A method for separating and purifying 3-fucosyllactose

Abstract

The present invention belongs to the field of separation and purification technology, and specifically relates to a method for separation and purification of 3-fucosyllactose. The present invention can achieve efficient separation and purification of 3-FL through two crystallization processes by controlling a suitable crystallization temperature, associating the crystallization sugar content with the timing of adding seed crystals twice, and the cooling crystallization rate, so as to obtain a high-quality 3-FL crystalline product. And the method is simple and convenient, more suitable for industrial production, and only simple and conventional pre-treatment steps are required to extract and purify high-quality 3-FL by the method; more widely, the method can be used to extract high-quality 3-FL from a crude 3-fucosyllactose solution prepared by enzymatic method and chemical synthesis method.

Description

Technical Field

[0001] The invention belongs to the technical field of separation and purification, and specifically relates to a method for separating and purifying 3-fucosyllactose. Background Art

[0002] Human milk oligosaccharides (HMOs) are important bioactive components in breast milk and are believed to be beneficial to infant health. They have received increasing attention in recent years. It is reported that breast milk contains about 35-60% fucosylated oligosaccharides, which are of great benefit to both infants and adults. In particular, 2'-fucosyllactose (2'-FL) and 3-fucosyllactose (3-FL), two isomeric trisaccharides composed of L-fucose and D-lactose, are the two main components of fucosylated HMOs. In 2'-FL, L-fucose is linked to the C-2 unit of galactose in lactose, while in 3-FL, L-fucose is linked to the C-3 unit of glucose in lactose. Therefore, the two have many similar chemical properties, but many different physical properties and biological characteristics. As the second most abundant fucosyl HMOs, some studies have confirmed that 3-FL can not only effectively protect the host from certain viruses or bacteria, but also can selectively promote the growth of beneficial microorganisms and inhibit the growth of harmful microorganisms as a prebiotic, thereby establishing a healthy intestinal microbial environment. Moreover, this effect is specific, and 2'-FL or other HMOs do not have the corresponding performance. Therefore, 3-FL has attracted much attention as a nutritional health food ingredient. However, 3-FL is a naturally occurring carbohydrate substance, and its content in breast milk is relatively low, and it does not exist in cow's milk, which to some extent limits the development and application of 3-FL.

[0003] In recent years, with the development of synthetic biology, more and more related technologies have shown that high-titer 3-FL can be produced by modifying different chassis strains, such as Escherichia coli and Saccharomyces cerevisiae, thus laying the foundation for the artificial synthesis of cheap 3-FL. At present, the maximum yield of 3-FL does not exceed 50 g / L, which is still at a relatively low level compared with the more mature biosynthesis of 2'-FL (greater than 120 g / L). Because in the synthetic system of synthetic biology, the difference in the original components of the culture medium in the fermentation broth is small, that is, the concentration of impurities is certain, and the higher the concentration of the product, the greater the ratio of product to impurities in the initial fermentation broth, which is conducive to greatly simplifying the traditional downstream purification process and reducing the purification cost, or obtaining higher quality products under the same process. However, the current maximum yield of 3-FL is still at a low level, which makes the downstream purification of 3-FL more difficult.

[0004] At present, the extraction and purification processes of oligosaccharides commonly use silica gel chromatography, ion exchange chromatography, macroporous resin nonspecific adsorption or simulated fluidized bed, etc., which basically require chromatography technology. It is well known that the low drug loading capacity of chromatography technology will inevitably involve the use of a large amount of fillers to fill the chromatography column once large-scale production is carried out. In addition, the chromatography fillers need to be regenerated after use. A large amount of acid and alkali solution or organic solvent will also be a large expense, resulting in high purification costs and serious environmental pollution. Summary of the invention

[0005] The object of the present invention is to provide a method for separating and purifying 3-fucosyllactose. The method provided by the present invention can not only obtain a crystalline product of 3-fucosyllactose with high purity, but also the crystalline product of 3-fucosyllactose is safe and reliable; at the same time, the cost of the separation and purification method is significantly reduced, it is green, environmentally friendly and low-pollution, and is suitable for industrial application.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a method for separating and purifying 3-fucosyllactose, comprising the following steps:

[0008] The crystallization stock solution is subjected to a primary crystallization to obtain a primary crystallization product, wherein the mass concentration of 3-fucosyllactose in the crystallization stock solution is ≥500 g / L; the primary crystallization product is dissolved in water to obtain a primary crystallization product solution, and the primary crystallization product solution is subjected to recrystallization to obtain a 3-fucosyllactose crystallization product;

[0009] The primary crystallization or recrystallization comprises the following steps:

[0010] Concentrating the crystallization stock solution or the primary crystallization product solution for the first time to obtain a first concentrated solution, wherein the first concentrated solution has a Brix degree of 65-75%;

[0011] performing a first mixing of the first concentrated solution and the first seed crystal to obtain a first mixed solution;

[0012] Concentrating the first mixed solution for the second time to obtain a second concentrated solution, wherein the second concentrated solution has a Brix degree of 78-83%;

[0013] performing a second mixing of the second concentrated solution and the second seed crystal to obtain a second mixed solution;

[0014] The second mixed solution is cooled and crystallized, the initial temperature of the cooling and crystallization is 70-75°C, the terminal temperature of the cooling and crystallization is 50-60°C, and in the process of cooling from the initial temperature to the terminal temperature, the cooling rate in the time interval at least close to the terminal temperature is ≤4°C / h.

[0015] Preferably, the crystallization stock solution is obtained by pre-treating the production raw material solution; the production raw material solution includes one or more of fermentation solution, enzymatic production solution, chemical synthesis production solution and natural latex; the pre-treatment includes desalting, decolorizing and concentrating in sequence;

[0016] The mass concentration of 3-fucosyllactose in the crystallization stock solution is 500-550 g / L.

[0017] Preferably, the cooling crystallization is carried out under stirring, and the stirring speed is 10-20 rpm;

[0018] The cooling crystallization includes sequentially performing a first stage cooling crystallization and a second stage cooling crystallization, wherein the terminal temperature of the first stage cooling crystallization is 63-67°C, the cooling rate of the first stage cooling crystallization is 5-9°C / h, and the cooling rate of the second stage cooling crystallization is 1.75-3.75°C / h.

[0019] Preferably, after the cooling crystallization is completed, the method further comprises: performing heat preservation crystallization under the terminal temperature condition of the cooling crystallization, and the heat preservation crystallization time is 18 to 24 hours.

[0020] Preferably, the cooling crystallization further comprises: performing a heat preservation treatment at the terminal temperature of the first stage cooling crystallization, and the time of the heat preservation treatment is 1 to 2 hours.

[0021] Preferably, the mass of the first seed crystal accounts for 0.1-1w / v% of the volume of the first concentrated solution;

[0022] The mass of the second seed crystal accounts for 0.1-1 w / v % of the volume of the second concentrated solution.

[0023] Preferably, the temperature of the first concentration, the first mixing, and the second concentration are independently 60-70° C.;

[0024] The temperature of the second mixing is 70-75°C.

[0025] Preferably, the first mixing is carried out under stirring conditions, and the stirring speed of the first mixing is 15~30rpm; the first mixing includes the following steps: under the temperature conditions of the first mixing, adding the first seed crystal to the first concentrated solution, and then performing heat-insulating stirring and mixing to obtain the first mixed solution; the heat-insulating stirring and mixing time is 0.5~1h.

[0026] Preferably, the second mixing is carried out under stirring conditions, and the stirring speed of the second mixing is 10~20rpm; the second mixing includes the following steps: heating the second concentrated solution from the second concentrating temperature to the second mixing temperature, and then performing insulation treatment, and then adding the second seed crystal to the second concentrated solution after the insulation treatment to obtain a second mixed solution; the insulation treatment time is 1~2h.

[0027] Preferably, the primary crystallization directly obtains a primary crystal slurry, further comprising: separating the primary crystal slurry from solid and liquid to obtain a primary crystal solid phase product; performing a first crystal washing on the primary crystal solid phase product to obtain the primary crystal product;

[0028] The recrystallization directly obtains a recrystallization slurry, and further comprises: separating the recrystallization slurry into solid and liquid to obtain a recrystallized solid phase product; performing a second crystal washing on the recrystallized solid phase product to obtain the 3-fucosyllactose crystal product;

[0029] The reagent used in the first crystal washing or the second crystal washing is an ethanol aqueous solution.

[0030] The invention provides a separation and purification method of 3-fucosyllactose, comprising the following steps: subjecting the crystallization stock solution to a primary crystallization to obtain a primary crystallization product, wherein the mass concentration of 3-fucosyllactose in the crystallization stock solution is ≥500g / L; dissolving the primary crystallization product in water to obtain a primary crystallization product solution, and recrystallizing the primary crystallization product solution to obtain a 3-fucosyllactose crystallization product; the primary crystallization or recrystallization comprises the following steps: subjecting the crystallization stock solution or the primary crystallization product solution to a first concentration to obtain a first concentrated solution, wherein the Brix degree of the first concentrated solution is 65-7 5%; performing a first mixing of the first concentrated solution and the first seed crystal to obtain a first mixed solution; performing a second concentration of the first mixed solution to obtain a second concentrated solution, wherein the Brix of the second concentrated solution is 78-83%; performing a second mixing of the second concentrated solution and the second seed crystal to obtain a second mixed solution; performing cooling crystallization of the second mixed solution, wherein the initial temperature of the cooling crystallization is 70-75°C, the terminal temperature of the cooling crystallization is 50-60°C, and during the process of cooling from the initial temperature to the terminal temperature, the cooling rate in the time interval at least close to the terminal temperature is ≤4°C / h. Due to the unique physical and chemical properties of 3-FL, it has stronger polarity and greater solubility in water than its isomer 2'-FL. Therefore, 3-FL will show extremely high viscosity at lower temperatures or higher concentrations. On a macroscopic level, the greater the viscosity of the sugar solution, the more difficult it is to form large crystals on a microscopic level (it becomes a paste and difficult to filter), and the larger the metastable zone of its supersaturated solution. Therefore, the supersaturated solution of 3-FL needs to be at a higher temperature and lower sugar content to form larger crystals that can be separated. However, too high a temperature will narrow the metastable zone of the supersaturated solution, thereby rapidly forming more microcrystals, causing the already low supersaturation to drop rapidly, making it difficult for the microcrystals to grow into larger crystals that can be separated. The present invention controls: a suitable crystallization temperature (the initial temperature of the cooling crystallization is 70-75°C), relates the crystallization sugar content to the timing of adding two crystal seeds (the Brix of the first concentrated solution is 65-75% and the Brix of the second concentrated solution is 78-83%), and the cooling crystallization rate, and can achieve efficient separation and purification of 3-FL through two crystallization processes (i.e., primary crystallization and recrystallization) to obtain a high-quality 3-FL crystal product. As can be seen from the results of the embodiment, the purity of the 3-FL crystal product obtained by separation and purification of the present invention is greater than 98%.

[0031] At the same time, the separation and purification method provided by the present invention has the following positive effects compared with the prior art:

[0032] Compared with the existing methods, the present invention is simple and easy to implement, and has strong adaptability. The crystallization separation and purification method of the present invention is specially designed for the physical and chemical properties of 3-FL, and uses crystallization to extract and purify high-quality and high-purity 3-FL products, which is the most suitable process for large-scale industrial production. The embodiments of the present invention prove that the crystallization separation and purification method provided by the present invention only needs to scale the system in proportion, whether from laboratory scale or large-scale production, and almost no other changes are required.

[0033] The present invention is more economical and practical, and has lower purification and extraction costs. The crystallization process used in the present invention only requires investment in a tank and supporting cooling and heating equipment, and both fixed investment and unit cost are significantly lower than those of chromatography processes.

[0034] The present invention is more environmentally friendly and has low pollution. The traditional chromatography process must use a large amount of acid and alkali solution to regenerate the chromatographic filler, or use organic solvents of different concentrations such as ethanol to elute the material, and the waste acid, waste alkali and waste alcohol produced in the purification process will be a big environmental problem. The present invention uses the crystallization separation purification method as the main purification means to directly eliminate the use of acid, alkali and organic solvent eluents, which is environmentally friendly and low-pollution.

[0035] The products produced by the present invention are safer and more reliable. Due to the unique physical and chemical properties of the product 3-FL, the conventional cooling crystallization method can hardly be used to produce 3-FL. However, since its supersaturated solution has high viscosity at low temperatures, its supersaturated solution is also stable at low temperatures and is not easy to crystallize. The related art often uses the method of adding organic solvents, but 3-FL products are often added to infant formula milk powder as a functional factor or used as special medical food, and their safety requirements are extremely high. The present invention does not use any organic solvents or crystal aids in the crystallization process, and only uses a very small amount of food-grade ethanol during washing. In addition, the complete volatilization of ethanol can be guaranteed in the subsequent drying process, which is very safe and reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 is the liquid chromatogram of the crystallization stock solution used in Example 2;

[0037] Figure 2 is a liquid chromatogram of the primary crystallization product obtained in Example 2;

[0038] Figure 3 The liquid chromatogram of the recrystallized product obtained in Example 2. DETAILED DESCRIPTION

[0039] The present invention provides a method for separating and purifying 3-fucosyllactose, comprising the following steps:

[0040] The crystallization stock solution is subjected to a primary crystallization to obtain a primary crystallization product, wherein the mass concentration of 3-fucosyllactose in the crystallization stock solution is ≥500 g / L; the primary crystallization product is dissolved in water to obtain a primary crystallization product solution, and the primary crystallization product solution is subjected to recrystallization to obtain a 3-fucosyllactose crystallization product;

[0041] The primary crystallization or recrystallization comprises the following steps:

[0042] Concentrating the crystallization stock solution or the primary crystallization product solution for the first time to obtain a first concentrated solution, wherein the first concentrated solution has a Brix degree of 65-75%;

[0043] performing a first mixing of the first concentrated solution and the first seed crystal to obtain a first mixed solution;

[0044] Concentrating the first mixed solution for the second time to obtain a second concentrated solution, wherein the second concentrated solution has a Brix degree of 78-83%;

[0045] performing a second mixing of the second concentrated solution and the second seed crystal to obtain a second mixed solution;

[0046] The second mixed solution is cooled and crystallized, the initial temperature of the cooling and crystallization is 70-75°C, the terminal temperature of the cooling and crystallization is 50-60°C, and in the process of cooling from the initial temperature to the terminal temperature, the cooling rate in the time interval at least close to the terminal temperature is ≤4°C / h.

[0047] In the present invention, unless otherwise specified, all preparation raw materials / components are commercially available products well known to those skilled in the art.

[0048] The present invention performs a crystallization on the crystallization stock solution to obtain a crystallization product. In the present invention, the crystallization stock solution is obtained by pre-treating the production raw material solution. The production raw material solution preferably includes one or more of a fermentation liquid, an enzymatic production liquid, a chemical synthesis production liquid and a natural latex, and is specifically a fermentation liquid in the embodiment. The pre-treatment preferably includes desalting, decolorizing and concentrating in sequence. When the production raw material liquid is specifically a fermentation liquid, the pre-treatment preferably includes sterilizing, desalting, decolorizing and concentrating in sequence. There are no special requirements for the specific implementation of the sterilization of the present invention, and filtration or centrifugation well known to those skilled in the art can be used. The filtration can specifically be plate and frame filtration, vacuum filtration or normal pressure filtration. Before the sterilization, the present invention preferably performs flocculation treatment or non-flocculation treatment on the fermentation liquid. When the fermentation liquid is flocculated, the flocculation treatment preferably includes high temperature denaturation, flocculant flocculation or acid-base flocculation. After the fermentation liquid is sterilized, a sterile clarified fermentation liquid is obtained. The present invention desalts the sterile clarified fermentation liquid. The present invention has no special requirements for the specific implementation of the desalination. Conventional desalination methods such as nanofiltration, electrodialysis or ion exchange known to those skilled in the art can be used to remove most of the inorganic salts in the sterile clarified fermentation liquid. After the sterile clarified fermentation liquid is desalted, a sterile low-salt fermentation liquid is obtained. In the present invention, the conductivity of the sterile low-salt fermentation liquid is preferably <1000 μS / cm, more preferably <400 μS / cm. The present invention preferably uses activated carbon to decolorize the sterile low-salt fermentation liquid, the number of decolorization is preferably 1 to 2 times, and the mass of the activated carbon used for each decolorization accounts for 2 to 5% of the volume of the sterile low-salt fermentation liquid. The decolorization preferably includes the following steps: mixing the sterile low-salt fermentation liquid and the activated carbon, stirring decolorization and standing decolorization in turn, the stirring decolorization time is preferably 10 to 30 min, and the standing decolorization time is preferably 1 to 3 h. After the decolorization, a decolored sterile low-salt fermentation liquid is obtained. The present invention preferably concentrates the decolorized sterile low-salt fermentation broth to obtain a crystallization stock solution. The present invention has no special requirements for the specific implementation process of the concentration, and can be carried out by osmotic filtration or vacuum concentration or atmospheric pressure heating concentration well known to those skilled in the art. The mass concentration of 3-fucosyllactose in the crystallization stock solution is preferably 500-550 g / L, and specifically 510 g / L or 530 g / L in the embodiment. The crystallization stock solution also contains lactose and difucosyllactose. The mass concentration of lactose in the crystallization stock solution is preferably 45-65 g / L, and specifically 53 g / L or 64 g / L in the embodiment, and the mass concentration of difucosyllactose is preferably 8-15 g / L, and specifically 8 g / L or 11 g / L in the embodiment.

[0049] The primary crystallization process is described in detail below: In the present invention, the primary crystallization comprises the following steps:

[0050] Concentrating the crystallization stock solution or the primary crystallization product solution for the first time to obtain a first concentrated solution, wherein the first concentrated solution has a Brix degree of 65-75%;

[0051] performing a first mixing of the first concentrated solution and the first seed crystal to obtain a first mixed solution;

[0052] Concentrating the first mixed solution for the second time to obtain a second concentrated solution, wherein the second concentrated solution has a Brix degree of 78-83%;

[0053] performing a second mixing of the second concentrated solution and the second seed crystal to obtain a second mixed solution;

[0054] The second mixed solution is cooled and crystallized, the initial temperature of the cooling and crystallization is 70-75°C, the terminal temperature of the cooling and crystallization is 50-60°C, and in the process of cooling from the initial temperature to the terminal temperature, the cooling rate in the time interval at least close to the terminal temperature is ≤4°C / h.

[0055] In the present invention, during the primary crystallization process: the temperature of the first concentration is preferably 60-70°C, and specifically 65°C in the embodiment. The Brix of the first concentrated solution is preferably 71-73%. The first concentration is preferably vacuum concentration, and the first concentration is performed under stirring, and the stirring speed is preferably 10-100 rpm. After obtaining the first concentrated solution, the first concentrated solution and the first seed crystal are first mixed to obtain a first mixed solution. In the present invention, the first mixing is performed under stirring, and the stirring speed of the first mixing is preferably 15-30 rpm, and specifically 15 rpm, 18 rpm, 20 rpm or 25 rpm in the embodiment. The first mixing preferably includes the following steps: under the temperature condition of the first mixing, the first seed crystal is added to the first concentrated solution, and then the mixture is stirred and mixed under heat preservation to obtain the first mixed solution. The temperature of the first mixing is preferably 60-70°C, and specifically 65°C in the embodiment. The first seed crystal is a 3-fucosyllactose crystal product. The mesh number of the first seed crystal is preferably ≥100 mesh, and more preferably ≥200 mesh. The mass of the first seed crystal accounts for a percentage of the volume of the first concentrated solution of preferably 0.1-1w / v%, preferably 0.5-0.95w / v%. In the present invention, the first seed crystal is preferably added once or added in batches multiple times. In industrial applications, when the amount of the first seed crystal is large, the present invention preferably adds the first seed crystal in batches multiple times, and the total time of the multiple additions in batches is ≤10min. The time for the heat preservation and stirring mixing is preferably 0.5-1h, and the speed of the heat preservation and stirring mixing is preferably 15-30 rpm, specifically 15 rpm, 18 rpm, 20 rpm or 25 rpm in the embodiment. The temperature of the second concentration is preferably 60-70°C, and specifically 65°C in the embodiment. The second concentration is preferably vacuum concentration, and the second concentration is carried out under stirring, and the stirring speed is preferably 10-100 rpm. The Brix degree of the second concentrated solution is preferably 80-83%.

[0056] After obtaining the second concentrated solution, the present invention performs a second mixing of the second concentrated solution and the second seed crystal to obtain a second mixed solution. The second mixing is preferably performed under stirring, and the speed of the second mixing stirring is preferably 10-20 rpm, and specifically 15 rpm, 20 rpm or 12 rpm in the embodiment. The second mixing preferably includes the following steps: heating the second concentrated solution from the temperature of the second concentration to the temperature of the second mixing, and then performing a heat preservation treatment, and then adding the second seed crystal to the second concentrated solution after the heat preservation treatment to obtain a second mixed solution; the heat preservation treatment time is preferably 1-2h. The temperature of the second mixing is preferably 70-75°C, and specifically 72°C in the embodiment. The second seed crystal is a 3-fucosyllactose crystal product. The mesh number of the second seed crystal is preferably ≥100 mesh, and more preferably ≥200 mesh. The mass of the second seed crystal is preferably 0.1-1w / v% of the volume of the second concentrated solution, and more preferably 0.2-1w / v%. In the present invention, the second seed crystal is preferably added once or in batches for multiple times. In industrial applications, when the amount of the second seed crystal is large, the present invention preferably adds the second seed crystal in batches for multiple times, and the total time of the batches for multiple additions is ≤10min.

[0057] After obtaining the second mixed solution, the present invention cools and crystallizes the second mixed solution to obtain a primary crystallization product. In the present invention, the cooling crystallization is carried out under stirring, and the stirring speed is preferably 10-20 rpm, and in the embodiment, it is specifically 15 rpm, 20 rpm or 12 rpm. The initial temperature of the cooling crystallization is preferably 72°C, and the terminal temperature of the cooling crystallization is preferably 53-56°C. The cooling crystallization includes sequentially performing the first stage cooling crystallization and the second stage cooling crystallization, and the terminal temperature of the first stage cooling crystallization is preferably 63-67°C, and in the embodiment, it is specifically 65°C. The cooling rate of the first stage cooling crystallization is preferably 5-9°C / h, and in the embodiment, it is specifically 5-7°C / h. The cooling rate of the second stage cooling crystallization is preferably 1.75-3°C / h, and more preferably 1.8-2.4°C / h. The cooling crystallization preferably also includes: heat preservation treatment at the terminal temperature of the first stage cooling crystallization, and the time of the heat preservation treatment is preferably 1-2h. After the cooling crystallization is completed, the present invention preferably further comprises: performing heat preservation crystallization under the terminal temperature condition of the cooling crystallization, and the heat preservation crystallization time is preferably 18 to 24 hours.

[0058] In a specific embodiment of the present invention, the primary crystallization comprises the following steps: the crystallization stock solution is first concentrated at 60-65°C to obtain a first concentrated solution, the first concentrated solution has a Brix of 71-73%; the first concentrated solution and the first seed crystal are mixed, and then the mixture is stirred for 0.5-1h, and then the second concentrated solution is continued at 60-65°C to obtain a second concentrated solution, the second concentrated solution has a Brix of 80-83%, and in the embodiment, it is 82% or 83%; the second concentrated solution is heated to 70-72°C and kept warm for 1h to obtain a first warm solution, the first warm solution and the second seed crystal are mixed and then cooled to 65°C within 1h and kept warm for 1h to obtain a second warm solution, the second warm solution is cooled to 53-56°C within 5h, and then kept warm for crystallization at 53-56°C for 18-24h to obtain the primary crystal product.

[0059] In the present invention, the primary crystallization directly obtains a primary crystal slurry. The present invention preferably further comprises: separating the primary crystal slurry from solid and liquid to obtain a primary crystal solid phase product; and performing a first crystal washing on the primary crystal solid phase product to obtain the primary crystal product. The solid-liquid separation is preferably centrifugal separation. In a specific embodiment of the present invention, the acceleration of the centrifugal separation is preferably 6000 g and the time is 10 min. The reagent used for the first crystal washing is preferably an ethanol aqueous solution. The mass percentage of ethanol in the ethanol aqueous solution is preferably 80-85%, and the number of the first crystal washing is preferably 1-2 times. The specific implementation of the crystal washing is preferably centrifugal washing. After the first crystal washing, the present invention preferably further comprises drying the obtained crystal washing product to obtain the primary crystal product. The present invention has no special requirements for the specific implementation of the drying. It can be vacuum drying or hot air drying or re-dissolving in pure water and spray drying, which are well known to those skilled in the art. In a specific embodiment of the present invention, the specific implementation of the drying is preferably vacuum drying, and the temperature of the vacuum drying is preferably 45-50°C, and the vacuum degree is 200 mbar.

[0060] In the present invention, the purity of the primary crystallization product is greater than 90%, specifically 93.3%, 91.1%, and 90.2% in the embodiments. The primary crystallization product also contains lactose and bifucosyllactose. The mass content of lactose in the primary crystallization product is 3-5%, specifically 3.9%, 4.3%, and 4.6% in the embodiments; the mass content of bifucosyllactose is 0.3-0.5%, specifically 0.3%, 0.4%, and 0.5% in the embodiments.

[0061] After obtaining the primary crystallization product, the present invention dissolves the primary crystallization product in water to obtain a primary crystallization product solution. The present invention has no special requirements on the mass concentration of the primary crystallization product in the primary crystallization product solution, as long as the primary crystallization product is completely dissolved.

[0062] After obtaining the primary crystallization product solution, the present invention recrystallizes the primary crystallization product solution to obtain a 3-fucosyllactose crystal product.

[0063] The recrystallization process is described in detail below: In the present invention, the recrystallization comprises the following steps:

[0064] Concentrating the crystallization stock solution or the primary crystallization product solution for the first time to obtain a first concentrated solution, wherein the first concentrated solution has a Brix degree of 65-75%;

[0065] performing a first mixing of the first concentrated solution and the first seed crystal to obtain a first mixed solution;

[0066] Concentrating the first mixed solution for the second time to obtain a second concentrated solution, wherein the second concentrated solution has a Brix degree of 78-83%;

[0067] performing a second mixing of the second concentrated solution and the second seed crystal to obtain a second mixed solution;

[0068] The second mixed solution is cooled and crystallized, the initial temperature of the cooling and crystallization is 70-75°C, the terminal temperature of the cooling and crystallization is 50-60°C, and in the process of cooling from the initial temperature to the terminal temperature, the cooling rate in the time interval at least close to the terminal temperature is ≤4°C / h.

[0069] In the present invention, during the recrystallization process: the temperature of the first concentration is preferably 60-70°C, and specifically 65°C in the embodiment. The Brix of the first concentrated solution is preferably 69-71%. After obtaining the first concentrated solution, the present invention performs a first mixing of the first concentrated solution and the first seed crystal to obtain a first mixed solution. In the present invention, the first mixing is performed under stirring conditions, and the speed of the first mixing stirring is preferably 15-30 rpm, and specifically 15 rpm, 18 rpm, 20 rpm or 25 rpm in the embodiment. The first mixing preferably includes the following steps: under the temperature condition of the first mixing, the first seed crystal is added to the first concentrated solution, and then the mixture is stirred and mixed under heat preservation to obtain the first mixed solution. The temperature of the first mixing is preferably 60-70°C, and specifically 65°C in the embodiment. The first seed crystal is a 3-fucosyllactose crystal product. The mesh number of the first seed crystal is preferably ≥100 mesh, and more preferably ≥200 mesh. The mass of the first seed crystal accounts for a percentage of the volume of the first concentrated solution of preferably 0.1-1%, and more preferably 0.2-1w / v%. In the present invention, the first seed is preferably added once or in batches for multiple times. In industrial applications, when the amount of the first seed is large, the present invention preferably adds the first seed in batches for multiple times, and the total time of the multiple additions in batches is ≤10min. The time for the heat preservation and stirring is preferably 0.5~1h, and the speed of the heat preservation and stirring is preferably 15~30 rpm, and specifically 15rpm, 18 rpm, 20 rpm or 25 rpm in the embodiment. The temperature of the second concentration is preferably 60~70℃, and specifically 65℃ in the embodiment. The second concentration is preferably vacuum concentration, and the second concentration is carried out under stirring, and the stirring speed is preferably 10~100 rpm. The Brix degree of the second concentrate is preferably 79~82%.

[0070] After obtaining the second concentrated solution, the present invention performs a second mixing of the second concentrated solution and the second seed crystal to obtain a second mixed solution. The second mixing is preferably performed under stirring, and the speed of the second mixing stirring is preferably 10-20 rpm, and specifically 15 rpm, 20 rpm or 12 rpm in the embodiment. The second mixing preferably includes the following steps: heating the second concentrated solution from the temperature of the second concentration to the temperature of the second mixing, and then performing a heat preservation treatment, and then adding the second seed crystal to the second concentrated solution after the heat preservation treatment to obtain a second mixed solution; the heat preservation treatment time is preferably 1-2h. The temperature of the second mixing is preferably 70-75°C, and specifically 70°C in the embodiment. The second seed crystal is a 3-fucosyllactose crystal product. The mesh number of the second seed crystal is preferably ≥100 mesh, and more preferably ≥200 mesh. The mass of the second seed crystal is preferably 0.1-1% of the volume of the second concentrated solution, and more preferably 0.2-1w / v%. In the present invention, the second seed crystal is preferably added once or in batches for multiple times. In industrial applications, when the amount of the first seed crystal is large, the present invention preferably adds the second seed crystal in batches for multiple times, and the total time of the batches for multiple additions is ≤10min.

[0071] After obtaining the second mixed solution, the present invention cools and crystallizes the second mixed solution to obtain a 3-fucosyllactose crystal product. In the present invention, the cooling crystallization is carried out under stirring, and the stirring speed is preferably 10-20 rpm, and in the embodiment, it is specifically 15 rpm, 20 rpm or 12 rpm. The initial temperature of the cooling crystallization is preferably 70°C, and the terminal temperature of the cooling crystallization is preferably 50-53°C. The cooling crystallization includes sequentially performing the first stage cooling crystallization and the second stage cooling crystallization, and the terminal temperature of the first stage cooling crystallization is preferably 63-67°C, and in the embodiment, it is specifically 65°C. The cooling rate of the first stage cooling crystallization is preferably 5-9°C / h, and in the embodiment, it is specifically 5-7°C / h. The cooling rate of the second stage cooling crystallization is 3-3.75°C / h. The cooling crystallization also preferably includes: heat preservation treatment at the terminal temperature of the first stage cooling crystallization, and the time of the heat preservation treatment is preferably 1-2h. After the cooling crystallization is completed, the present invention preferably further comprises: performing heat preservation crystallization under the terminal temperature condition of the cooling crystallization, and the heat preservation crystallization time is preferably 18 to 24 hours.

[0072] In a specific embodiment of the present invention, the recrystallization comprises the following steps: performing a first concentration on the primary crystallization product solution at 60-65°C to obtain a first concentrated solution, wherein the first concentrated solution has a Brix of 69-71%; mixing the first concentrated solution and the first seed crystal, and then performing a second concentration at 60-65°C to obtain a second concentrated solution, wherein the Brix of the second concentrated solution is 79-82%; heating the second concentrated solution to 70°C and performing the heat preservation for 1 hour to obtain a first heat preservation solution, mixing the first heat preservation solution and the second seed crystal, and then cooling the mixture to 65°C within 1 hour and performing the heat preservation for 1 hour to obtain a second heat preservation solution, cooling the second heat preservation solution to 50-53°C within 4 hours, and then performing heat preservation crystallization at 50-53°C for 18-24 hours to obtain the 3-fucosyllactose crystal product.

[0073] In the present invention, the recrystallization directly obtains a recrystallization slurry, and the present invention preferably further comprises: separating the recrystallization slurry from solid and liquid to obtain a recrystallized solid phase product; and performing a second crystal washing on the recrystallized solid phase product to obtain the 3-fucosyllactose crystalline product. The solid-liquid separation is preferably centrifugal separation, and the reagent used for the second crystal washing is preferably an ethanol aqueous solution. The mass percentage of ethanol in the ethanol aqueous solution is preferably 85-90%, and the number of the second crystal washing is preferably 1-2 times. After the second crystal washing, the present invention preferably further comprises drying the obtained crystal washing product to obtain the 3-fucosyllactose crystalline product. The present invention has no special requirements for the specific implementation of the drying, and vacuum drying or hot air drying or re-dissolving in pure water and spray drying, which are well known to those skilled in the art, can be used.

[0074] In the present invention, the purity of the 3-fucosyllactose crystalline product is greater than 98%, specifically 98.6% or 98.4% in the embodiment, and the 3-fucosyllactose crystalline product also contains a small amount of impurities, including lactose and difucosyllactose, wherein the mass content of lactose is ≤1.4%, and the mass content of difucosyllactose is ≤0.1%.

[0075] The present invention preferably uses the 3-fucosyllactose crystalline product obtained by recrystallization as the seed crystal in the above-mentioned primary crystal or recrystallization process. When used as the first seed crystal or the second seed crystal, the present invention preferably grinds the 3-fucosyllactose crystalline product, and the grinding is preferably wet grinding, and ethanol is preferably used as the grinding medium during the grinding. The mesh number of the first seed crystal and the second seed crystal is ≥200 mesh. The present invention has no special requirements for the specific implementation of the grinding.

[0076] In order to further illustrate the present invention, the technical solutions provided by the present invention are described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0077] In the following examples: 3-fucosyllactose, lactose, difucosyllactose and other sugars were quantitatively determined by high performance liquid chromatography, the instrument model was Waters Binary HPLC Pump 1525 - Detector 2414 / 2489, the chromatographic column was Phenomenex Rezex ROA-Organic Acid H+ (8%), and the 3-fucosyllactose standard was 98% standard of Japan Glycarbo.

[0078] In the following examples: the purity of 3-fucosyllactose is the percentage of the mass of 3-fucosyllactose in the feed solution or product to the mass of all dry matter.

[0079] In the following examples: the sugar content of the crystallization solution is determined by a handheld digital refractometer, the instrument model is ATAGO PAL-2, and the meaning is the percentage of the mass of the total sugar in the solution to the total mass of the solution.

[0080] In the following examples: In the following examples 2 and 3, the pre-treatment method for obtaining the 3-FL crystal stock solution is specifically the following steps:

[0081] The bacteria in the fermentation liquid are removed from the fermentation liquid at the end of fermentation to obtain a sterile clarified fermentation liquid; the method for obtaining the sterile clarified fermentation liquid can use plate and frame filtration, vacuum filtration, normal pressure filtration or centrifugation. Before removing the bacteria in the fermentation liquid, the fermentation liquid can be flocculated or not flocculated by high temperature denaturation, flocculant flocculation or acid-base flocculation to facilitate subsequent filtration to obtain a sterile clarified fermentation liquid.

[0082] After obtaining the sterile clarified fermentation broth, most of the inorganic salts in the sterile clarified fermentation broth are removed by conventional desalination processes such as nanofiltration, electrodialysis or ion exchange to obtain a sterile low-salt fermentation broth; the conductivity of the sterile low-salt fermentation broth obtained in the embodiment is less than 1400 μS / cm.

[0083] After obtaining the sterile low-salt fermentation broth, the sterile low-salt fermentation broth was decolorized with activated carbon in the embodiment. The activated carbon decolorization was performed up to 2 times according to the color quality of the sterile low-salt fermentation broth. The mass of the activated carbon added for each decolorization was 2-5% of the volume of the sterile low-salt fermentation broth. After adding the activated carbon to the sterile low-salt fermentation broth, the mixture was stirred for 10-30 min and then allowed to stand for 1-3 h to complete the decolorization.

[0084] After obtaining the decolorized sterile low-salt fermentation broth, the 3-FL in the decolorized sterile low-salt fermentation broth is finally concentrated to 500-550 g / L by reverse osmosis filtration or vacuum concentration or normal pressure heating concentration to obtain a crystallization stock solution.

[0085] In the following embodiments: the added seed crystals can be obtained by grinding the 3-FL recrystallized product obtained from the previous crystallization, and the grinding can be performed by dry grinding in a mortar or by grinding in a ball mill with 95% ethanol added as a solvent to a size of more than 200 mesh.

[0086] In the examples, the recrystallized product can be dried by vacuum drying or hot air drying to obtain the final product, or redissolved in pure water and spray-dried to obtain the final product.

[0087] Example 1

[0088] Determination of the solubility curve of 3-FL in water:

[0089] Take some high-quality (purity greater than 98%) 3-FL dried overnight at 105°C, weigh 150 g for the first time and dissolve it in a 250 mL sealed conical flask filled with 100 mL pure water, then place the conical flask in a constant temperature water bath with magnetic stirring at 25°C, 40°C, 55°C, 70°C and 85°C, stir continuously at 200 rpm, then slowly add dry 3-FL to the flask, 1 g each time, continue to add after it is completely dissolved, until it is insoluble within 2 hours, record the mass of 3-FL added up to this moment, repeat the experiment three times, and take the average value as the solubility of 3-FL in water at this temperature. The experimental results are shown in Table 1:

[0090] Table 1 Solubility of 3-FL in water

[0091]

[0092] Example 2

[0093] (1) Small-scale primary crystallization of 3-FL

[0094] After pretreatment, 580 mL of 3-FL crystallization stock solution was obtained, in which the concentration of 3-FL was 468 g / L, the concentration of lactose was 70 g / L, and the concentration of difucosyllactose was 6 g / L (the liquid chromatogram of the crystallization stock solution is shown in Figure 1The liquid chromatography detection data of the crystallization stock solution are shown in Table 2). The crystallization stock solution was placed in a vacuum stirring kettle at 65°C and concentrated to 73% Brix. At this time, the solution volume was 270 mL, and 2.5 g of seed powder was added (the first time the seed was added). When the seed was added for the first time, low-speed stirring was maintained at 25 rpm. After the first seed was added, low-speed stirring was continued at 25 rpm for 0.5 h, and then the concentration was continued to obtain a concentrated solution with a sugar content of 82% Brix, and then the temperature was raised to 72°C. After maintaining for 1 h, 2.5 g of seed powder was added (the second seed was added, and the volume change during the second concentration process was negligible, that is, the volume of the first concentrated solution was basically the same as the volume of the second concentrated solution). When the seed was added for the second time, low-speed stirring was maintained at 15 rpm. After the second seed was added, the temperature was slowly lowered to 65°C within 1 h. After low-speed constant-temperature stirring at 15 rpm at 65°C for 1 h, the temperature was slowly lowered to 56°C for 5 h, and crystallized at constant temperature for 18 h (the stirring speed of the entire process from the second seed addition to the end of constant-temperature crystallization was 15 rpm), and centrifuged at 6000 g for 10 min, remove the upper layer of liquid, add 15 mL of 85% ethanol aqueous solution to wash the crystals, centrifuge at 2000 g for 10 min, remove the upper layer of washing liquid, add 15 mL of 85% ethanol to wash the crystals again, centrifuge and dry in a vacuum at 45°C, 200 mbar overnight to obtain a primary crystal product.

[0095] After testing, the obtained primary crystal product was about 25.5 g, the single recovery rate was 9.4%, the purity was 93.3%, and it contained 3.9% lactose and 0.3% difucosyllactose. Figure 2 Table 3 is the liquid chromatography data of the primary crystallization product obtained in this example.

[0096] Table 2 Liquid chromatography test data of the crystallization stock solution in Example 2

[0097]

[0098] Table 3 Liquid chromatography test data of the primary crystal product prepared in Example 2

[0099]

[0100] (2) Small-scale recrystallization of 3-FL

[0101] Take 25.5g of the primary crystallization product, add 19mL of purified water to completely dissolve the sample, and measure the sugar content to be about 59.0% Brix. Concentrate to 71% Brix at 65℃ in a vacuum stirring kettle, add 0.4g of seed powder (the first time to add seed), maintain low-speed stirring at 25 rpm when adding seed for the first time, and continue to maintain low-speed stirring at 25 rpm for 0.5h after the first seed is added, and then continue to concentrate to a sugar content of 82% Brix in the concentrate, then heat to 70℃, keep for 1h, and then add 0.4g of powder seed (the second time to add seed), maintain low-speed stirring at 20 rpm when adding seed, and immediately after the second seed is added, slowly cool to 65℃ within 1h, keep warm at 20 rpm for 1h, then slowly cool to 53℃ for 4h, and crystallize at constant temperature for 18h (the stirring speed is 20 rpm from the second time to the end of constant temperature crystallization), centrifuge at 6000g for 10min, remove the upper layer of liquid, and add 3mL The crystals were washed with 90% ethanol aqueous solution, centrifuged at 2000 g for 10 min, the upper layer of the washing solution was removed, and 3 mL of 90% ethanol was added to repeat the washing once. After centrifugation, the recrystallized product was obtained by vacuum drying at 45°C and 200 mbar overnight.

[0102] After testing, the obtained recrystallized product was about 4.6 g, the single recovery rate was 18.0%, the purity was 98.6%, and it contained 1.4% lactose. The liquid chromatogram of the recrystallized product obtained in this example is as follows: Figure 3 Table 4 is the liquid chromatography detection data of the recrystallized product obtained in this example.

[0103] Table 4 Liquid chromatography test data of the recrystallized product prepared in Example 2

[0104]

[0105] Example 3

[0106] (1) Large-scale primary crystallization of 3-FL

[0107] After pretreatment, 48.6 L of 3-FL concentrate was obtained, in which the concentration of 3-FL was 530 g / L, the concentration of lactose was 64 g / L, and the concentration of difucosyllactose was 11 g / L. The concentrate was placed in a vacuum concentration stirring tank at 65°C and concentrated to 72% Brix. 203 g of seed powder (the first seed addition) was added in several times within 10 min. When the seed was added for the first time, low-speed stirring was maintained at 18 rpm. After the first seed addition, low-speed stirring was continued at 25 rpm for 0.5 h. Then, the concentration was continued to obtain a concentrate with a sugar content of 83% Brix. The temperature was then raised to 72°C and maintained for 1 h. 176 g of seed powder (the second seed addition) was added in several times within 10 min. When the seed was added for the second time, low-speed stirring was maintained at 12 rpm. After the second seed addition, the temperature was immediately slowly lowered to 65°C within 1 h. After low-speed constant-temperature stirring at 12 rpm at 65°C for 1 h, the temperature was lowered to 56°C for 5 h, and crystallized at constant temperature for 20 h (the stirring speed was 12 rpm throughout the entire process from the second seed addition to the end of the constant-temperature crystallization). Centrifuged at 4000 g for 30 min, the upper layer of liquid was removed, and 4 L The crystals were washed with 85% ethanol aqueous solution, centrifuged at 1500 g for 30 min, the upper layer of the washing solution was removed, and 4 L of 85% ethanol was added to repeat the washing once. After centrifugation, the crystals were vacuum dried at 45°C and 200 mbar overnight to obtain a primary crystal product.

[0108] After testing, the obtained primary crystallization product was about 6075 g, with a single recovery rate of 23.6% and a purity of 90.2%, which contained 4.6% lactose and 0.5% difucosyllactose.

[0109] (2) Large-scale recrystallization of 3-FL

[0110] 6075 g of the primary crystallization product was taken, 4 L of purified water was added to completely dissolve the sample, and the sugar content was measured to be about 60.1% Brix. It was concentrated to 70.9% Brix at 65°C in a vacuum concentration stirring tank, and 43 g of seed powder was added several times within 10 min (the first addition of seed crystals). When the seed crystals were added for the first time, low-speed stirring was maintained at 20 rpm. After the first seed crystal addition, low-speed stirring was continued at 20 rpm for 0.5 h, and the concentrate was continued to have a sugar content of 81.8% Brix. Then the temperature was raised to 70°C, and after maintaining for 1 h, 37 g of powder seed crystals were added several times within 10 min (the second addition of seed crystals). Low-speed stirring was maintained at 15 rpm when adding seed crystals. After the second seed crystal addition was completed, the temperature was slowly lowered to 65°C for 1 h, and low-speed constant temperature stirring was performed at 15 rpm for 1 h. Then the temperature was lowered to 53°C for 4 h, and constant temperature crystallization was performed for 19 h (the stirring speed of the entire process from the second addition of seed crystals to the end of constant temperature crystallization was 15 rpm), 4000 The mixture was centrifuged at 1500 g for 10 min, the upper layer of the solution was removed, 0.8 L of 90% ethanol aqueous solution was added to wash the crystals, and the mixture was centrifuged at 1500 g for 10 min, the upper layer of the washing solution was removed, and 0.8 L of 90% ethanol was added to wash the crystals again. After centrifugation, the mixture was vacuum dried at 45 °C and 200 mbar overnight to obtain the recrystallized product.

[0111] After testing, the obtained recrystallized product was about 1079 g, with a single recovery rate of 17.8% and a purity of 98.4%, which contained 1.1% lactose and 0.1% difucosyllactose.

[0112] Comparative Example 1: Single variable comparison based on "(1) small-scale primary crystallization 3-FL" in Example 2

[0113] After pretreatment, 580 mL of 3-FL crystallization stock solution was obtained, in which the concentration of 3-FL was 468 g / L, the concentration of lactose was 70 g / L, and the concentration of difucosyllactose was 6 g / L. The crystallization stock solution was placed in a vacuum stirring kettle and concentrated to 78% Brix at 65°C. At this time, the solution volume was 250 mL, and 2 g of seed powder (the first addition of seed crystals) was added. When the seed crystals were added for the first time, low-speed stirring was maintained at 25 rpm. After the first addition of seed crystals, low-speed stirring was continued at 25 rpm for 0.5 h. At this time, a small amount of milky turbidity appeared in the crystallization kettle. The sugar content of the concentrated solution was continued to 82% Brix, and then the temperature was raised to 72°C. After maintaining for 1 h, a large amount of milky turbidity appeared in the crystallization kettle, and 3-FL microcrystals had precipitated in large quantities. 2 g of seed powder (the second addition of seed crystals) was added. When the seed crystals were added for the second time, low-speed stirring was maintained at 15 rpm. Immediately after the second addition of seed crystals, the temperature was slowly lowered to 65°C for 1 h. After low-speed constant-temperature stirring at 15 rpm for 1 h, the temperature was slowly lowered to 56°C for 5 h, and constant-temperature crystallization was performed for 18 h (the stirring speed of the entire process from the second addition of seed crystals to the end of constant-temperature crystallization was 15 rpm). The entire crystallization solution was in a creamy state and could not be centrifuged and filtered, and the crystallization failed.

[0114] Comparative Example 2: Single variable comparison based on "(1) small-scale primary crystallization 3-FL" in Example 2

[0115] After pretreatment, 580 mL of 3-FL crystallization stock solution was obtained, in which the concentration of 3-FL was 468 g / L, the concentration of lactose was 70 g / L, and the concentration of difucosyllactose was 6 g / L. The crystallization stock solution was placed in a vacuum stirring kettle at 65°C and concentrated to 73% Brix. At this time, the solution volume was 270 mL, and 2.5 g of seed powder (the first addition of seed crystals) was added. When the seed crystals were added for the first time, low-speed stirring was maintained at 25 rpm. After the first addition of seed crystals, low-speed stirring was continued at 25 rpm for 0.5 h, and then the concentration was continued to obtain a concentrated solution with a sugar content of 86% Brix. The temperature was then raised to 72°C and maintained for 1 h. Then 2 g of seed powder (the second addition of seed crystals) was added. When the seed crystals were added for the second time, low-speed stirring was maintained at 15 rpm. Immediately after the second addition of seed crystals, the temperature was slowly lowered to 65°C within 1 h. After low-speed constant temperature stirring at 15 rpm at 65°C for 1 h, a large amount of milky turbidity appeared in the crystallization kettle, and a large amount of 3-FL microcrystals had precipitated. Then 2.5 g of seed powder was added, the temperature was slowly lowered to 65°C within 1 h, and low-speed constant temperature stirring at 15 rpm for 1 h was maintained. The temperature was slowly lowered to 56°C within 5 h, and constant temperature crystallization was performed for 18 After h, the entire crystallization liquid became creamy and could not be centrifuged and filtered, and crystallization failed.

[0116] Comparative Example 3: Single variable comparison based on "(1) small-scale primary crystallization 3-FL" in Example 2

[0117] After pretreatment, 580 mL of 3-FL crystallization stock solution was obtained, in which the concentration of 3-FL was 468 g / L, the concentration of lactose was 70 g / L, and the concentration of difucosyllactose was 6 g / L. The crystallization stock solution was placed in a vacuum stirring kettle at 65°C and concentrated to 73% Brix. The stirring was maintained at 25 rpm for 0.5h. The sugar content of the concentrated solution was continued to be 82% Brix. The temperature was then raised to 72°C and maintained for 1h. Then 5 g of seed powder (adding seeds) was added. The stirring was maintained at 15 rpm when adding seeds. After the second seed addition, the temperature was slowly lowered to 65°C within 1h. After low-speed constant temperature stirring at 15 rpm at 65°C for 1h, the temperature was slowly lowered to 56°C within 5h. Constant temperature crystallization was performed for 18h (the stirring speed was 15 rpm from the beginning of seed addition to the end of constant temperature crystallization). The feed solution was centrifuged at 6000 g for 10 min. The feed solution was stratified. The top feed solution was relatively clear, while the middle feed solution was emulsified. Some crystals were precipitated at the bottom, indicating that the size distribution of 3-FL crystals in the feed solution was wide, microcrystals, small crystals and larger crystals existed at the same time, and the crystallization quality was poor.

[0118] Comparative Example 4: Single variable comparison based on "(1) small-scale primary crystallization 3-FL" in Example 2

[0119] After pretreatment, 580 mL of 3-FL crystallization stock solution was obtained, in which the concentration of 3-FL was 468 g / L, the concentration of lactose was 70 g / L, and the concentration of difucosyllactose was 6 g / L. The crystallization stock solution was placed in a vacuum stirring kettle at 65°C and concentrated to 73% Brix. At this time, the solution volume was 270 mL, and 2.5 g of seed powder (the first addition of seed) was added. When the seed was added for the first time, low-speed stirring was maintained at 25 rpm. After the first addition of seed, low-speed stirring was continued at 25 rpm for 0.5 h, and then the concentration was continued to obtain a concentrated solution with a sugar content of 82% Brix. The temperature was then raised to 72°C and maintained for 1 h. Then 2.5 g of seed powder (the second addition of seed) was added. When the seed was added for the second time, low-speed stirring was maintained at 15 rpm. After the second addition of seed, the temperature was immediately lowered to 65°C within 1 h. After low-speed constant temperature stirring at 15 rpm at 65°C for 1 h, the temperature was lowered to 56°C for 2 h. At this time, a small amount of milky turbidity appeared in the crystallization kettle. After constant temperature crystallization for 18 h, centrifugation was performed at 6000 g for 10 min, the feed liquid showed stratification, the top feed liquid was relatively clear while the middle feed liquid was emulsified, and some crystals were precipitated at the bottom, indicating that the size distribution of 3-FL crystals in the feed liquid was wide, microcrystals, small crystals and larger crystals existed at the same time, and the crystallization quality was poor.

[0120] Comparative Example 5: Single variable comparison based on “(2) Small-scale recrystallization of 3-FL” in Example 2

[0121] Take 25.5 g of the primary crystallization product in Example 2, add 19 mL of purified water to completely dissolve the sample, measure the sugar content to be about 59.0% Brix, concentrate to 71% Brix at 65 ° C in a vacuum stirred kettle, add 0.4 g seed powder (the first time to add seed), maintain low-speed stirring at 25 rpm when adding seed for the first time, continue to maintain low-speed stirring at 25 rpm for 0.5 h after the first seed is added, then continue to concentrate to 82% Brix, then heat to 70 ° C, keep for 1 h, then add 0.4 g powder seed (second seed), maintain low-speed stirring at 20 rpm when adding seed, immediately after the second seed is added, slowly cool to 65 ° C within 1 h, stir at 20 rpm for 1 h, then slowly cool to 30 ° C within 4 h, crystallize at constant temperature for 18 h (the stirring speed of the entire process from the second seed addition to the end of constant temperature crystallization is 20 rpm), centrifuge at 6000 g for 10 min, the feed liquid was too viscous on the surface, which was not conducive to centrifugal crystal extraction, and the crystal content was small. The upper layer of feed liquid was removed, 3 mL of 90% ethanol aqueous solution was added to wash the crystals, and the crystals were centrifuged at 2000g for 10 min. The upper layer of crystal washing liquid was removed, and 3 mL of 90% ethanol was added to wash the crystals again. After centrifugation, the recrystallized product was obtained after vacuum drying at 45℃ and 200mbar overnight.

[0122] After testing, the obtained recrystallized product was about 1.6 g, and the single recovery rate was 6.3%. The recovery rate was too low and did not meet the conditions for large-scale production.

[0123] Comparative Example 6: Single variable comparison based on “(2) Small-scale recrystallization of 3-FL” in Example 2

[0124] Take 25.5 g of the primary crystallization product in Example 2, add 19 mL of purified water to completely dissolve the sample, measure the sugar content to be about 59.0% Brix, concentrate to 71% Brix at 65 ° C in a vacuum stirred kettle, add 0.4 g of seed powder (the first time to add seed), maintain low-speed stirring at 25 rpm when adding seed for the first time, continue to maintain low-speed stirring at 25 rpm for 0.5 h after the first seed is added, and then continue to concentrate to 82% Brix, then heat to 70 ° C, keep for 1 h, and then add 0.4 g of powder seed (second seed), maintain low-speed stirring at 60 rpm when adding seeds, immediately after the second seed is added, slowly cool to 65 ° C within 1 h, stir at 60 rpm for 1 h, then slowly cool to 53 ° C within 4 h, and crystallize at constant temperature for 18 h (the stirring speed of the entire process from the second seed addition to the end of constant temperature crystallization is 60 rpm), the crystallization liquid was a suspension, the 3-FL products were all microcrystals and small crystals, centrifugation was difficult, and recrystallization failed.

[0125] From the above examples, it can be seen that the present invention can achieve efficient separation and purification of 3-FL through two crystallization processes by strictly controlling the operating steps and operating parameters of primary crystallization and recrystallization, and obtain high-quality 3-FL crystalline products. When the user does not have high requirements for the purity of the 3-FL product, it is also possible to choose to use a single crystallization, or when the quality of the crystallization stock solution is high, a high-quality 3-FL product can be obtained by only using a single crystallization or recrystallization. The present invention uses two crystallizations for the general best situation of the liquid obtained by most fermentation methods, and is not limited to the use of two crystals. And the method is simple and convenient, more suitable for industrial production, only simple conventional pre-treatment steps are required, and the method can be used to extract and purify high-quality 3-FL; more widely, the method can be used to extract high-quality 3-FL from crude 3-fucosyllactose solutions prepared by enzymatic methods and chemical synthesis methods.

[0126] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for separating and purifying 3-fucosyllactose, characterized in that: The following steps are involved: The crystallization stock solution is subjected to a primary crystallization, wherein the mass concentration of 3-fucosyllactose in the crystallization stock solution is 500-550 g / L, to obtain a primary crystallization product; dissolving the primary crystallization product in water to obtain a primary crystallization product solution, and recrystallizing the primary crystallization product solution to obtain a 3-fucosyllactose crystallization product; The primary crystallization or recrystallization comprises the following steps: Concentrating the crystallization stock solution or the primary crystallization product solution for the first time to obtain a first concentrated solution, wherein the first concentrated solution has a Brix degree of 65-75%; performing a first mixing of the first concentrated solution and first seed crystals, wherein the first seed crystals are 3-fucosyllactose crystal products, to obtain a first mixed solution; Concentrating the first mixed solution for the second time to obtain a second concentrated solution, wherein the second concentrated solution has a Brix degree of 78-83%; performing a second mixing of the second concentrated solution and second seed crystals, wherein the second seed crystals are 3-fucosyllactose crystal products, to obtain a second mixed solution; The second mixed solution is cooled and crystallized, the initial temperature of the cooling crystallization is 70~75°C, the terminal temperature of the cooling crystallization is 50~60°C, the cooling crystallization includes sequentially performing a first stage cooling crystallization and a second stage cooling crystallization, the terminal temperature of the first stage cooling crystallization is 63~67°C, the cooling rate of the first stage cooling crystallization is 5~9°C / h, and the cooling rate of the second stage cooling crystallization is 1.75~3.75°C / h; the cooling crystallization is carried out under stirring, and the stirring speed is 10~20 rpm.

2. The separation and purification method according to claim 1, characterized in that The crystallization stock solution is obtained by pre-treating the production raw material solution; the production raw material solution includes one or more of fermentation solution, enzymatic production solution, chemical synthesis production solution and natural latex; the pre-treatment includes desalting, decolorizing and concentrating in sequence.

3. The separation and purification method according to claim 1, characterized in that: After the cooling crystallization is completed, the method further comprises: performing heat preservation crystallization under the terminal temperature condition of the cooling crystallization, and the heat preservation crystallization time is 18 to 24 hours.

4. The separation and purification method according to claim 1, characterized in that: The cooling crystallization further comprises: performing a heat preservation treatment at the terminal temperature of the first stage cooling crystallization, and the heat preservation treatment time is 1 to 2 hours.

5. The separation and purification method according to claim 1, characterized in that: The mass of the first seed crystal accounts for a percentage of 0.1-1 w / v of the volume of the first concentrated solution; The mass of the second seed crystal accounts for 0.1-1 w / v % of the volume of the second concentrated solution.

6. The separation and purification method according to claim 1, characterized in that: The temperatures of the first concentration, the first mixing, and the second concentration are independently 60-70° C.; The temperature of the second mixing is 70-75°C.

7. The separation and purification method according to claim 1 or 6, characterized in that: The first mixing is carried out under stirring conditions, and the stirring speed of the first mixing is 15~30 rpm; the first mixing includes the following steps: under the temperature conditions of the first mixing, adding the first seed crystal to the first concentrated solution, and then performing heat preservation and stirring to obtain the first mixed solution; the heat preservation and stirring time is 0.5~1h.

8. The separation and purification method according to claim 1 or 6, characterized in that: The second mixing is carried out under stirring conditions, and the stirring speed of the second mixing is 10-20 rpm; the second mixing includes the following steps: heating the second concentrated solution from the second concentration temperature to the second mixing temperature, and then performing a heat preservation treatment, and then adding the second seed crystal to the second concentrated solution after the heat preservation treatment to obtain a second mixed solution; the heat preservation treatment time is 1-2h.

9. The separation and purification method according to claim 1, characterized in that: The primary crystallization directly obtains a primary crystal slurry, further comprising: separating the primary crystal slurry into solid and liquid to obtain a primary crystal solid phase product; performing a first crystal washing on the primary crystal solid phase product to obtain the primary crystal product; The recrystallization directly obtains a recrystallization slurry, and further comprises: separating the recrystallization slurry into solid and liquid to obtain a recrystallized solid phase product; performing a second crystal washing on the recrystallized solid phase product to obtain the 3-fucosyllactose crystal product; The reagent used in the first crystal washing or the second crystal washing is an ethanol aqueous solution.

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