A method for preparing high-quality 2'-fucosylated lactose

Abstract

This invention discloses a method for preparing 2'-fucosyllactose (2'-FL) crystals. First, amorphous crude 2'-fucosyllactose is dissolved in water by heating, stirred, and filtered to obtain a filtrate. Then, the filtrate is stirred and cooled to a first temperature, at which point isopropanol is added. When oil precipitation occurs, seed crystals are added and the mixture is sonicated, maintaining the first temperature for crystal growth for a certain time. The temperature is then further lowered to a second temperature and maintained for a certain time to continue crystal precipitation. The resulting system containing crystals is filtered, washed, and the solid portion is dried to obtain the final product. This application yields high-quality 2'-fucosyllactose crystals with good particle size, good flowability, high yield, and high purity.

Description

Technical Field

[0001] This invention belongs to the field of food additive refining, and relates to a method for preparing high-quality 2'-fucosylated lactose crystals. Background Technology

[0002] 2'-Fucosyllactose (abbreviated as 2'-FL or 2'-FLH) is a natural polysaccharide extracted from marine brown algae. It possesses various biological activities, such as anti-tumor, antiviral, antioxidant, and anti-inflammatory effects. 2'-Fucosyllactose is the third largest solid component in breast milk, after lactose and fat, and is an important functional factor with significant biological functions. It plays a crucial role in the growth and development of newborns and is a probiotic oligosaccharide widely used in food and pharmaceutical fields. In general, 2'-Fucosyllactose, as a novel functional food ingredient, has broad market prospects and application value. However, the synthesis of 2'-FL using chemical or enzymatic methods suffers from drawbacks such as cumbersome reaction processes, high costs, and low product yields, failing to meet market demands. With technological advancements and increased emphasis on health, the research and application of fucosyllactose will receive wider attention and promotion.

[0003] 2'-Fucose-based lactose currently faces significant challenges in purification, such as poor stability in amorphous form, while crystalline products are expensive and have poor flowability. Summary of the Invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide a method for preparing high-quality 2'-fucosylated lactose crystals, which addresses the shortcomings of the prior art. The method uses crude amorphous 2'-fucosylated lactose as raw material to prepare high-quality 2'-fucosylated lactose crystals with good particle size, good flowability, high yield and high purity.

[0005] To address the aforementioned technical problems, this invention discloses a method for preparing 2'-fucosylated lactose crystals, characterized by comprising the following steps:

[0006] (1) Dissolve the crude amorphous product of 2'-fucosylation lactose in water by heating, stir and mix well, filter and obtain the filtrate;

[0007] (2) Stir and cool the filtrate obtained in step (1), preferably by using a water bath circulation to cool it down. After cooling down to the first temperature, add isopropanol to the system; when oil precipitation occurs, add seed crystals and perform ultrasound, maintain the first temperature to grow crystals for a certain time; then continue to cool down to the second temperature and maintain the temperature for a certain time to continue to precipitate crystals. The first temperature is 20-35℃ and the second temperature is 0-5℃.

[0008] (3) Filter the system containing crystals obtained in step (2), rinse it, and dry the solid part to obtain the final product.

[0009] In step (1), the purity of the crude 2'-fucosylated lactose is ≥93.5%; the water is any one of tap water, pure water, or ultrapure water; and the heating and dissolving temperature is 50-55℃.

[0010] In step (1), the concentration of the crude 2'-fucosylated lactose in water is 400-500 g / L.

[0011] In step (1), the stirring rate is 200-300 r / min; in step (2), the stirring rate is 200-400 r / min.

[0012] In step (2), the amount of isopropanol added is 3 to 6 times the volume of the filtrate. In some specific embodiments, the isopropanol is added slowly. Adding the isopropanol binary solvent before crystal nucleation is beneficial for the nucleation of 2'-fucosylated lactose crystals and shortens the crystallization induction period.

[0013] In step (2), the amount of 2'-fucosylated lactose seed crystals used is 0.1% to 0.4% of the amorphous crude 2'-fucosylated lactose; the ultrasonic frequency is 20 to 60 kHz.

[0014] In step (2), maintain the first temperature for crystal growth for 150-200 minutes.

[0015] In step (2), the cooling rate is 0.3–0.7 °C / min, and after cooling to the second temperature, the temperature is maintained for 1–2 hours to continue crystal precipitation. The cooling rate is controlled to avoid the generation of nucleation bursts during cooling crystallization.

[0016] In step (3), the rinsing solution is an ethanol solution; the drying is a heat drying process with a drying temperature of 40-55°C and a drying time of 6-12 hours.

[0017] In a preferred embodiment, in step (1), the concentration of crude 2'-fucosylated lactose in water is 400-500 g / L, and the stirring rate is 200-300 r / min; in step (2), the stirring rate is 200-400 r / min, the amount of 2'-fucosylated lactose seed crystals used is 0.1%-0.4% of the mass of amorphous crude 2'-fucosylated lactose, and the ultrasonic amplitude is 20%-50%.

[0018] The crystal has at least XPRD characteristic peaks selected from those with 2θ values ​​of 13.62°±0.2°, 15.21°±0.2°, 16.98°±0.2°, 18.29°±0.2°, 20.61°±0.2°, and 25.31°±0.2°, and has essentially the same characteristics as... Figure 4 The TG-DSC spectrum shown.

[0019] This application uses a solution with a high initial concentration, employs ultrasound and the addition of seed crystals to promote nucleation, and screens crystallization systems including solutions such as ethanol, methanol, water, isopropanol, n-butanol, ethyl acetate, and acetone. After screening, the water and isopropanol system was selected for experiments, and the resulting product has advantages such as high yield and good bulk density.

[0020] Beneficial effects: Compared with the prior art, this application has the following advantages:

[0021] (1) This invention affects the nucleation and growth process of 2'-fucosylated lactose crystals by regulating the stirring rate, dropping rate and solvent synergy of the system, and uses cooling crystallization to increase the yield of 2'-fucosylated lactose crystals, thus preparing high-quality 2'-fucosylated lactose crystal products.

[0022] (2) This invention optimizes the quality of crystal particles by exploring the initial concentration of the solution, different crystallization systems, the amount of seed crystals added, and the ultrasonic amplitude;

[0023] (3) The 2'-fucosylated lactose crystals prepared by the present invention have the characteristics of high crystallinity, large crystal size and good fluidity, which significantly improves the product quality. Attached Figure Description

[0024] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0025] Figure 1 Microscopic image of amorphous 2'-fucosylated lactose (magnification 10×10);

[0026] Figure 2 Microscopic image of 2'-fucosylated lactose crystals from Example 1 (magnification 10×10);

[0027] Figure 3 X-ray diffraction (XRPD) pattern of 2'-fucosylated lactose crystals;

[0028] Figure 4 TG-DSC image of 2'-fucosylated lactose crystals. Detailed Implementation

[0029] The characterization method and instruments for 2'-fucosylated lactose crystals of this invention are as follows:

[0030] Powder X-ray diffraction: Approximately 0.100 g of the ground 2'-fucosylated lactose crystal powder was collected using a powder X-ray diffractometer (Rigaku Smartlab or Bruker D8 Advance) at room temperature. The light source was Cu Kα rays, the scanning step was 0.02°, the scanning voltage was set to 40 kV, the current to 40 mA, the scanning rate to 0.2 s / 0.02°, and the scanning range 2θ to 5–50°. The data were analyzed using JADE software and plotted using Origin software.

[0031] Thermogravimetric analysis and differential scanning calorimetry: Approximately 0.030 g of the ground 2'-fucosylated lactose crystal powder was placed in the sample pans of the thermogravimetric analyzer and the differential scanning calorimeter, respectively, and the weight change was tracked from room temperature to 500℃. The atmosphere used was nitrogen, and the heating rate was 20℃ / min.

[0032] Particle size analysis: Approximately 0.100 g of crystalline solid 2'-fucosylated lactose was taken and its particle size was determined using a Malvern instrument at room temperature. The dispersant was ethanol.

[0033] Yield Calculation: Yield refers to the ratio of the actual product output obtained from a unit quantity of raw materials input to the theoretically calculated product output in a chemical reaction or related chemical industrial production. In this experiment, the yield calculation formula is as follows:

[0034]

[0035] Among them, Y R The yield of 2'-fucosylation lactose is represented by m0, which refers to the mass of 2'-fucosylation lactose crystals calculated theoretically, and m1 refers to the mass of 2'-fucosylation lactose crystals actually obtained.

[0036] The crude amorphous 2'-fucosylation lactose used in the examples was obtained from Hubei Huizepu Pharmaceutical Technology Co., Ltd., and the purity of the crude 2'-fucosylation lactose was ≥93.5%. The 2'-fucosylation lactose seed crystals used in the examples were purchased from Hubei Huizepu Pharmaceutical Technology Co., Ltd., and the purity was ≥98%.

[0037] Example 1

[0038] 50g of crude 2'-fucosyllactose (95.5%) was dissolved in 100mL of pure water. The solution was stirred at 200r / min at 55℃ until completely dissolved. After stabilizing for 10min, the solution was hot-filtered, and the filtrate was poured into a 500mL water bath circulation jacket. Stirring was started at 400r / min, and the initial temperature (first temperature) was set to 25℃ using a programmed temperature control mode. Isopropanol, an organic solvent four times the volume of the filtrate, was pumped into the system at a dropping rate of 0.3mL / min. When oil separation occurred, 0.3g of 2'-fucosyllactose seed crystals were slowly added to the system, and ultrasonication with an amplitude of 50% was used to promote nucleation. The temperature was then lowered to 5±0.5℃ (second temperature) at a cooling rate of 0.5℃ / min and maintained at this temperature for 120min, resulting in an increase in the number of crystals. Stirring was continued for 2 hours, and the product was observed under a microscope. (See photograph for details.) Figure 2 .

[0039] The crystal slurry was filtered and washed with 90 mL of ethanol, then heated and dried at 55°C for 6 hours to obtain white crystalline solid particles with good flowability.

[0040] The corresponding XRPD spectrum is as follows: Figure 3 As shown, the characteristic peaks are located at 13.62°±0.2°, 15.21°±0.2°, 16.98°±0.2°, 18.29°±0.2°, 20.61°±0.2°, and 25.31°±0.2°; the TG-DSC thermal analysis curve is shown below. Figure 4 As shown, the melting point is 247.5℃, indicating that the crystal is relatively stable at room temperature.

[0041] Example 2

[0042] 50g of crude 2'-fucosylated lactose (95.5%) was dissolved in 100mL of pure water. The solution was stirred at 200r / min at 55℃ until completely dissolved. After stabilizing for 10min, the solution was hot-filtered, and the filtrate was poured into a 500mL water bath circulation jacket. Stirring was started at 350r / min, and the initial temperature (first temperature) was set to 25℃ using a programmed temperature control mode. Isopropanol, an organic solvent four times the volume of the filtrate, was pumped into the system at a dropping rate of 0.3mL / min. When oil separation occurred, 0.3g of 2'-fucosylated lactose seed crystals were slowly added to the system, and ultrasonication with an amplitude of 50% was used to promote nucleation. The temperature was then lowered to 5±0.5℃ (second temperature) at a cooling rate of 0.5℃ / min and maintained at this temperature for 120min, resulting in an increase in the number of crystals. Stirring was continued for 2 hours, and the product was observed under a microscope. (See photograph for details.) Figure 2 .

[0043] The crystal slurry was filtered and washed with 90 mL of ethanol, then heated and dried at 55°C for 6 hours to obtain white crystalline solid particles with good flowability.

[0044] Example 3

[0045] 50g of crude 2'-fucosylated lactose (95.5%) was dissolved in 100mL of pure water. The solution was stirred at 200r / min at 55℃ until completely dissolved. After stabilizing for 10min, the solution was hot-filtered, and the filtrate was poured into a 500mL water bath circulation jacket. Stirring was started at 400r / min, and the initial temperature (first temperature) was set to 25℃ using a programmed temperature control mode. Isopropanol, an organic solvent four times the volume of the filtrate, was pumped into the system at a dropping rate of 0.5mL / min. When oil separation occurred, 0.3g of 2'-fucosylated lactose seed crystals were slowly added to the system, and ultrasonication with an amplitude of 50% was used to promote nucleation. The temperature was then lowered to 5±0.5℃ (second temperature) at a cooling rate of 0.5℃ / min and maintained at this temperature for 120min, resulting in an increase in the number of crystals. Stirring was continued for 2 hours, and the product was observed under a microscope. (See photograph for details.) Figure 2 .

[0046] The crystal slurry was filtered and washed with 90 mL of ethanol, then heated and dried at 55°C for 6 hours to obtain white crystalline solid particles with good flowability.

[0047] Example 4

[0048] 50g of crude 2'-fucosylated lactose (95.5%) was dissolved in 100mL of pure water. The solution was stirred at 200r / min at 55℃ until completely dissolved. After stabilizing for 10min, the solution was hot-filtered, and the filtrate was poured into a 500mL water bath circulation jacket. Stirring was started at 350r / min. A programmed temperature control mode was used, with the initial temperature (first temperature) set at 25℃. Isopropanol, an organic solvent four times the volume of the filtrate, was pumped into the system at a dropping rate of 0.5mL / min. When oil precipitation occurred, 0.3g of 2'-fucosylated lactose seed crystals were slowly added to induce precipitation. The temperature was then lowered to 50±0.5℃ (second temperature) at a cooling rate of 0.5℃ / min and maintained at this temperature for 120min. An increase in the number of crystals was observed. Stirring was continued for 2 hours, and the crystals were observed under a microscope.

[0049] The crystal slurry was filtered and washed with 90 mL of ethanol, then heated and dried at 55°C for 6 hours to obtain white crystalline solid particles with good flowability.

[0050] Comparative Example 1

[0051] The process of this comparative example is the same as that of Example 1, except that the stirring rate is different when using isopropanol as the antisolvent. Specifically, the stirring rate is adjusted to 300 r / min.

[0052] Comparative Example 2

[0053] The process of this comparative example is the same as that of Example 1, except that the stirring rate is different when using isopropanol as the antisolvent. Specifically, the stirring rate is adjusted to 250 r / min.

[0054] Comparative Example 3

[0055] The process of this comparative example is the same as that of Example 3, except that the stirring rate is different when using isopropanol as the antisolvent. Specifically, the stirring rate is adjusted to 300 r / min.

[0056] Comparative Example 4

[0057] The process of this comparative example is the same as that of Example 4, except that the stirring rate is different when using isopropanol as the antisolvent. Specifically, the stirring rate is adjusted to 250 r / min.

[0058] Comparative Example 5

[0059] The comparative example uses the same process as Example 1, except that the amplitude during ultrasonication is different, specifically 25%.

[0060] Comparative Example 6

[0061] The comparative example uses the same process as Example 1, except that the amount of seed crystals added is different, specifically 0.2g.

[0062] Comparative Example 7

[0063] The comparative example uses the same process as Example 1, except that the amount of seed crystals added is different, specifically 0.1g.

[0064] Comparative Example 8

[0065] The amount of seed crystals added in this comparative example is the same as that in CN201980083196.6, and the initial concentration of the 2'-fucosylated lactose solution is the same. The specific antisolvent is methanol.

[0066] Comparative Example 9

[0067] The amount of seed crystals added and the initial concentration of the 2'-fucosylated lactose solution are the same as in CN201980083196.6, with ethanol as the antisolvent.

[0068] Comparative Example 10

[0069] In this comparative example, the amount of seed crystals added is the same as that in CN201980083196.6, and the initial concentration of the 2'-fucosylated lactose solution is the same. Specifically, the antisolvent is butanol.

[0070] Comparative Example 11

[0071] In this comparative example, the amount of seed crystals added is the same as that in CN201980083196.6, and the initial concentration of the 2'-fucosylated lactose solution is the same. Specifically, the antisolvent is acetic acid.

[0072] Comparative Example 12

[0073] In this comparative example, the amount of seed crystals added and the initial concentration of the 2'-fucosylated lactose solution are the same as in CN201980083196.6, with butyric acid as the antisolvent.

[0074] Comparative Example 13

[0075] The process of this comparative example is the same as that of Example 1, except that the good solvent and the anti-solvent are different. Specifically, the good solvent is methanol and the anti-solvent is n-butanol.

[0076] Comparative Example 14

[0077] The comparative example uses the same process as Example 1, except that the good solvent and the antisolvent are different. Specifically, the good solvent is methanol and the antisolvent is ethyl acetate.

[0078] Comparative Example 15

[0079] The process of this comparative example is the same as that of Example 1, except that the initial concentration of the solution is different, specifically 200 g / L.

[0080]

[0081]

[0082]

[0083] Comparative Example 16

[0084] The comparative example follows the same process as Example 1, except for the cooling rate, which is specifically controlled at 0.3℃ / min.

[0085] The purified 2'-fucosylated lactose crystals obtained in the above examples were tested and compared under different experimental conditions, and the results are shown in Table 1.

[0086] Table 1 Comparison of experimental parameters for 2-fucosylated lactose obtained under different conditions

[0087] This invention provides a method for preparing high-quality 2-fucosylated lactose crystals. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A method for preparing 2'-fucosylated lactose crystals, characterized in that, Includes the following steps: (1) Dissolve the crude amorphous 2'-fucosylated lactose in water by heating, stir and mix well, filter and obtain filtrate, wherein the purity of the crude 2'-fucosylated lactose is ≥93.5%; the water is any one of tap water, pure water, or ultrapure water; the heating and dissolving temperature is 50~55℃; (2) Stir and cool the filtrate obtained in step (1). After cooling to the first temperature, add isopropanol to the system. The amount of isopropanol added is 3 to 6 times the volume of the filtrate. When oil precipitation occurs, add seed crystals and perform ultrasound. Maintain the first temperature for crystal growth for a certain period of time. Then continue to cool to the second temperature and maintain the temperature for a certain period of time to continue crystal precipitation. The first temperature is 20 to 35°C and the second temperature is 0 to 5°C. The amount of seed crystals used is 0.1% to 0.4% of the crude amorphous 2'-fucosylated lactose. The ultrasound frequency is 20 to 60 kHz. (3) Filter the system containing crystals obtained in step (2), rinse it, and dry the solid part to obtain the final product.

2. The preparation method according to claim 1, characterized in that, In step (1), the concentration of the crude 2'-fucosylated lactose in water is 400~500 g / L.

3. The preparation method according to claim 1, characterized in that, In step (1), the stirring rate is 200~300 r / min; in step (2), the stirring rate is 200~400 r / min.

4. The preparation method according to claim 1, characterized in that, In step (2), maintain the first temperature for crystal growth for 150~200 min.

5. The preparation method according to claim 1, characterized in that, In step (2), the cooling rate is 0.3~0.7℃ / min, and after cooling to the second temperature, the temperature is maintained for 1~2 h to continue crystal precipitation.

6. The preparation method according to claim 1, characterized in that, In step (3), the rinsing solution is an ethanol solution; the drying is a heat drying process with a drying temperature of 40~55℃ and a drying time of 6~12 h.

7. A 2'-fucosylated lactose crystal, characterized in that, The crystal has the XPRD characteristic peaks shown in Figure 3 and the TG-DSC spectrum shown in Figure 4.

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