Fucosyllactose and a method for purifying the same

Abstract

The present application relates to the technical field of separation engineering, and provides a kind of fucosyl lactose and its purification method, the purification method includes: after removing production bacteria and macromolecular substance, decoloring, desalination treatment to fucosyl lactose fermentation broth, obtain the concentrated solution with fucosyl lactose content of 35~55%, carry out continuous chromatography separation, the filler in the chromatographic column used in the continuous chromatography separation is calcium type gel resin, the temperature of chromatographic column is 50~65 DEG C, and pure water is used as eluent.The present application can obtain high yield, high purity, low impurity, no solvent residue fucosyl lactose by removing production bacteria and macromolecular substance, decoloring, desalination treatment and continuous chromatography separation under specific conditions.

Description

Technical Field

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

[0002] 2'-fucosyllactose (2'-FL) is a non-reducing trisaccharide composed of lactose and L-fucose, with the molecular formula C 18 H 32 O 15 2'-FL, with the CAS number 41,263-94-9, is one of the most abundant oligosaccharides in human milk. It possesses numerous beneficial physiological functions, such as regulating intestinal flora, resisting pathogen adhesion, immune regulation, and promoting nervous system development and repair. 2'-FL comprises approximately 31% (by mole) of human milk oligosaccharides (HMOs) and is a key ingredient in infant formula.

[0003] Since fucosyllactose produced by extraction or chemical synthesis methods cannot meet its needs, microbial fermentation is currently the mainstream production technology. It uses the microorganism's own metabolic pathways to synthesize 2'-FL. This method is more environmentally friendly and efficient. 2'-FL produced by microbial fermentation must undergo a rigorous 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, pigment removal, spray drying and granulation, which not only require thorough sterilization, but also the removal of other impurities such as soluble proteins, polysaccharides, nucleic acids, pigments, etc.

[0004] The prior art has proposed 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. Microfiltration and ultrafiltration remove insoluble components and proteins, and then two membranes of different sizes are used to remove high and low molecular weight impurities. On this basis, two electroosmotic desalination and activated carbon adsorption steps are performed, and the solution is concentrated and spray-dried. CN109705175A uses simulated moving bed chromatography technology to purify 2'-FL from a particle-free solution. Prior to this, microfiltration, nanofiltration, and electrodialysis steps are still required. The chromatographic column used in this chromatography technology is a cation exchange resin, the eluent is an ethanol-water solution, and the purified solution needs to be adjusted with NaOH for pH. Therefore, electrodialysis is required for desalination again after chromatographic purification. The purity of the purified oligosaccharides 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, macromolecules are removed by ultrafiltration. Then, after two desalination steps, one nanofiltration step, and two activated carbon decolorization steps, spray drying is performed to obtain a sample with a purity of 95%. CN118027115A sequentially removes bacteria from the fucosyllactose fermentation broth, decolorizes it, undergoes ion exchange desalination, undergoes chromatographic separation, and spray-dries it to obtain fucosyllactose with a purity of 98%.

[0005] While the aforementioned solutions increase the purity of 2′-FL to over 93%, even reaching 98%, they focus solely on the overall purity of the product and fail to strictly control the content of certain impurities. For example, the EU requires that the content of 2′-fucosyl-D-lactulose, an isomer of 2′-FL, must be controlled below 1%. Existing technologies rarely address this metric, nor do they offer purification methods to achieve this goal. Summary of the Invention

[0006] In view of the shortcomings of the existing technology, 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′-fucosyllactose is less than 1%, and the content of 3-fucosyllactulose in 3-fucosyllactose is less than 1%.

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

[0008] In existing technologies, desalted liquid is typically concentrated to a certain concentration before being directly spray-dried to obtain the finished product. However, this method produces a low-purity product with a high content of unsweetened sugars, which lacks competitive advantages. Alternatively, to remove unsweetened sugars, crystallization is performed using acetic acid or alcoholic solvents. While this method can produce a higher-purity product, the use of acetic acid imparts a pungent odor, forcing subsequent applications to be restricted to wet processes. Crystallization using alcoholic solvents requires an explosion-proof workshop design, resulting in high investment in equipment and plant space, the risk of residual solvent in the product, and a low crystallization yield.

[0009] The present invention uses continuous chromatography to remove miscellaneous sugars from the feed solution, offering advantages such as high efficiency, high yield, high product purity, and minimal investment in equipment and plant. Furthermore, the present invention has found that to control the 2′-fucosyllactulose content to below 1%, continuous chromatography is very effective after removing the producing bacteria and macromolecular substances, decolorizing, and desalting. The chromatographic column, fucosyllactose feed concentration, column temperature, and eluent are key parameters for continuous chromatography, and these parameters must be within the above-defined ranges.

[0010] More preferably, the calcium type gel resin has a cross-linking 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, the content of fucosyllactose in the concentrated solution is 35-55% before loading on the column.

[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 can be a 2′-fucosyllactose fermentation broth, a 3-fucosyllactose fermentation broth, or a fermentation broth containing both 2′-fucosyllactose and 3-fucosyllactose. When the fermentation broth is a 3-fucosyllactose fermentation broth, the content of 3-fucosyllactulose can be controlled according to the method of the present invention. When the fermentation broth contains both 2′-fucosyllactose and 3-fucosyllactose, the content of both 2′-fucosyllactulose and 3-fucosyllactulose can be controlled according to the method of the present invention.

[0014] Currently, there are two main pathways for the fermentative synthesis of 2′-FL. One is the salvage pathway, which requires exogenous L-fucose. This pathway utilizes the bifunctional enzyme fkp to convert exogenous L-fucose into GDP-L-fucose, which is then combined with exogenous lactose by a fucosyltransferase to produce 2′-FL. The other is the de novo pathway, which uses glucose or glycerol as a carbon source and synthesizes the precursor GDP-L-fucose through multiple metabolic steps in the host cell. Ultimately, 2′-FL is obtained through the reaction of expressed exogenous fucosyltransferase with lactose. Both pathways require a large amount of GDP-L-fucose as a product synthesis precursor to combine with exogenous lactose to produce 2′-FL. Therefore, the accumulation of this precursor within the production strain is crucial. The main methods include the following three: (1) using a strong promoter or high-copy expression strategy to enhance the GDP-L-fucose synthesis pathway, or overexpressing the positive transcription factor gene of the colanic acid synthesis pathway (such as rcsA); (2) knocking out genes related to the downstream metabolism of GDP-L-fucose (such as wcaJ) to achieve intermediate accumulation; (3) regulating the synthesis of the limiting cofactors GTP and NADPH of key enzymes. Among them, the production strain, i.e., the chassis microorganism, is generally Escherichia coli. Specific strain selection, fermentation conditions, and optimization methods can refer to the existing technology. 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] Currently, fermentation broths containing 2'-fucosyllactose reported domestically and internationally generally contain 40-200 g / L of 2'-fucosyllactose. In the embodiments 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 producing bacteria and macromolecules from the fucosyllactose fermentation broth comprises: pre-treating the fucosyllactose fermentation broth to denature the protein, and then removing the producing bacteria and macromolecules by membrane filtration or mechanical separation.

[0017] Furthermore, the pretreatment conditions are instantaneous heating to 85-100° C. and maintaining the temperature for 0.5-30 minutes to denature the protein, and then instantaneously cooling the temperature 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, and the rotation speed is 5000-120000 rpm.

[0020] In the prior art, the fermentation broth is usually centrifuged or filtered through a ceramic membrane to remove the productive bacteria, and the resulting clear fluid is then subjected to ultrafiltration to remove macromolecular substances (proteins, nucleic acids, endotoxins, etc.). However, due to the high protein content in the slurry after the production bacteria are removed, the ultrafiltration area required is very large, and the ultrafiltration membrane is easily clogged, shortening the membrane's service life.

[0021] In the present invention, the fermentation broth is pre-treated by heating, and the protein in the feed liquid is denatured by the heating. After centrifugation or ceramic membrane filtration, the protein is retained in the concentrated liquid, resulting in a low protein content in the resulting clear liquid. This reduces the area required for subsequent ultrafiltration equipment and reduces equipment investment. Furthermore, the fermentation broth after heat treatment does not cause workplace environmental pollution, such as the production of bacteriophages, 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. The ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 3000 to 5000 Da.

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

[0024] Existing technologies usually use activated carbon for decolorization, which requires a large amount of activated carbon, thereby generating a large amount of waste activated carbon, causing an environmental burden. In addition, 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, does not pollute the workshop environment, and does not generate hazardous waste.

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

[0027] Further preferably, the decolorization adopts one or more of 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 to 9 BV / h, the temperature does not exceed 40° C., and the discharge conductivity is ≤200 μs / cm.

[0029] Specifically, the continuous ion exchange resin desalination 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 then enters the continuous chromatography 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 with a feed temperature of 130-190° C. and an exhaust temperature of 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:

[0035] The present invention provides a method for purifying fucosyllactose. After removing the producing bacteria and macromolecular substances, decolorizing, and desalting, continuous chromatographic separation under specific conditions is performed to obtain high-yield, high-purity, and low-impurity fucosyllactose. The purification method of the present invention is simple, does not require excessive equipment and plant investment, and is conducive to industrial-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The present invention provides a process flow chart of the method for purifying fucosyllactose. DETAILED DESCRIPTION

[0037] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0038] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0039] 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:

[0040] 1. The chromatographic conditions of the hydrophilic retention column are as follows:

[0041] 1.1 Chromatographic column: hydrophilic retention column, 250mm×4.6mm, 3.5μm or equivalent;

[0042] 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;

[0043] 1.3 Column temperature: 25°C;

[0044] 1.4 Differential refractive index detector temperature: 35°C;

[0045] 1.5 Flow rate: 1 mL / min;

[0046] 1.6 Injection volume: 5 μL;

[0047] 1.7 Running time: 45min.

[0048] 2. Amide bonded column chromatography conditions are as follows:

[0049] 2.1 Chromatographic column: Amide bonded column, 150 mm × 4.6 mm, 3 μm or equivalent;

[0050] 2.2 Mobile phase: acetonitrile: water = 64:36 (v / v);

[0051] 2.3 Column temperature: 25°C;

[0052] 2.4 Differential refractive index detector temperature: 37°C;

[0053] 2.5 Flow rate: 1.1 mL / min;

[0054] 2.6 Injection volume: 5 μL;

[0055] 2.7 Running time: 8min.

[0056] In the following examples, 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.

[0057] The calcium type gel resin used in the present invention has a cross-linking degree of 4-8%, a pore diameter of 0.5-5 nm, and a porosity of 30-40%.

[0058] Example 1

[0059] This embodiment provides a method for purifying 2′-fucosyllactose (2′-FL). The process flow chart is as follows: Figure 1 The specific steps are as follows:

[0060] (1) 10 L of 2′-fucosyllactose fermentation broth (2′-fucosyllactose content: 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. The resulting supernatant was calculated to have a yield of 98.5%.

[0061] (2) The clear solution obtained in step (1) was passed through a 5000Da ultrafiltration membrane, circulated to the minimum volume, and then top-washed with pure water three times until the sugar content of the clear solution was less than 1%. The 2′-fucosyllactose content was detected to be 8.7%, and the yield of this process was calculated to be 98.9%;

[0062] (3) 10 L of the supernatant from step (2) was decolorized by resin (LXT 036) at a flow rate of 3 BV / h to obtain a decolorized solution. The content of 2′-fucosyllactose was detected to be 7.8%, and the calculated yield was 95.5%;

[0063] (4) The decolorized solution obtained in step (3) was subjected to adsorption desalination by cation exchange resin LX101 (Lanxiao Bio) and anion exchange resin LXT203 (Lanxiao Bio) 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%;

[0064] (5) The desalted liquid in step (4) was concentrated by reverse osmosis high-pressure membrane and evaporation to obtain a concentrate having a 2′-fucosyllactose content of 46.5% and a calculated yield of 96.9%;

[0065] (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 55° C.; the purity of 2′-fucosyllactose in the eluate is 97.7%, and the yield is 90.3%;

[0066] (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%.

[0067] Example 2

[0068] This example provides a method for purifying 2′-fucosyllactose (2′-FL), and the specific steps are as follows:

[0069] (1) 10 L of 2′-fucosyllactose fermentation broth (2′-fucosyllactose content: 18%) was heated to 85°C for 30 min, cooled to 30°C, and filtered through a 200 nm filter membrane to remove the production bacteria and protein. The resulting supernatant was calculated to have a yield of 98.9%.

[0070] (2) The clear solution obtained in step (1) was passed through a 3000 Da ultrafiltration membrane, circulated to the minimum volume, and then top-washed with pure water three times until the sugar content of the clear solution was less than 1%. The 2′-fucosyllactose content was detected to be 12.3%, and the yield of this process was calculated to be 97.9%;

[0071] (3) 10 L of the clear solution from step (2) was decolorized by resin (LXT 067) at a flow rate of 6 BV / h to obtain a decolorized solution. The content of 2′-fucosyllactose was detected to be 11.7%, and the calculated yield was 95.3%;

[0072] (4) The decolorized solution obtained in step (3) was subjected to adsorption desalination by cation exchange resin LXT110 (Lanxiao Bio) and anion exchange resin LX 207 (Lanxiao Bio) 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%;

[0073] (5) The desalted liquid in step (4) was concentrated by high-pressure membrane and evaporation to obtain a concentrate having a 2′-fucosyllactose content of 55% and a calculated yield of 95.3%;

[0074] (6) The concentrated solution obtained in step (5) was separated using a continuous chromatography column (Ca-type gel resin) and eluted with pure water to obtain an eluate, wherein the column temperature of the chromatography column was 50° C.; the purity of 2′-fucosyllactose in the eluate was 98.2%, and the yield was 94.8%;

[0075] (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%.

[0076] Example 3

[0077] This example provides a method for purifying 2′-fucosyllactose (2′-FL), and the specific steps are as follows:

[0078] (1) 10 L of 2′-fucosyllactose fermentation broth (2′-fucosyllactose content: 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. The resulting supernatant was calculated to have a yield of 99.1%.

[0079] (2) The clear solution 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 solution was less than 1%. The 2′-fucosyllactose content was detected to be 11.3%, and the yield of this process was calculated to be 98.3%;

[0080] (3) 10 L of the clear solution from step (2) was decolorized by resin (LXT 067) at a flow rate of 2 BV / h to obtain a decolorized solution. The content of 2′-fucosyllactose was detected to be 10.4%, and the calculated yield was 94.7%;

[0081] (4) The decolorized solution obtained in step (3) was subjected to adsorption desalination by cation exchange resin D001 (Lanxiao Bio) and anion exchange resin D301 (Lanxiao Bio) 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%;

[0082] (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%;

[0083] (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 65° C.; the purity of 2′-fucosyllactose in the eluate is 97.9%, and the yield is 90.8%;

[0084] (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%.

[0085] Example 4

[0086] This example provides a method for purifying 3-fucosyllactose. The difference between this example and Example 1 is that the fermentation broth used is 3-fucosyllactose fermentation broth, and in step (1), instead of ceramic membrane filtration, centrifugation 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%.

[0087] The content of 3-fucosyllactose in the concentrated solution in step (5) is 45.9%.

[0088] 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%.

[0089] Example 5

[0090] This example provides a method for purifying 2′-fucosyllactose (2′-FL). The difference from Example 1 is that in step (1), ceramic membrane filtration is not used, but centrifugation is used to remove production bacteria and proteins, etc., at a rotation speed of 9000 rpm to obtain a filtered clear liquid. The yield of this process is calculated to be 90.1%.

[0091] The content of 2′-fucosyllactose in the concentrated solution in step (5) is 48.5%.

[0092] 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%.

[0093] Example 6

[0094] This example provides a method for purifying 2′-fucosyllactose (2′-FL). The difference between this method and Example 1 is that activated carbon is used for decolorization in step (3) to obtain a decolorized solution. The content of 2′-fucosyllactose is detected to be 7.5%, and the calculated yield is 90.5%.

[0095] The content of 2′-fucosyllactose in the concentrated solution in step (5) was 52.3%. The purity of the finally obtained 2′-fucosyllactose was 98.2%, and the total yield was 75.4%. Among them, the content of 2′-fucosyllactulose was 0.37%.

[0096] Example 7

[0097] This example provides a method for purifying 2′-fucosyllactose (2′-FL). The method differs from Example 1 in that, in step (4), the decolorized solution 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 solution. The conductivity of the desalted solution is 59.3 μs / cm, the content of 2′-fucosyllactose in the desalted solution is 7.59%, and the calculated yield is 97.1%.

[0098] The content of 2′-fucosyllactose in the concentrated solution in step (5) is 35%.

[0099] 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%.

[0100] Comparative Example 1

[0101] This comparative example provides a method for purifying 2′-fucosyllactose (2′-FL). The difference between the method and Example 1 is 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%.

[0102] Comparative Example 2

[0103] This comparative example provides a method for purifying 2′-fucosyllactose (2′-FL). The difference between the comparative example and 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 three 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%.

[0104] Comparative Example 3

[0105] This comparative example provides a method for purifying 2′-fucosyllactose (2′-FL). The method differs from Example 1 in that the conditions for the 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%. The 2′-fucosyllactulose content is 1.31%.

[0106] Comparative Example 4

[0107] 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%.

[0108] 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%.

[0109] Comparative Example 5

[0110] 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%.

[0111] 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%.

[0112] In this comparative example, the content of 2′-fucosyllactose in the concentrated solution of step (5) was greater than 55%, the poor fluidity of the material in the chromatographic column affected the separation effect, the purity and total yield of 2′-fucosyllactose were not as good as those in Example 2, and the time required for concentration and chromatographic separation was long, which affected the production efficiency of 2′-fucosyllactose.

[0113] 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.

[0114] (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.

[0115] (3) Conventional technical means of the prior art can be used to remove the production bacteria and macromolecular substances from the fermentation broth, and to perform decolorization and desalination. As long as the content of fucosyllactose in the concentrate is maintained at 35-55% before continuous chromatographic separation on the column, a final product containing less than 1% fucosyllactulose in the fucosyllactose can be obtained by chromatographic separation using a calcium-type gel resin.

[0116] It should be noted that the endpoints of the ranges and any values ​​disclosed herein 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 endpoints of each range, the endpoints of each range and 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 considered to be specifically disclosed herein.

[0117] In the description of this specification, the reference terms "one embodiment", "some embodiments", "specific implementation methods", or "some specific implementation methods" and the like mean 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 embodiments 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 can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0118] 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 various 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 liquid with a fucosyllactose content of 35-55% is obtained, and continuous chromatographic separation is performed. The chromatographic column used for the continuous chromatographic separation is filled with a calcium-type gel resin, the chromatographic column temperature 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 is selected from one or more of 2′-fucosyllactose fermentation broth and 3-fucosyllactose fermentation broth.

3. The method for purifying fucosyllactose according to claim 1, wherein: The step of removing the production bacteria and macromolecular substances from the fucosyllactose fermentation broth comprises: The fucosyllactose fermentation broth is pretreated to denature the protein, and then filtered through a filter membrane or mechanically separated to remove the production bacteria and macromolecular substances.

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 to 200 nm; And / or, the mechanical separation is centrifugal separation with a rotation speed of 5000 to 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 cut-off of 3000 to 5000 Da.

6. The method for purifying fucosyllactose according to claim 1, characterized in that: The decolorization adopts resin decolorization, the feeding speed 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 to 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, wherein: After the desalting process, the feed liquid is subjected to membrane concentration and evaporation concentration processes to make the fucosyllactose content in the concentrate 35-55%, and then enters the continuous chromatography separation process; 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 at a feed temperature of 130-190° C. and an exhaust temperature of 80-95° C. to obtain purified fucosyllactose.

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