A crystalline form of a trisaccharide

Abstract

This invention provides a lactose- N Lacto-N-triose II crystal form A belongs to the field of separation and purification technology. The lactose-... N The trisaccharide crystal form A exhibits characteristic diffraction peaks in its powder X-ray diffraction pattern at diffraction angles (2θ ± 0.2°) at positions of 4.80, 8.32, 9.62, 12.72, 18.67, 19.90, 21.05, and 21.59. This invention provides a stable lactose- N Lacto-N-triose II crystal form A and its preparation method were achieved, realizing lactose- N The iterative development of lacto-N-triose II preparation technology has provided a reliable technology for large-scale industrial production.

Description

Technical Field

[0001] This invention relates to a lactose-N-trisaccharide crystal, belonging to the field of separation and purification technology. Background Technology

[0002] Human milk oligosaccharides (HMOs) are a class of complex oligosaccharides, abundant in human milk and possessing unique functions. They are composed of monosaccharides and their derivatives, sialic acid, and other structural units linked by glycosidic bonds. To date, more than 200 different oligosaccharides with different structures have been discovered. However, our understanding of them has taken more than a century.

[0003] As early as 1886, Australian pediatrician and microbiologist Escherich discovered a link between infant gut bacteria and digestive function. In 1900, researchers Moro and Tissier separately confirmed that the bacterial composition of the feces of breastfed and formula-fed infants differed. Could there be components in breast milk that cause these differences in infant gut bacteria? In 1926, It was discovered that whey in breast milk contains growth-promoting factors that enhance the growth of Bifidobacteria. So what exactly in whey promotes the growth of Bifidobacteria? In 1930, French scientists Polonowski and Lespagnol detected a carbohydrate-like component in breast milk whey, naming it "gynolactose." In 1954, It was confirmed that the factor "bifidus factor" promoting the growth of Bifidobacteria is actually an oligosaccharide. In the same year, scientists Polonowski and Montreuil used two-dimensional paper chromatography to isolate oligosaccharides from lactose oligosaccharides. In 1983, Egge et al. confirmed HMOs using rapid atomic bombardment mass spectrometry. In 1999, Coppa et al. detected 30-50% of HMOs in the feces of breastfed infants in their original structural form. In 2000, some scholars found that HMOs can withstand hydrolysis by enzymes in the infant's digestive tract. This phenomenon fully demonstrates that the biological function of human lactose oligosaccharides is not simply to provide substances and energy for infants.

[0004] Since the beginning of the 21st century, advancements in research methods have revealed that the concentration of total HMOs (acidic and neutral oligosaccharides) in breast milk decreases with prolonged lactation. Furthermore, HMOs have been found to be crucial for infant growth and development, both now and in the long term. They promote gut microbiota balance, foster the proliferation of beneficial bacteria, inhibit the growth of harmful bacteria, resist pathogenic infections, prevent the colonization of pathogenic bacteria, prevent inflammatory bowel disease and gastroenteritis, regulate the immune system, and promote infant cognitive development. Therefore, timely supplementation with HMOs is beneficial for maintaining the healthy growth of infants and young children. However, carbohydrate synthesis has always been a significant challenge for synthetic scientists. From the perspective of carbohydrate synthesis in living organisms, the synthesis of these molecules is not a simple replication of a single template but is regulated by multiple glycosyltransferases and glycosidases, thus determining the complexity, diversity, and microscopic heterogeneity of carbohydrate structures. Encouragingly, with the rapid development of glycochemistry and recombinant enzymology, glycochemists and glycobiologists have conducted extensive research in the field of carbohydrate synthesis, reporting a series of new methods and strategies for synthesizing oligosaccharides.

[0005] Lacto-N-triose (English name: Lacto-N-trioseII, abbreviated as LNTII, also known as GlcNAc-β1,3-Gal-β1,4-Glc, CAS: 75645-27-1, chemical structure as shown in Formula 1).

[0006]

[0007] LNTII is a main-chain precursor of HMOs, currently generally prepared by chemical or biological methods. According to literature, its solid form can be obtained through evaporation and concentration followed by filtration and drying, freeze-drying, or spray drying. Chinese invention patent CN112154150A (application number 201980034406.2) describes the synthesis of human milk oligosaccharides via enzymatic fermentation, followed by enzymatic treatment, ultrafiltration, nanofiltration, and separation and purification by chromatographic column drying to obtain solid oligosaccharides. This patent provides a general method for obtaining the solid form but does not mention purification by crystallization. Multiple experiments have confirmed that this method yields an amorphous solid. Chinese invention patent CN109705175A (application number 201811603666.0) describes a method for purifying neutral human milk oligosaccharides from a crude solution containing neutral human milk oligosaccharides, including the use of simulated moving bed chromatography and purification steps such as concentration, dialysis, and / or filtration, finally spray-drying to obtain an amorphous powder with a particle size of 5-500 micrometers. CN104428307 (A) (WO2013 / 185780) discloses a method for removing or at least significantly reducing the amount of organic solvent residues in HMO, which includes the step of spray drying an aqueous solution of HMO, the method providing HMO in an amorphous solid form.

[0008] In spray drying, raw materials tend to stick to the inner wall of the equipment during the spraying process, resulting in some material loss. Compared with crystallization separation and purification methods, it has problems such as high energy consumption, large labor input, and low purification effect. In addition, the high temperature process can easily cause changes in the physicochemical properties of some raw materials, and the resulting amorphous powder has strong hygroscopicity, which increases the difficulty of product storage and use.

[0009] CN110483652A (application number 201910501997.1) describes a technology for obtaining human milk oligosaccharides (HMOs) by freeze drying. However, the freeze-dried powder (amorphous) obtained by freeze drying technology also requires freezing and / or low temperature in its storage and transportation, which increases the cost and difficulty of product storage and use. Moreover, the freeze drying process has the disadvantages of large equipment investment, huge energy consumption and low production efficiency.

[0010] In summary, to provide the market with a stable and cost-effective solid form of LNTII and meet the needs of product iteration, there is an urgent need to develop an efficient and large-scale industrially applicable LNTII separation method, and to provide LNTII solid forms with more advantages to facilitate the storage of industrial products and accelerate their application in various products such as dairy products, food, pharmaceuticals, cosmetics, and feed, thereby meeting the needs of the public. Summary of the Invention

[0011] Purpose of the invention: To provide a stable new crystal form of LNTII and its preparation method, to achieve the iteration of LNTII separation technology and solid product morphology, and to provide a reliable technology for large-scale industrial production.

[0012] Through experiments, the applicant obtained a new crystal form of LNTII, which was named LNTII crystal form A. Details are as follows:

[0013] The technical solution of the present invention is: an LNTII crystal form A, whose powder X-ray diffraction pattern has characteristic peaks at 2θ values ​​(2θ±0.2°) of 4.80, 8.32, 9.62, 12.72, 18.67, 19.90, 21.05 and 21.59.

[0014] Preferably, the LNTII crystal form A has a powder X-ray diffraction pattern with characteristic peaks at 2θ values ​​(2θ±0.2°) of 4.80, 7.24, 8.32, 9.62, 12.72, 16.67, 18.67, 19.28, 19.90, 21.05, 21.59, 22.14 and 26.93.

[0015] Preferably, the crystal form A of the present invention has characteristic peaks in its powder X-ray diffraction pattern at 2θ values ​​(2θ±0.2°) of 4.80, 7.24, 8.32, 9.62, 12.72, 16.67, 17.35, 18.67, 19.28, 19.90, 20.72, 21.05, 21.59, 22.14, 23.17, 23.71, 24.15, 25.20, 26.93, 29.12, and 29.49.

[0016] Preferably, the crystal form A of the present invention has characteristic peaks in its powder X-ray diffraction pattern at 2θ values ​​(2θ±0.2°) of 4.80, 7.24, 8.32, 9.62, 9.89, 11.65, 12.72, 14.42, 16.67, 17.35, 18.67, 19.28, 19.90, 20.72, 21.05, 21.59, 22.14, 23.17, 23.71, 24.15, 25.20, 26.93, 28.68, 29.12, 29.49, 30.34, 31.08, 31.88, and 34.10.

[0017] The melting point of LNTII crystal form A described in this invention is in the range of 191-196°C; while the melting point of amorphous powder LNTII disclosed in the prior art is in the range of 130-134°C.

[0018] The preparation method of LNTII crystal form A includes the following steps:

[0019] Step 1. Prepare a solution containing LNTII for later use.

[0020] Preferably, the concentration of the LNTII solution is 0.2-0.5 g / ml;

[0021] Step 2. Cool the LNTII-containing solution obtained in Step 1. Preferably, cool it to 25°C or below. More preferably, the cooling rate should be 0.5°C / min, and should not be too fast. Set aside for later use.

[0022] Step 3. Add acetone to the solution from step 2 while stirring to allow crystallization.

[0023] Preferably, the amount of acetone added is equal to or greater than the volume of water. More preferably, the volume ratio of water to acetone is 1:2-3; more preferably, it is 1:2.4-2.7; and even more preferably, it is 1:2.5.

[0024] Preferably, acetone is added under stirring at a flow rate of 0.07-0.25 ml / min; after the addition is complete, crystals are grown under stirring.

[0025] Seed crystals can also be added in this step of the process;

[0026] Preferably, the stirring rate during the crystallization process is 100 r / min or higher.

[0027] Preferably, after the acetone is added, stirring should continue, and the crystal growth time should be no less than 12 hours.

[0028] The method for preparing LNTII crystal form A includes separating the crystal in a solution containing LNTII, wherein the solvent of the solution contains water and acetone.

[0029] Preferably, the LNTII crystal form A of the present invention can be obtained by adding acetone to a fermentation broth containing LNTII after purification by decolorization, desalting, etc., and then cooling and crystallizing; or it can be obtained by preparing LNTII solid into an aqueous solution, adding acetone to it, and then cooling and crystallizing. Cooling and crystallizing refers to cooling to a temperature below 25°C.

[0030] The lactose-N-triose II crystal form A described in this invention can be used as an intermediate in the synthesis of other sugars or compounds, or as an end product.

[0031] Beneficial effects:

[0032] This invention provides a stable LNTII crystal form A and its preparation method, achieving an iteration of LNTII preparation technology and providing a reliable technology for large-scale industrial production. Compared with existing amorphous powders and preparation methods, the described crystal form A and its preparation method have the following advantages:

[0033] (1) The stability of LNTII crystal form A described in this invention is superior to that of amorphous powder, which is beneficial for the long-term storage of LNTII products and their application in food and pharmaceuticals. It can reduce the packaging cost of LNTII and extend the shelf life of the product;

[0034] (2) The preparation method of crystal form A is beneficial to the separation of industrial LNTII products, reducing production costs and improving production efficiency.

[0035] In summary, this invention has achieved the first LNTII crystal form, realizing a historic leap in human milk oligosaccharide separation technology, and will benefit the public. Attached Figure Description

[0036] Figure 1 X-ray diffraction pattern of amorphous powder of lacto-N-triose II.

[0037] Figure 2 X-ray diffraction pattern of Lacto-N-triose II crystal form A.

[0038] Figure 3 Scanning electron microscope (SEM) image of amorphous powder of lactose-N-triose II.

[0039] Figure 4 SEM image of Lacto-N-triose II A crystal form.

[0040] Figure 5 The morphology of amorphous powder and crystal form A of lactose-N-triose II placed in a constant temperature and humidity chamber (the left side is the amorphous powder, and the right side is LNTII crystal form A).

[0041] Figure 6 The morphology of amorphous powder and crystal form A of lactose-N-triose II after absorbing moisture in a constant temperature and humidity chamber (25℃, 75% relative humidity) (the left side is the amorphous powder, and the right side is LNTII crystal form A). Detailed Implementation

[0042] The solid form of LNTII described in the examples can be prepared according to published literature, and can be a spray-dried powder, a lyophilized powder, or a solid obtained by other methods. The LNTII fermentation broth is a fermentation broth obtained according to existing technology.

[0043] Example 1. Acetylglucosyltransferase gene lgtA Construction of the (18S::HPLgtA) expression box:

[0044] 1.1 Obtaining the fusion sequences of the HPLgtA coding region, ADH1 termination region, and galactosidase LAC4 promoter region:

[0045] HPLgtA was synthesized from the whole genome, and its amino acid sequence is shown in SEQ ID NO:1.

[0046] The sequence of the LAC4 promoter region of galactosidase is shown in SEQ ID NO:2. The sequence of the ADH1 termination region is shown in SEQ ID NO:3.

[0047] Based on the above sequence, primers were designed as shown in Table 1.

[0048] Table 1

[0049] .

[0050] PCR amplification was performed using the HPLgtA gene, Kluyveromyces lactis genome, and Saccharomyces cerevisiae genome as templates, respectively. The amplified components are shown in Table 2.

[0051] Table 2

[0052] .

[0053] The PCR amplification procedure is shown in Table 3.

[0054] Table 3

[0055] .

[0056] After the PCR reaction was completed, agarose gel electrophoresis was performed for identification. Bands of the expected size were purified and recovered using a gel extraction kit. The recovered product was then used to measure the DNA concentration using a micro-volume analyzer and diluted to 1 ng / μL with ddH2O. Using the diluted DNA fragment as a template, fusion PCR amplification was performed using ADH1-Not1 and LAC4Pro-Nde1 / LAC4Pro-S as primers. The amplified components are shown in Table 4.

[0057] Table 4

[0058] .

[0059] The PCR amplification procedure is shown in Table 5.

[0060] Table 5

[0061] .

[0062] After the PCR reaction was completed, agarose gel electrophoresis was performed to identify the bands that met the expected size. The bands were purified and recovered using a gel recovery kit to obtain the fusion sequences of the HPLgtA coding region, ADH1 termination region, and galactosidase LAC4 promoter region.

[0063] 1.2 The primers for obtaining the upstream and downstream homologous arm sequences, PUG6 vector sequence, and PUG6 vector resistance sequence by PCR amplification are shown in Table 6.

[0064] Table 6

[0065] .

[0066] PCR amplification was performed using Kluyveromyces lactis genome and PUG6 vector as templates. The amplification components are shown in Table 7.

[0067] Table 7

[0068] .

[0069] The PCR amplification procedure is shown in Table 8.

[0070] Table 8

[0071] .

[0072] After the PCR reaction procedure is completed, agarose gel electrophoresis is performed for identification, and bands of the expected size are purified and recovered using a gel recovery kit.

[0073] 1.3 Acquisition of Expression Boxes

[0074] The fusion sequence obtained in step 1.1 was mixed with the upstream and downstream homologous arm sequences, backbone sequence, and resistance sequence obtained in step 1.2 at a total volume of 5 μL and a molar ratio of 1:1:1:1:1, and then ligated into 5 μL of seamless cloning MIX. The ligation temperature was 50℃, and the time was 30-60 min. After ligation, E. coli transformation was performed immediately, and the correct transformants were picked for plasmid extraction to obtain the expression vector 18S:HPLgtA carrying the HPLgtA expression cassette.

[0075] The sources of the reagents and kits used in this embodiment are shown in Table 9.

[0076] Table 9

[0077] .

[0078] The solvent and buffer formulations used in this embodiment are as follows:

[0079] 50×TAE solution: prepared using ddH2O, containing 2 M Tris, 100 mM Na2EDTA·H2O, 2% SDS, and adjusted to pH 8.5.

[0080] Genome extraction buffer: prepared using ddH2O containing 200 mM Tris-HCl, 250 mM NaCl, 2% SDS, 25 mM EDTA, and adjusted to pH 8.0.

[0081] E. coli culture medium: prepared with 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride and water. Solid culture medium requires the addition of 20 g / L agar powder. Sterilize at 121℃ for 20 min before use.

[0082] Yeast culture medium: prepared by adding water to 20 g / L tryptone, 10 g / L yeast extract, and 20 g / L glucose. Solid culture medium requires the addition of 20 g / L agar powder. Sterilize at 115℃ for 30 minutes before use.

[0083] The culture conditions used in this embodiment are as follows:

[0084] Escherichia coli was cultured on solid plates in a constant temperature incubator at 37 ℃ and in shake flasks in a shaker at 37 ℃ and 200 rpm.

[0085] Yeast was cultured on solid plates in a constant temperature incubator at 30 ℃ and in shake flasks in a shaker at 30 ℃ and 200 rpm.

[0086] The yeast genome extraction method in this embodiment:

[0087] (1) Pick a single colony of Kluyveromyces lactis into 1 mL YPD (10 mL centrifuge tube) medium and incubate overnight at 30°C and 200 rpm;

[0088] (2) Take 600 μL of bacterial culture into a 1.5 mL EP tube, centrifuge at 10000 rpm for 1 min, and discard the supernatant;

[0089] (3) Add 600-800 μL of genome extraction buffer and 100 μL of quartz sand, and shake thoroughly for 5 min;

[0090] (4) Water bath at 65℃ for 30 minutes, inverting the container every 10 minutes;

[0091] (5) Take the supernatant into a new 1.5 ml EP tube, add an equal volume of DNA extraction solution, and mix by pipetting;

[0092] (6) Centrifuge at 13000 rpm for 10 min. Transfer 400 μL of the supernatant to a new 1.5 mL EP tube;

[0093] (7) Add 0.6 times the volume of isopropanol and 40 μL of 3M sodium acetate, mix well, and let stand at -20℃ for 30 min;

[0094] (8) Centrifuge at 13000 rpm for 10 min, discard the supernatant, and obtain the yeast genome;

[0095] (9) Wash twice with 70% ethanol, and after the ethanol evaporates, dissolve in ddH2O and store at -20℃.

[0096] Example 2: Construction of recombinant strains of Kluyveromyces lactis:

[0097] 1. After the ΔLAC4::HPLgt A expression cassette was successfully constructed, yeast transformation was performed to obtain recombinant strains.

[0098] The specific methods for yeast conversion are as follows:

[0099] (1) First, prepare competent yeast cells: streak a small amount of frozen yeast strain onto a plate of solid culture medium and incubate upside down at 30°C for 2 days. Pick a single yeast colony and incubate it in 50 mL of liquid culture medium at 30°C and 220 rpm until OD reaches 100%. 600 The concentration should be between 0.8 and 1.5. Collect the bacterial cells, wash with 25 mL of sterile water, centrifuge at 1500 × g for 10 min at room temperature, and discard the supernatant. Add 1 mL of 100 mM lithium chloride buffer, resuspend the precipitate, centrifuge at 12000 rpm for 30 s, and discard the supernatant. Add 400 μL of 100 mM lithium chloride buffer again, resuspend the precipitate, and obtain competent yeast cells. Aliquot into 50 μL tubes for transformation.

[0100] Meanwhile, boil 1 mL of salmon sperm DNA for 5 min and then quickly ice bath to prepare single-stranded carrier DNA.

[0101] (2) Transformation: Centrifuge the competent yeast cells prepared above and remove residual lithium chloride solution with Tips. For each transformation, add the following in order: 50% PEG3350 (240 μL); 1M LiCl (36 μL); 2 mg / mL single-stranded Salmon sperm DNA (25 μL); 5-10 μg / 50 μL H2O plasmid DNA (50 μL), vortex vigorously until the precipitated cells are completely and evenly distributed; incubate at 30 ℃ for 30 min; heat shock at 42 ℃ for 20-25 min; centrifuge at 8000 rpm for 10 min and collect the yeast cells; then, resuspend the yeast in 500 μL of liquid medium and incubate at 30 ℃ on a shaker; after 1-4 h, take 25-100 μL of the bacterial solution and spread it on selective medium plates and incubate upside down at 30 ℃.

[0102] The plasmid DNA is the ΔLAC4 knockout cassette constructed in Example 1, or the ΔLAC4::HPLgt A expression cassette.

[0103] 2. Validation of recombinant strains of Kluyveromyces lactis:

[0104] After incubating the plate statically for 2-3 days, verify the emergence of single colonies of transformants using the following method:

[0105] Single colonies of transformants were picked and cultured overnight at 30°C with shaking at 200 rpm in 1.5 mL liquid medium 1 and liquid medium 2, respectively. Afterward, 50 μL of the bacterial culture was transferred to both liquid medium 1 and liquid medium 2 and cultured overnight at 30°C with shaking at 200 rpm. Growth was observed, and transformants that grew normally in solid medium 1 but barely grew in solid medium 2 were selected for preservation and considered recombinant strains.

[0106] The specific details of the culture medium in this embodiment are as follows:

[0107] Solid culture medium 1: 20 g / L tryptone, 10 g / L yeast extract, 20 g / L glucose, and 20 g / L agar powder were mixed with water and then sterilized at 115℃ for 30 min before use.

[0108] Liquid culture medium 1: 20 g / L tryptone, 10 g / L yeast extract, and 20 g / L glucose were added to water and then sterilized by steam at 115℃ for 30 min before use.

[0109] Solid culture medium 2: Prepared by adding water to 20 g / L tryptone, 10 g / L yeast extract, 20 g / L lactose, and 20 g / L agar powder, and then sterilized by steaming at 115℃ for 30 min before use.

[0110] Liquid culture medium 2: Prepared by adding 20 g / L tryptone, 10 g / L yeast extract and 20 g / L lactose to water, and then sterilized by steaming at 115℃ for 30 min before use.

[0111] Example 3. Two-stage culture of recombinant strains of Kluyveromyces lactis to catalyze the synthesis of LNTII.

[0112] The first stage is the bacterial growth period:

[0113] Cells were cultured using glucose as a carbon source to accumulate cell mass and enzyme levels until the cells entered the late logarithmic or stationary phase. The strain was streaked onto solid medium and incubated at 30°C for 2-3 days. Single colonies were then picked and inoculated into 1.5 mL of liquid medium and cultured at 30°C and 200 rpm until OD500 reached the target growth phase. 600 =1, inoculate 2% of the culture medium into 5L of liquid medium (10L fermenter), and incubate overnight at 30℃ with shaking at 200 rpm. Then, inoculate 2% of the culture medium into 50mL shake flasks of liquid medium and incubate at 30℃ with shaking at 200 rpm to accumulate cell volume.

[0114] The second stage is the product synthesis period: after 40 h of shaking culture at 30℃ and 200 rpm, fed-batch fermentation begins, with a total fermentation time of 72 h. After 72 h of fermentation, the fermentation broth is centrifuged, and the cells are disrupted using a high-pressure homogenizer. Proteins are removed by centrifugation, and the broth and supernatant are collected. The resulting liquid containing LNTII is then decolorized and desalted using anion and cation exchange resins, and purified using chromatography resins to obtain a purified solution. The purified solution is concentrated, and the LNTII concentration in the concentrated solution is 466 g / L, which is then used for later use; alternatively, solid powder LNTII can be obtained by spray drying, which is also used for later use.

[0115] The specific details of the culture medium are as follows:

[0116] Solid culture medium: 20 g / L tryptone, 10 g / L yeast extract, 20 g / L glucose, 20 g / L agar powder, sterilized by steam at 115℃ for 30 min before use.

[0117] Liquid culture medium: 20 g / L tryptone, 10 g / L yeast extract, 20 g / L glucose, 8 g / L lactose, and 5 mM dipotassium hydrogen phosphate, magnesium sulfate, ammonium sulfate and manganese sulfate, sterilized by steam at 115℃ for 30 min before use.

[0118] Feeding: 2 g / L lactose, 20 g / L glucose, and 5 mM dipotassium hydrogen phosphate, magnesium sulfate, ammonium sulfate, and manganese sulfate.

[0119] The following examples use the purified fermentation broth obtained in Example 1, or the solid LNTII obtained from the fermentation broth obtained by spray drying.

[0120] Example 4. Take 5g of solid LNTII obtained in Example 1, add 10ml of purified water, and dissolve completely at 50℃. After dissolution, cool to 25℃ at a cooling rate of 0.5℃ / min, add 0.06g of seed crystals, add 30ml of acetone at a flow rate of 0.25ml / min, and culture for 12h with stirring. Filter, wash with acetone pre-cooled at 0℃, and dry at 55℃ to obtain LNTII crystals, which are called crystal form A. The purity was determined by HPLC to be 97.98%, and the yield was 90.89%.

[0121] X-ray diffraction (XRD) was performed on the uncrystallized solid LNTII (obtained by spray drying in Example 1) and the crystal form obtained in this example, according to the X-ray diffraction method (method II, powder X-ray diffraction) specified in Appendix 0451 of the Chinese Pharmacopoeia (2020 edition). The XRD pattern of the uncrystallized solid LNTII is shown in the appendix. Figure 1 It is an amorphous solid. The X-ray diffraction pattern of the crystal form obtained in this embodiment is shown in the appendix. Figure 2 It has characteristic peaks of crystals, and the peak positions and peak intensities are shown in Table 10.

[0122] The morphology of solid LNTII before crystallization and the crystal form obtained in this invention were examined according to JY / T0584-2020 "General Rules for Scanning Electron Microscopy Analysis". The scanning electron microscope images of solid LNTII powder before crystallization are attached. Figure 3 The electron micrograph of LNTII crystal form A obtained in this embodiment is attached. Figure 4 .

[0123] According to the method specified in GB / T21781-2008 "Test Methods for Melting Point and Melting Range of Chemicals - Capillary Method", the melting point of the crystal form obtained in this example was tested and found to be 194℃.

[0124] Table 10

[0125] .

[0126] Example 5. Take 5g of the solid powder LNTII obtained from the spraying in Example 3, add 25ml of purified water, and dissolve completely at 40℃. After dissolution, cool to 15℃, add 0.02g of seed crystals, add 50ml of acetone, and allow the mixture to crystallize at a flow rate of 0.07ml / min for 20 hours with stirring. Filter, wash with pre-cooled acetone at 8℃, and dry at 60℃ to obtain LNTII crystal form. Its X-ray diffraction pattern is the same as that of the crystal form obtained in Example 4, which is LNTII crystal form A. Melting point: 196℃. HPLC determination shows a purity of 98.18% and a yield of 92.6%.

[0127] Example 6. Take 5g of the solid powder LNTII obtained from the spraying in Example 3, add 20ml of purified water, and dissolve completely at 55℃. After dissolution, cool to 10℃, add 50ml of acetone, with a flow rate of 0.20ml / min, and crystallize for 48h with stirring. Filter, wash with acetone pre-cooled at 0-8℃, and dry at 65℃ to obtain LNTII crystal form. Its X-ray diffraction pattern is the same as that of the crystal form obtained in Example 4, which is LNTII crystal form A, with a melting point of 194℃, a purity of 97.87% determined by HPLC, and a yield of 93.89%.

[0128] Example 7. The purified solution obtained in Example 3 (LNTII concentration of 466 g / L) was concentrated, cooled to 25°C, and 10 ml was taken. 0.04 g of seed crystals were added, along with 20 ml of acetone. The mixture was stirred at a flow rate of 0.25 ml / min and allowed to stand for 16 h to allow crystals to grow. The solution was filtered, washed with acetone pre-cooled at 5°C, and dried at 55°C to obtain LNTII crystal form. Its X-ray diffraction pattern was the same as that of the crystal form obtained in Example 4, indicating LNTII crystal form A. The melting point was 191°C, and the purity was determined by HPLC to be 92.3%, with a yield of 90.89%.

[0129] Example 8. Take the concentrated solution containing LNTII obtained in Example 3 (LNTII concentration of 466 g / L), cool it to 15°C, take 10 ml, add 0.06 g of seed crystals, add 30 ml of acetone, stir, with a flow rate of 0.18 ml / min, and allow it to stand for crystal growth for 16 h. Filter, wash with acetone pre-cooled at 0°C, and dry at 60°C to obtain LNTII crystal form. Its X-ray diffraction pattern is the same as that of the crystal form obtained in Example 4, which is LNTII crystal form A, melting point 193°C, purity determined by HPLC is 92.57%, and yield is 92.6%.

[0130] Example 9. Take the concentrated solution containing LNTII obtained in Example 3 (LNTII concentration of 466 g / L), cool it to 20°C, take 10 ml, add 0.06 g of seed crystals, add 25 ml of acetone, stir, and allow it to stand for crystal growth at a flow rate of 0.20 ml / min for 12 h. Filter, wash with acetone pre-cooled at 0°C, and dry at 65°C to obtain LNTII crystal form. Its X-ray diffraction pattern is the same as that of the crystal form obtained in Example 4, which is LNTII crystal form A, melting point 192°C, purity determined by HPLC is 91.98%, and yield is 93.39%.

[0131] Example 10. Take 5g of solid LNTII obtained in Example 3, add 15ml of purified water, and dissolve completely at 45℃. After dissolution, cool to 25℃, add 0.06g of seed crystals, and crystallize for 0.5-1h; cool to 15℃ at a cooling rate of 0.1℃ / min; at this temperature, add 40ml of acetone to the solution at a flow rate of 0.15ml / min, and crystallize for 18h with stirring. Filter, wash with acetone pre-cooled at 5℃, and dry at 55℃ to obtain LNTII crystal form. Its X-ray diffraction pattern is the same as that of the crystal form obtained in Example 4, which is LNTII crystal form A, melting point 194℃, purity determined by HPLC is 98.58%, and yield is 94.28%.

[0132] Example 11. Hygroscopicity determination

[0133] Step 1. Treatment of glass petri dishes

[0134] The day before the constant temperature and humidity experiment, the glass petri dish (without a lid) was placed in a constant temperature and humidity chamber at 25±1℃ and 75±2% relative humidity for 4 hours. It was then removed and weighed precisely. The experiment was repeated until a constant weight was achieved. The data were recorded in Table 12.

[0135] Step 2. Weigh the spray-dried LNTII solid powder obtained in Example 3 and the LNTII crystal form A obtained in Example 4, and place them in the open glass petri dishes that were kept at constant weight in Step 1. Place them in a constant temperature and humidity chamber at 25°C and 75% relative humidity. After 5 days, remove them to observe the appearance changes, weigh them, and calculate the moisture absorption. The results are recorded in Tables 11 and 12, respectively. The morphology of the samples before moisture absorption is shown in the appendix. Figure 5 The morphology of the sample after moisture absorption is shown in the attached image. Figure 6 .

[0136] Table 11

[0137] .

[0138] Table 12

[0139] .

[0140] The phenomena in Table 11 and the results in Table 12 show that the stability of LNTII crystal form A described in this invention is better than that of amorphous powder, which is beneficial for the long-term storage of LNTII products and their application in food and pharmaceuticals. It can also reduce the packaging cost of LNTII and extend the shelf life of the products.

[0141] It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the given examples, those skilled in the art can modify or make equivalent substitutions to the technical solutions of the present invention as needed, without departing from the spirit and scope of the technical solutions of the present invention. sequence list <110> Shandong Henglu Biotechnology Co., Ltd. <120> A new crystalline form of a trisaccharide <160> 19 <170> SIPOSequenceListing 1.0 <210> 1 <211> 333 <212> PRT <213> Neisseria meningitidis <400> 1 Met Gln Pro Leu Val Ser Val Leu Ile Cys Ala Tyr Asn Val Glu Lys 1 5 10 15 Tyr Phe Ala Gln Ser Leu Ala Ala Val Val Asn Gln Thr Trp Arg Asn 20 25 30 Leu Asp Ile Leu Ile Val Asp Asp Gly Ser Thr Asp Gly Thr Leu Ala 35 40 45 Ile Ala Gln Arg Phe Gln Glu Gln Asp Gly Arg Ile Arg Ile Leu Ala 50 55 60 Gln Pro Arg Asn Ser Gly Leu Ile Pro Ser Leu Asn Ile Gly Leu Asp 65 70 75 80 Glu Leu Ala Lys Ser Gly Gly Gly Gly Glu Tyr Ile Ala Arg Thr Asp 85 90 95 Ala Asp Asp Ile Ala Ala Pro Asp Trp Ile Glu Lys Ile Val Gly Glu 100 105 110 Met Glu Lys Asp Arg Ser Ile Ile Ala Met Gly Ala Trp Leu Glu Val 115 120 125 Leu Ser Glu Glu Lys Asp Gly Asn Arg Leu Ala Arg His His Glu His 130 135 140 Gly Lys Ile Trp Lys Lys Pro Thr Arg His Glu Asp Ile Ala Asp Phe 145 150 155 160 Phe Pro Phe Gly Asn Pro Ile His Asn Asn Thr Met Ile Met Arg Arg 165 170 175 Ser Val Ile Asp Gly Gly Leu Arg Tyr Asn Thr Glu Arg Asp Trp Ala 180 185 190 Glu Asp Tyr Gln Phe Trp Tyr Asp Val Ser Leu Gly Arg Leu Ala 195 200 205 Tyr Tyr Pro Glu Ala Leu Val Lys Tyr Arg Leu His Ala Asn Gln Val 210 215 220 Ser Ser Lys Tyr Ser Ile Arg Gln His Glu Ile Ala Gln Ile Gln 225 230 235 240 Lys Thr Ala Arg Asn Asp Phe Leu Gln Ser Met Gly Phe Lys Thr Arg 245 250 255 Phe Asp Ser Leu Glu Tyr Arg Gln Ile Lys Ala Val Ala Tyr Glu Leu 260 265 270 Leu Glu Lys His Leu Pro Glu Glu Asp Phe Glu Leu Ala Arg Arg Phe 275 280 285 Leu Tyr Gln Cys Phe Lys Arg Thr Asp Thr Leu Pro Ala Gly Ala Trp 290,295,300 Leu Asp Phe Ala Ala Asp Gly Arg Met Arg Arg Leu Phe Thr Leu Arg 305 310 315 320 Gln Tyr Phe Gly Ileu His Arg Ileu Lys Asn Arg 325 330 <210> 2 <211> 600 <212> DNA <213> Kluyveromyces lactis <400> 2 tgtcttgcat gttaataata gcctagcctg tgagccgaaa cttagggtag gcttagtgtt 60 ggaacgtaca tatgtatcac gttgacttgg tttaaccagg cgacctggta gccagccata 120 cccacacacg ttttttgtat cttcagtata gttgtgaaaa gtgtagcgga aatttgtggt 180 ccgagcaaca gcgtcttttt ctagtagtgc ggtcggttac ttggttgaca ttggtatttg 240 gactttgttg ctacaccatt cactacttga agtcgagtgt gaagggtatg atttctagtg 300 gtgaacacct ttagttacgt aatgttttca ttgctgtttt acttgagatt tcgattgaga 360 aaaaggtatt tatagctcg aatcaatgtg ttatcattgt gaagatgttc ttccctaact 420 cgaaaggtat atgaggcttg tgtttcttag gagaattatt attctttgt tatgttgcgc 480 ttgtagttgg aaaaggtgaa gagacaaaag cgcttaacac ttgaaattta ggaaagagca 540 gaatttggca aaaaaaataa aaaaaaata aacacacata ctcatcgaga actgaaagat 600 <210> 3 <211> 188 <212> DNA <213> artificial sequence <400> 3 gcgaatttct tatgatttat gatttttatt attaaataag ttataaaaaa aataagtgta 60 tacaaatttt aaagtgactc ttaggtttta aaacgaaaat tcttattctt gagtaactct 120 ttcctgtagg tcaggttgct ttctcaggta tagcatgagg tcgctcttat tgaccacacc 180 tctaccgg 188 <210> 4 <211> 35 <212> DNA <213> artificial sequence <400> 4 ggaattccat atgtatcacg ttgacttggt ttaac 35 <210> 5 <211> 38 <212> DNA <213> artificial sequence <400> 5 atcataagaa attcgcttag gacttcttca acaacttc 38 <210> 6 <211> 36 <212> DNA <213> artificial sequence <400> 6 tcgagaactg aaagatatga cctccgcttc ctctca 36 <210> 7 <211> 44 <212> DNA <213> artificial sequence <400> 7 gttgaagaag tcctaagcga atttcttatg attatgatt tta 44 <210> 8 <211> 32 <212> DNA <213> artificial sequence <400> 8 agaatgcggc cgcccggtag aggtgtggtc aa 32 <210> 9 <211> 34 <212> DNA <213> artificial sequence <400> 9 acgaggaaca actaacgaga tcatggtatc ctca 34 <210> 10 <211> 35 <212> DNA <213> artificial sequence <400> 10 gaagcggagg tcatatcttt cagttctcga tgagt 35 <210> 11 <211> 35 <212> DNA <213> artificial sequence <400> 11 cgagaactga aagatatgac ctccgcttcc tctca 35 <210> 12 <211> 36 <212> DNA <213> artificial sequence <400> 12 ctatagacat atgatgcttg tctcaaagat taagcc 36 <210> 13 <211> 36 <212> DNA <213> artificial sequence <400> 13 catgatctcg ttagttgttc ctcgttaagg tattta 36 <210> 14 <211> 33 <212> DNA <213> artificial sequence <400> 14 gatatcagat ccacttgtct gcttaattgc gat 33 <210> 15 <211> 34 <212> DNA <213> artificial sequence <400> 15 gcataggcca ctataaatga ccaagtttga ccag 34 <210> 16 <211> 31 <212> DNA <213> artificial sequence <400> 16 cctctaccgg tgcaggtcga caacccttaa t 31 <210> 17 <211> 32 <212> DNA <213> artificial sequence <400> 17 taagcagaca agtggatctg atatcaccta at 32 <210> 18 <211> 29 <212> DNA <213> artificial sequence <400> 18 tggtcattta tagtggccta tgcggccgc 29 <210> 19 <211> 40 <212> DNA <213> artificial sequence <400> 19 ctttgagaca agcatcatat gtctatagtg tcacctaaat 40

Claims

1. A lactose-N-trisaccharide II crystal form A, characterized in that, Its powder X-ray diffraction pattern has characteristic peaks at 2θ±0.2° values ​​of 4.80, 7.24, 8.32, 9.62, 12.72, 16.67, 18.67, 19.28, 19.90, 21.05, 21.59, 22.14 and 26.

93.

2. The lactose-N-trisaccharide II crystal form A as described in claim 1, characterized in that, Its powder X-ray diffraction pattern has characteristic peaks at 2θ±0.2° at 4.80, 7.24, 8.32, 9.62, 12.72, 16.67, 17.35, 18.67, 19.28, 19.90, 20.72, 21.05, 21.59, 22.14, 23.17, 23.71, 24.15, 25.20, 26.93, 29.12 and 29.

49.

3. The lactose-N-trisaccharide II crystal form A as described in claim 1, characterized in that, Its powder X-ray diffraction pattern has characteristic peaks at 2θ±0.2° at 4.80, 7.24, 8.32, 9.62, 9.89, 11.65, 12.72, 14.42, 16.67, 17.35, 18.67, 19.28, 19.90, 20.72, 21.05, 21.59, 22.14, 23.17, 23.71, 24.15, 25.20, 26.93, 28.68, 29.12, 29.49, 30.34, 31.08, 31.88 and 34.

10.

4. The lactose-N-trisaccharide II crystal form A according to any one of claims 1-3, characterized in that, Its melting point is 191-196℃.

5. The method for preparing lactose-N-trisaccharide II crystal form A according to any one of claims 1-3, characterized in that, A solution containing lactose-N-trisaccharide II is mixed with an organic solvent to obtain crystal form A, wherein the volume ratio of water to organic solvent added in the crystallization solution is 1:2.4-2.7, and the organic solvent is selected from acetone.

6. The method for preparing lactose-N-trisaccharide II crystal form A as described in claim 5, characterized in that, The volume ratio of water to organic solvent added is 1:2.

5.

7. The method for preparing lactose-N-trisaccharide II crystal form A as described in claim 5, characterized in that, Seed crystals are added to a solution containing lactose-N-trisaccharide, and the crystals are cultured for more than 0.5 h. After mixing with an organic solvent, crystal form A is obtained. The organic solvent is selected from acetone.

Images