Fucosyllactose and purification method thereof

Abstract

The invention relates to the technical field of separation engineering, and provides fucosyllactose and a purification method thereof, and the purification method comprises the following steps: removing production bacteria and macromolecular substances from a fucosyllactose fermentation liquor, decolorizing, desalting to obtain a concentrated solution with the fucosyllactose content of 35-55%, and carrying out continuous chromatographic separation to obtain the fucosyllactose. A filler in a chromatographic column adopted in the continuous chromatographic separation is calcium type gel resin, the temperature of the chromatographic column is 50-65 DEG C, and pure water is adopted as an eluent. According to the method, the fucosyllactose with high yield, high purity, low impurity content and no solvent residue can be obtained by adopting continuous chromatographic separation under specific conditions after removal of production bacteria and macromolecular substances, decoloration and desalination treatment.

Description

Technical Field

[0001] The invention relates to the technical field of separation engineering, and in particular to fucosyllactose and a purification method thereof. Background Art

[0002] 2′-fucosyllactose (2′-FL) is a L -fucose is a non-reducing trisaccharide with the molecular formula C 18 H 32 O 15 , CAS number is 41 263-94-9, is one of the oligosaccharides with the highest content in human milk, it has a variety of physiological functions beneficial to the human body, such as regulating intestinal flora, resisting the adhesion of pathogens, immune regulation, and promoting the development and repair of the nervous system. 2'-FL accounts for about 31% (molar fraction) in human milk oligosaccharides (HMOs) and is an important ingredient in infant formula.

[0003] Since fucosyllactose produced by extraction or chemical synthesis methods cannot meet its needs, microbial fermentation is the current mainstream production technology, which uses the microorganism's own metabolic pathway to synthesize 2'-FL. This method is more environmentally friendly and efficient. 2'-FL prepared by microbial fermentation must undergo a strict downstream separation and extraction process to become a qualified product, so downstream separation and extraction is extremely important. The basic process steps include sterilization, desalination, depigmentation, spray drying and granulation, which not only require thorough sterilization, but also require the removal of other impurities such as soluble proteins, polysaccharides, nucleic acids, pigments, etc.

[0004] The prior art proposes a variety of solutions for the separation and purification of 2'-FL. For example, CN113811537A uses membrane filtration technology to purify oligosaccharides from bacterial fermentation broth, removes insoluble components and proteins through microfiltration and ultrafiltration, and then removes high molecular weight and low molecular weight impurities through two membranes of different sizes. On this basis, two electroosmotic desalination and activated carbon adsorption steps are performed, and spray drying is performed after concentration. CN109705175A uses simulated moving bed chromatography technology to purify 2'-FL in particle-free solution. Before this, it still needs to go through microfiltration, nanofiltration, and electrodialysis steps. The chromatographic column used in this chromatography technology is a cation exchange resin, and the eluent is an ethanol aqueous solution. The purified liquid needs to be adjusted with NaOH for pH, so electrodialysis is required for desalination again after chromatographic purification. The purity of oligosaccharides after purification is up to 93%. CN106132977A uses a combination of cation and anion exchange desalination, nanofiltration, and electrodialysis steps to purify neutral human milk oligosaccharides. After the fermentation broth is treated with glucosidase, the macromolecules are removed by ultrafiltration, and then after two desalting steps, one nanofiltration, and two activated carbon decolorization steps, the sample purity is 95% after spray drying. CN118027115A sequentially treats the fucosyllactose fermentation broth with bacterial removal, decolorization, ion exchange desalination, chromatographic separation, and spray drying to obtain fucosyllactose with a purity of up to 98%.

[0005] Although the above schemes increase the purity of 2′-FL to more than 93%, or even 98%, they only focus on the overall purity of the product and do not strictly control the content of certain impurities. For example, 2′-fucosyl lactulose (2'-fucosyl-D-lactulose), which is an isomer of 2′-FL, is required by the EU to have a content of 2′-fucosyl lactulose in 2′-FL that must be controlled below 1%. Existing technologies rarely pay attention to this indicator, and no relevant purification methods are given to achieve this goal. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention provides a method for purifying fucosyllactose to prepare fucosyllactose with high purity, low miscellaneous sugars, no solvent residue, good smell and taste, and high yield, wherein the content of 2′-fucosyllactulose in 2′-fucosylfrucose is less than 1%, and the content of 3′-fucosyllactulose in 3′-fucosylfrucose is less than 1%.

[0007] The invention provides a method for purifying fucosyllactose, comprising: removing production bacteria and macromolecular substances from fucosyllactose fermentation broth, decolorizing and desalting to obtain a concentrated solution with a fucosyllactose content of 35-55%, and performing continuous chromatographic separation, wherein the filler in the chromatographic column used in the continuous chromatographic separation is a calcium-type gel resin, the temperature of the chromatographic column is 50-65°C, and pure water is used as an eluent.

[0008] In the prior art, after obtaining the desalted liquid, it is usually concentrated to a certain concentration and then directly spray-dried to obtain the finished product. However, the product obtained in this way has low purity and high content of impure sugars, and does not have a competitive advantage. Alternatively, in order to remove impure sugars, acetic acid and alcohol solvents are used for crystallization. Although this can produce a product with higher purity, the use of acetic acid will make the product contain a pungent odor of acetic acid, and it can only be used as a wet method in subsequent applications; and the use of alcohol solvents for crystallization requires the use of explosion-proof workshop design, high investment in equipment and plant, and the product has the risk of solvent residues, and the crystallization yield is not high.

[0009] The present invention uses continuous chromatography to remove the miscellaneous sugars in the feed liquid, which has the advantages of high efficiency, high yield, high product purity, and low investment in equipment and plant. At the same time, the present invention has found that in order to control the 2′-fucosyllactulose content below 1%, it is very effective to adopt continuous chromatography separation after removing the production bacteria and macromolecular substances, decolorization, and desalting. Among them, the chromatographic column, fucosyllactose feed concentration, column temperature and eluent of continuous chromatography separation are key parameters, and the above-defined ranges need to be adopted.

[0010] Further preferably, the calcium type gel resin has a crosslinking degree of 4-8%, a pore size of 0.5-5 nm, and a porosity of 30-40%.

[0011] In order to obtain good purification results with the chromatographic column filler, the feed concentration of fucosyllactose is controlled at 35~55%, that is, before loading on the column, the fucosyllactose content in the concentrate is 35~55%.

[0012] In an embodiment of the present invention, the fucosyllactose fermentation broth includes one or more of 2′-fucosyllactose and 3′-fucosyllactose.

[0013] It is understood that the fucosyllactose fermentation broth may be a 2′-fucosyllactose fermentation broth, a 3′-fucosyllactose fermentation broth, or a fermentation broth containing 2′-fucosyllactose and 3′-fucosyllactose. When it is a 3′-fucosyllactose fermentation broth, the content of 3′-fucosyllactulose can be controlled according to the method of the present invention. When it is a fermentation broth containing 2′-fucosyllactose and 3′-fucosyllactose, the content of 2′-fucosyllactulose and 3′-fucosyllactulose can be controlled according to the method of the present invention.

[0014] At present, there are two main ways to synthesize 2′-FL by fermentation. One method requires the use of exogenous L -The salvage pathway of fucose is the Salvage pathway, which uses the bifunctional enzyme fkp to convert exogenous L - Fucose to GDP - L-fucose, which is combined with exogenous lactose by fucosyltransferase to generate 2′-FL; the other is to use glucose or glycerol as the carbon source and synthesize the precursor GDP- L -fucose, and finally 2′-FL is obtained by the reaction of the expressed exogenous fucosyltransferase with lactose. This pathway is a de novo synthesis pathway. denovo Both pathways require a large amount of GDP- L -fucose is used as a precursor for product synthesis and combines with exogenous lactose to produce 2′-FL. Therefore, it is extremely important to accumulate this precursor in the production strain cells. The main methods include the following three: (1) Use a strong promoter or high copy expression strategy to regulate GDP- L - Enhancement of the fucose synthesis pathway, or overexpression of the positive transcription factor genes of the colanic acid synthesis pathway (e.g. rcS ); (2) Knock out GDP- L -Genes related to fucose downstream metabolism (e.g. wxya ) to achieve intermediate accumulation; (3) to regulate the synthesis of limiting cofactors GTP and NADPH of key enzymes. Among them, the production strain, i.e., the chassis microorganism, is generally Escherichia coli. The specific strain selection, fermentation conditions, and optimization methods can refer to the prior art. The present invention focuses on the steps after obtaining the fermentation broth containing 2′-fucosyllactose, so how to obtain the fermentation broth will not be described in detail.

[0015] At present, the fermentation broth containing 2′-fucosyllactose reported at home and abroad generally has a 2′-fucosyllactose content of 40-200 g / L. In the embodiment of the present invention, the mass content of 2′-fucosyllactose in the fucosyllactose fermentation broth is 4-18%.

[0016] In some embodiments of the present invention, the step of removing the production bacteria and macromolecules from the fucosyllactose fermentation broth comprises: pre-treating the fucosyllactose fermentation broth to denature the protein, and then removing the production bacteria and macromolecules by membrane filtration or mechanical separation.

[0017] Furthermore, the pretreatment conditions are to instantly heat the mixture to 85-100°C and maintain the temperature for 0.5-30 minutes to denature the protein, and then instantly cool the mixture to below 40°C.

[0018] In some embodiments of the present invention, the membrane filtration uses a ceramic membrane with a pore size of 50 to 200 nm.

[0019] In some embodiments of the present invention, the mechanical separation is centrifugal separation with a rotation speed of 5000-120000 rpm.

[0020] In the prior art, the fermentation liquid is usually centrifuged or filtered through a ceramic membrane to remove the production bacteria, and the clear liquid is separated, and then ultrafiltration is used to remove macromolecular substances (protein, nucleic acid, endotoxin, etc.). However, since the protein content in the liquid after the production bacteria is removed is high, the ultrafiltration area needs to be large, and the ultrafiltration membrane is easily blocked, which affects the service life of the membrane.

[0021] In the present invention, after the fermentation liquid is pre-treated by heating, the protein in the feed liquid is denatured by heating, and is retained in the concentrated liquid after centrifugation or ceramic membrane filtration, and the protein content in the obtained clear liquid is low, so that the subsequent ultrafiltration equipment area is small and the equipment investment is small. On the other hand, after the fermentation liquid is heated, it will not cause workshop environmental pollution, such as phage production, during the production process due to live bacteria filtration.

[0022] In order to further intercept a small amount of protein, nucleic acid, etc. in the material, ultrafiltration is performed after the membrane filtration or mechanical separation, and the ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 3000-5000Da.

[0023] In some embodiments of the present invention, the decolorization adopts resin decolorization, the feed rate is 1-9 BV / h, the temperature does not exceed 40°C, and more specifically, the feed rate is 2-6 BV / h.

[0024] The existing technology usually uses activated carbon for decolorization, which requires a large amount of activated carbon, thereby producing a large amount of waste activated carbon, causing an environmental burden, and the activated carbon will adsorb the target product in the feed liquid, resulting in a low yield.

[0025] The present invention adopts resin decolorization, has high yield, and will not pollute the workshop environment or generate hazardous waste.

[0026] If technological innovation produces activated carbon suitable for adsorbing impurities in fucosyllactose fermentation broth, using the activated carbon to decolorize the fucosyllactose fermentation broth is also within the protection scope of the present invention.

[0027] Further preferably, the decolorization adopts one or more of the resins LXT 036, LXT 067, and LXT 080.

[0028] In some embodiments of the present invention, the desalination adopts continuous ion exchange resin desalination, the feed rate is 1-9 BV / h, the temperature does not exceed 40° C., and the discharge conductivity is ≤200 μs / cm.

[0029] Specifically, the desalination using continuous ion exchange resin includes sequentially passing through a cation exchange resin and an anion exchange resin, wherein the cation exchange resin is selected from one or more of LX101, LXT110, and D001, and the anion exchange resin is selected from one or more of LX207, LXT203, and D301.

[0030] In some embodiments of the present invention, after the desalting treatment, the feed liquid is subjected to membrane concentration treatment so that the fucosyllactose content in the concentrated liquid is 35-55%, and enters the continuous chromatographic separation process; the membrane concentration is a reverse osmosis high-pressure membrane, the upper limit of the operating pressure can reach 60 bar, and the temperature does not exceed 40°C.

[0031] Compared with evaporation concentration, membrane concentration operates at low temperature throughout the entire process, which not only reduces energy consumption but also improves product quality.

[0032] In some embodiments of the present invention, after the continuous chromatographic separation is completed, the obtained eluate is spray dried, the feed temperature is 130-190° C., and the exhaust temperature is 80-95° C. to obtain purified fucosyllactose.

[0033] In a second aspect, the present invention provides a fucosyllactose product, wherein the fucosyllactose product is purified by the above-mentioned purification method, and the content of fucosyllactulose in the fucosyllactose product is less than 1%.

[0034] The beneficial effects of the present invention are: The present invention provides a method for purifying fucosyllactose, which can obtain high-yield, high-purity, low-impurity fucosyllactose by removing production bacteria and macromolecular substances, decolorizing and desalting, and then taking continuous chromatographic separation under specific conditions. The purification method of the present invention has a simple process, does not require excessive equipment and plant investment, and is conducive to industrial-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The present invention provides a process flow chart of the method for purifying fucosyllactose. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are 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.

[0037] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0038] In the following examples, the contents of 2′-fucosyllactose and 2′-fucosyllactulose were determined by high performance liquid chromatography (HPLC) under the following chromatographic conditions: 1. The chromatographic conditions of the hydrophilic retention column are as follows: 1.1 Chromatographic column: hydrophilic retention column, 250 mm × 4.6 mm, 3.5 µm or equivalent; 1.2 Mobile phase: Accurately weigh 582.8 g of acetonitrile, add appropriate amount of water to obtain 857.2 g of solution, and then add 10 mL of triethylamine; 1.3 Column temperature: 25 °C; 1.4 Differential refractive index detector temperature: 35 ℃; 1.5 Flow rate: 1 mL / min; 1.6 Injection volume: 5 μL; 1.7 Running time: 45min.

[0039] 2. Amide bonded column chromatography conditions are as follows: 2.1 Chromatographic column: amide bonded column, 150 mm × 4.6 mm, 3 µm or equivalent; 2.2 Mobile phase: acetonitrile: water = 64:36 (v / v); 2.3 Column temperature: 25 °C; 2.4 Differential refractive index detector temperature: 37 °C; 2.5 Flow rate: 1.1 mL / min; 2.6 Injection volume: 5 μL; 2.7 Running time: 8 min.

[0040] In the following embodiments, when the reverse osmosis high pressure membrane is in operation, the pressure does not exceed 60 bar and the temperature does not exceed 40°C.

[0041] The calcium type gel resin used in the present invention has a crosslinking degree of 4-8%, a pore size of 0.5-5 nm, and a porosity of 30-40%.

[0042] Example 1

[0043] This embodiment provides a method for purifying 2′-fucosyllactose (2′-FL). The process flow chart is as follows: Figure 1 As shown, the specific steps are as follows: (1) 10 L of 2′-fucosyllactose fermentation broth (mass content of 2′-fucosyllactose is 13%) was heated to 90°C for 10 min, then cooled to 30°C and filtered through a 50 nm filter membrane to remove the production bacteria and proteins to obtain a filtered clear liquid. The yield of this process was calculated to be 98.5%; (2) The clear liquid obtained in step (1) was passed through a 5000Da ultrafiltration membrane, circulated to the minimum volume, and then top-washed with pure water for 3 times until the sugar content of the clear liquid was less than 1%. The content of 2′-fucosyllactose was detected to be 8.7%, and the yield of this process was calculated to be 98.9%; (3) 10 L of the clear liquid from step (2) was decolorized by resin (LXT 036) at a flow rate of 3 BV / h to obtain a decolorized liquid. The content of 2′-fucosyllactose was detected to be 7.8%, and the calculated yield was 95.5%; (4) The decolorized solution obtained in step (3) was subjected to adsorption desalination by cation exchange resin LX101 (Lanxiao Biotechnology) and anion exchange resin LXT203 (Lanxiao Biotechnology) in sequence to obtain a desalted solution. The conductivity of the desalted solution was 103 μs / cm, the content of 2′-fucosyllactose in the desalted solution was 7.5%, and the calculated yield was 96.7%; (5) The desalted liquid in step (4) was concentrated by reverse osmosis high pressure membrane and evaporation to obtain a concentrated liquid with a 2′-fucosyllactose content of 46.5% and a calculated yield of 96.9%; (6) The concentrated solution obtained in step (5) is separated by using a continuous chromatographic column Ca-type gel resin and eluted with pure water to obtain an eluate, wherein the column temperature of the chromatographic column is 55° C. The purity of 2′-fucosyllactose in the eluate is 97.7%, and the yield is 90.3%; (7) The eluate obtained in step (6) was spray-dried at 165° C. to obtain 2′-fucosyllactose with a purity of 97.9% and a total yield of 78.1%. The content of 2′-fucosyllactulose was 0.59%.

[0044] Example 2

[0045] This example provides a method for purifying 2′-fucosyllactose (2′-FL), and the specific steps are as follows: (1) 10L of 2′-fucosyllactose fermentation broth (mass content of 2′-fucosyllactose is 18%) was heated to 85°C for 30 min, then cooled to 30°C and filtered through a 200nm filter membrane to remove the production bacteria and proteins to obtain a filtered clear liquid. The yield of this process was calculated to be 98.9%; (2) The clear liquid obtained in step (1) was passed through a 3000Da ultrafiltration membrane, circulated to the minimum volume, and then top washed with pure water three times until the sugar content of the clear liquid was less than 1%. The content of 2′-fucosyllactose was detected to be 12.3%, and the yield of this process was calculated to be 97.9%; (3) 10 L of the clear liquid from step (2) was decolorized by resin (LXT 067) at a flow rate of 6 BV / h to obtain a decolorized liquid. The content of 2′-fucosyllactose was detected to be 11.7%, and the calculated yield was 95.3%; (4) The decolorized solution obtained in step (3) was subjected to adsorption desalination by cation exchange resin LXT110 (Lanxiao Biotechnology) and anion exchange resin LX 207 (Lanxiao Biotechnology) in sequence to obtain a desalted solution. The conductivity of the desalted solution was 97.3 μs / cm, the content of 2′-fucosyllactose in the desalted solution was 10.5%, and the calculated yield was 97.1%; (5) The desalted liquid in step (4) was concentrated by high pressure membrane and evaporation to obtain a concentrated liquid with a 2′-fucosyllactose content of 55% and a calculated yield of 95.3%; (6) Separating the concentrated solution obtained in step (5) using a continuous chromatographic column (Ca-type gel resin) and eluting with pure water to obtain an eluate, wherein the column temperature of the chromatographic column is 50° C.; the purity of 2′-fucosyllactose in the eluate is 98.2%, and the yield is 94.8%; (7) The eluate obtained in step (6) was spray-dried at 185° C. to obtain 2′-fucosyllactose with a purity of 98.5% and a total yield of 80.9%. The content of 2′-fucosyllactulose was 0.31%.

[0046] Example 3

[0047] This example provides a method for purifying 2′-fucosyllactose (2′-FL), and the specific steps are as follows: (1) 10L of 2′-fucosyllactose fermentation broth (mass content of 2′-fucosyllactose is 15.9%) was heated to 100°C for 0.5 min, then cooled to 40°C and filtered through a 100 nm filter membrane to remove the production bacteria and proteins to obtain a clear filtrate. The yield of this process was calculated to be 99.1%. (2) The clear liquid obtained in step (1) was passed through a 4000Da ultrafiltration membrane, circulated to the minimum volume, and then top washed with pure water three times until the sugar content of the clear liquid was less than 1%. The content of 2′-fucosyllactose was detected to be 11.3%, and the yield of this process was calculated to be 98.3%; (3) 10 L of the clear liquid from step (2) was decolorized by resin (LXT 067) at a flow rate of 2 BV / h to obtain a decolorized liquid. The content of 2′-fucosyllactose was detected to be 10.4%, and the calculated yield was 94.7%; (4) The decolorized solution obtained in step (3) was subjected to adsorption desalination by cation exchange resin D001 (Lanxiao Biotechnology) and anion exchange resin D301 (Lanxiao Biotechnology) in sequence to obtain a desalted solution. The conductivity of the desalted solution was 63.1 μs / cm, the content of 2′-fucosyllactose in the desalted solution was 9.1%, and the calculated yield was 97.9%; (5) The desalted liquid in step (4) was concentrated by high pressure membrane and evaporated to obtain a membrane concentrate having a 2′-fucosyllactose content of 50.3% and a calculated yield of 95.5%; (6) The concentrated solution obtained in step (5) is separated by using a continuous chromatographic column Ca-type gel resin and eluted with pure water to obtain an eluate, wherein the column temperature of the chromatographic column is 65° C. The purity of 2′-fucosyllactose in the eluate is 97.9%, and the yield is 90.8%; (7) The eluate obtained in step (6) was spray-dried at 165° C. to obtain 2′-fucosyllactose with a purity of 98.1% and a total yield of 78.3%. The content of 2′-fucosyllactulose was 0.61%.

[0048] Example 4

[0049] This example provides a method for purifying 3′-fucosyllactose, which differs from Example 1 in that the fermentation broth used is 3′-fucosyllactose fermentation broth, and in step (1), instead of ceramic membrane filtration, centrifugal separation is used to remove the production bacteria and proteins to obtain a filtered clear liquid. The 3′-fucosyllactose yield of this step is calculated to be 90.3%.

[0050] The content of 3′-fucosyllactose in the concentrated solution in step (5) is 45.9%.

[0051] The final 3′-fucosyllactose obtained had a purity of 97.7% and a total yield of 72.5%, wherein the content of 3′-fucosyllactulose was 0.69%.

[0052] Example 5

[0053] This example provides a method for purifying 2′-fucosyllactose (2′-FL). The difference between this method and Example 1 is that in step (1), ceramic membrane filtration is not used, but centrifugal separation is used to remove production bacteria and proteins, etc. The rotation speed is 9000 rpm to obtain a filtered clear liquid. The yield of this process is calculated to be 90.1%.

[0054] The content of 2′-fucosyllactose in the concentrated solution in step (5) is 48.5%.

[0055] The final 2′-fucosyllactose obtained had a purity of 98.01% and a total yield of 71.8%, wherein the content of 2′-fucosyllactulose was 0.57%.

[0056] Example 6

[0057] This example provides a method for purifying 2′-fucosyllactose (2′-FL), which differs from Example 1 in that activated carbon is used for decolorization in step (3) to obtain a decolorized liquid, and the content of 2′-fucosyllactose is detected to be 7.5%, and the calculated yield is 90.5%.

[0058] The content of 2′-fucosyllactose in the concentrated solution in step (5) is 52.3%. The purity of the finally obtained 2′-fucosyllactose is 98.2%, and the total yield is 75.4%. Among them, the content of 2′-fucosyllactulose is 0.37%.

[0059] Example 7

[0060] This example provides a method for purifying 2′-fucosyllactose (2′-FL), which differs from Example 1 in that in step (4), the decolorized liquid obtained in step (3) is subjected to adsorption desalination by cation exchange resin LXT110 and anion exchange resin LXT203 in sequence to obtain a desalted liquid, the conductivity of the desalted liquid is 59.3 μs / cm, the content of 2′-fucosyllactose in the desalted liquid is 7.59%, and the calculated yield is 97.1%.

[0061] The content of 2′-fucosyllactose in the concentrated solution in step (5) is 35%.

[0062] The final 2′-fucosyllactose obtained had a purity of 97.3% and a total yield of 71.1%, wherein the content of 2′-fucosyllactulose was 0.83%.

[0063] Comparative Example 1

[0064] This comparative example provides a method for purifying 2′-fucosyllactose (2′-FL), which differs from Example 1 in that the continuous chromatographic separation in step (6) is not performed, and the concentrated solution obtained in step (5) is directly spray-dried at 165° C. to obtain 2′-fucosyllactose with a purity of 95.1% and a total yield of 86.9%, wherein the content of 2′-fucosyllactulose is 1.51%.

[0065] Comparative Example 2

[0066] This comparative example provides a method for purifying 2′-fucosyllactose (2′-FL). The difference between the comparative example and the example 1 is that the continuous chromatographic separation in step (6) is not performed, and the concentrated solution obtained in step (5) is first subjected to ethanol crystallization. The specific operation is as follows: adding alcohol in an amount of 3 times the weight of the feed solution, keeping the temperature at 60.5°C for crystallization for 4 hours, then cooling to room temperature, centrifuging to obtain wet crystals, and then drying the obtained crystals at 50°C to obtain 2′-fucosyllactose with a purity of 99.57% and a total yield of 67.8%, wherein the content of 2′-fucosyllactulose is 0.13%.

[0067] Comparative Example 3

[0068] This comparative example provides a method for purifying 2′-fucosyllactose (2′-FL), which differs from Example 1 in that the conditions for continuous chromatographic separation in step (6) are different. Specifically, the chromatographic column used is a potassium-type gel resin, and the column temperature is 50° C. The eluate obtained in step (6) is spray-dried at 165° C. to obtain 2′-fucosyllactose with a purity of 95.9% and a total yield of 78.0%, wherein the content of 2′-fucosyllactulose is 1.31%.

[0069] Comparative Example 4

[0070] This example provides a method for purifying 2′-fucosyllactose (2′-FL), which differs from Example 1 in that the content of 2′-fucosyllactose in the concentrated solution in step (5) is 30%.

[0071] The final 2′-fucosyllactose obtained had a purity of 96.9% and a total yield of 70.5%, wherein the content of 2′-fucosyllactulose was 1.27%.

[0072] Comparative Example 5

[0073] This example provides a method for purifying 2′-fucosyllactose (2′-FL), which differs from Example 1 in that the content of 2′-fucosyllactose in the concentrated solution in step (5) is 60.5%.

[0074] The final 2′-fucosyllactose obtained had a purity of 97.9% and a total yield of 76.5%, wherein the content of 2′-fucosyllactulose was 0.73%.

[0075] In this comparative example, the content of 2′-fucosyllactose in the concentrated solution of step (5) is greater than 55%, the poor fluidity of the material in the chromatographic column affects the separation effect, the purity and total yield of 2′-fucosyllactose are not as good as those in Example 2, and the time required for concentration and chromatographic separation is relatively long, which affects the production efficiency of 2′-fucosyllactose.

[0076] The above results show that: (1) After removing the production bacteria and macromolecular substances, decolorizing and desalting, continuous chromatographic separation of the concentrated solution with a fucosyllactose content of 35-55% using a chromatographic column filled with a calcium-type gel resin can effectively remove the fucosyllactulose component in the fucosyllactose concentrated solution. The fucosyllactulose content in the obtained fucosyllactose final product is less than 1%, which meets the EU and GRAS standards.

[0077] (2) The content of fucosyllactose in the concentrate before column loading and the type of chromatographic column used will significantly affect the separation of fucosyllactulose and fucosyllactose.

[0078] (3) The removal of production bacteria and macromolecular substances from the fermentation broth, decolorization and desalting can be carried out by conventional technical means. As long as the content of fucosyllactose in the concentrate is maintained at 35-55% before continuous chromatographic separation, the final product with less than 1% fucosyllactulose in the fucosyllactose can be obtained by calcium-type gel resin chromatography.

[0079] It should be noted that the endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in this article.

[0080] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "specific implementation methods", or "some specific implementation methods" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the embodiment of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0081] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for purifying fucosyllactose, characterized in that: include: After removing the production bacteria and macromolecular substances from the fucosyllactose fermentation liquid, decolorizing and desalting, a concentrated solution with a fucosyllactose content of 35-55% is obtained, and continuous chromatographic separation is performed. The filler of the chromatographic column used in the continuous chromatographic separation is a calcium-type gel resin, the temperature of the chromatographic column is 50-65°C, and pure water is used as the eluent.

2. The method for purifying fucosyllactose according to claim 1, characterized in that: The fucosyllactose fermentation broth includes one or more of 2′-fucosyllactose and 3′-fucosyllactose.

3. The method for purifying fucosyllactose according to claim 1, characterized in that: The step of removing production bacteria and macromolecular substances from the fucosyllactose fermentation broth comprises: The fucosyllactose fermentation liquid is pretreated to denature the protein, and then the production bacteria and macromolecular substances are removed by membrane filtration or mechanical separation.

4. The method for purifying fucosyllactose according to claim 3, characterized in that: The pretreatment conditions are: instantaneously heating to 85-100°C and maintaining the temperature for 0.5-30 minutes to denature the protein, and then instantaneously cooling to below 40°C; The membrane filtration uses a ceramic membrane with a pore size of 50-200 nm; And / or, the mechanical separation is centrifugal separation with a rotation speed of 5000-120000 rpm.

5. The method for purifying fucosyllactose according to claim 3, characterized in that: After the membrane filtration or mechanical separation, ultrafiltration is further performed, and the ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 3000-5000Da.

6. The method for purifying fucosyllactose according to claim 1, characterized in that: The decolorization adopts resin decolorization, the feed rate is 1-9 BV / h, and the temperature does not exceed 40° C.; the resin is selected from one or more of LXT036, LXT 067, and LXT080.

7. The method for purifying fucosyllactose according to claim 1, characterized in that: The desalination adopts continuous ion exchange resin desalination, the feed rate is 1-9 BV / h, the temperature does not exceed 40°C, and the discharge conductivity is ≤200μs / cm; The ion exchange resin includes a cation exchange resin and an anion exchange resin for desalination, the cation exchange resin is selected from one or more of LX101, LXT110, and D001, and the anion exchange resin is selected from one or more of LX207, LXT203, and D301.

8. The method for purifying fucosyllactose according to claim 1, characterized in that: After the desalting treatment, the feed liquid is subjected to membrane concentration and evaporation concentration treatment, so that the fucosyllactose content in the concentrated liquid is 35-55%, and enters the continuous chromatography separation step; The membrane concentrator is a reverse osmosis high-pressure membrane with an upper limit of operating pressure of 60 bar and a temperature not exceeding 40°C.

9. The method for purifying fucosyllactose according to any one of claims 1 to 8, characterized in that: After the continuous chromatographic separation is completed, the obtained eluate is spray-dried, the feed temperature is 130-190° C., and the exhaust temperature is 80-95° C. to obtain purified fucosyllactose.

10. A fucosyllactose product, characterized in that: The fucosyllactose product is purified by the purification method according to any one of claims 1 to 9, and the content of fucosyllactulose in the fucosyllactose product is less than 1%.

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