Summary and analysis:

[0157] The process optimizes the precipitation and ultrafiltration conditions, effectively improves the impurity removal efficiency, and avoids the loss of lactase activity at high temperature. The purity of the final product is increased to 83.4%, and the recovery rate is 85.9%. Compared with Example 1, the impurity removal is more thorough and the quality of the finished product is higher.

[0158] Embodiment 3:

[0159] Differences from Example 1:

[0160] The electrodialysis pH was adjusted to 2, and the conductivity was more strictly controlled (<1.5ms / cm), which improved the desalination efficiency.

[0161] Summary and analysis:

[0162] By further optimizing the electrodialysis conditions, this example significantly reduced salt residue and product loss, the purity of the finished product reached 84.6%, and the recovery rate increased to 88.7%. Compared with Example 1, the optimized electrodialysis conditions are the key to improving the purity and recovery rate of the finished product.

[0163] Embodiment 4:

[0164] Differences from Example 1:

[0165] The amount of secondary decolorizing powder carbon is increased to 2% of the liquid weight.

[0166] Summary and analysis:

[0167] Increasing the amount of powdered carbon effectively enhanced the decolorization effect, the color of the finished product was closer to pure white, the purity was 83.9%, and the recovery rate was increased to 89.6%. Compared with Example 1, enhanced decolorization was the main reason for improving the sensory quality.

[0168] Comparative test results:

[0169] sample Sensory requirements Residual odor Solubility Purity testing Yield calculation Comparative Example 1 White yellowish No special smell 10min 0.812 0.803 Comparative Example 2 White yellowish No special smell 10min 0.805 0.812 Comparative Example 3 White yellowish No special smell 9min 0.773 0.808 Example 1 light white No special smell 9min 0.828 0.82 Example 2 light white No special smell 10min 0.834 0.859 Example 3 White No special smell 10min 0.846 0.887 Example 4 White No special smell 9.5min 0.839 0.896

[0170] Summarize:

[0171] Compared with the control example, the optimized process flow of the embodiment significantly improved the purity and recovery rate of 6-SL, especially the optimization of ultrafiltration, nanofiltration and electrodialysis conditions was crucial to the product quality and yield. Through the adjustment of process parameters, each embodiment demonstrated the technical effects for different optimization targets, highlighting the advantages of the technical solution of the present invention.

[0172] 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 method for improving the purity of 6-SL, characterized in that: The method comprises the following steps: S1: removing bacteria, inactivating the fermentation broth, and filtering it through a microfiltration membrane with a pore size of 50 nm to 500 nm to obtain a supernatant; S2: enzymatic decolorization, adding 2% lactase by volume to the supernatant obtained in step S1 for enzymatic hydrolysis, adding a divalent metal salt to precipitate anions, wherein the divalent metal salt is calcium chloride, magnesium chloride or ferrous chloride, then adding 2% activated carbon by volume, stirring and mixing, heating to 65° C. for inactivation, and then performing plate and frame filtration; S3: ultrafiltration, filtering the liquid obtained in step S2 using an ultrafiltration membrane with a molecular weight cutoff of 3000-10000Da; S4: Nanofiltration, the liquid filtered in step S3 is concentrated by nanofiltration, and the molecular weight cutoff range of the nanofiltration membrane is 300-1000Da; S5: electrodialysis, adjusting the pH of the concentrate in step S4 to 2 to 4, and performing electrodialysis desalination; S6: ion exchange, purifying the liquid after desalination in step S5 by ion exchange, wherein the ion exchange uses a strong acid cation exchange resin and a strong base anion exchange resin; S7: Evaporation and concentration: the liquid obtained in step S6 is concentrated by rotary evaporation at 65°C to a sugar content of 30-40; S8: secondary decolorization, using powdered carbon to make a thin filter cake of the concentrated solution obtained in step S7 and then filtering and decolorizing with activated carbon; S9: spray drying, spray drying the liquid filtered in step S8 to obtain a 6-sialyllactose finished product with a purity of ≥80%.

2. A method for improving the purity of 6-SL according to claim 1, characterized in that: The fermentation liquid in step S1 is inactivated by heating, the inactivation temperature is 80-100° C., and the inactivation time is 10-20 minutes.

3. A method for improving the purity of 6-SL according to claim 1, characterized in that: In the step S2: After adding lactase, the enzymolysis time is 1-3 hours, and the enzymolysis temperature is 30-50°C; The mass of the added divalent metal salt is 1-3% of the liquid weight; The amount of activated carbon added is 1-3% of the liquid weight; The filtration accuracy after plate and frame filtration is 0.5μm to 5μm.

4. A method for improving the purity of 6-SL according to claim 1, characterized in that: The ultrafiltration in step S3 uses an ultrafiltration membrane with a molecular weight cutoff of 3000-10000Da, the filtration process temperature is 10-30°C, and the filtration pressure is 0.1-0.4MPa.

5. A method for improving the purity of 6-SL according to claim 1, characterized in that: The molecular weight cutoff of the nanofiltration membrane in step S4 is 300-1000Da, the concentration of the concentrated liquid is 50-100g / L, the nanofiltration process temperature is 10-30°C, and the filtration pressure is 0.2-0.6MPa.

6. A method for improving the purity of 6-SL according to claim 1, characterized in that: The electrodialysis conditions in step S5 are: Adjust pH to 2-4; The conductivity is controlled at 0.5-3ms / cm; The desalting time is 30-90 minutes.

7. A method for improving the purity of 6-SL according to claim 1, characterized in that: The ion exchange resin used in the S6 step is: Strong acid cation exchange resin, resin particle size is 50-300μm; Strongly basic anion exchange resin, with a particle size of 50-300 μm; The resin was regenerated after every two column volumes using a 4-6% acidic or alkaline solution.

8. A method for improving the purity of 6-SL according to claim 1, characterized in that: The rotary evaporation concentration temperature in step S7 is 65° C., the vacuum degree is 0.08-0.1 MPa, and the sugar content of the concentrated liquid is 30-40.

9. A method for improving the purity of 6-SL according to claim 1, characterized in that: The secondary decolorization in step S8 uses powdered carbon to prepare a thin filter cake, the amount of powdered carbon added is 0.5-2% of the weight of the liquid, and the residual pigment and bacteria are removed by filtering through a thin layer of activated carbon.

10. A method for improving the purity of 6-SL according to claim 1, characterized in that: The spray drying conditions in step S9 are: The inlet air temperature is 120-150℃; The air outlet temperature is 80-100℃; The purity of 6-SL finished product is ≥80%.

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