WO2014069625A1 - Crystal of oligosaccharide, and method for producing same - Google Patents

Crystal of oligosaccharide, and method for producing same Download PDF

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WO2014069625A1
WO2014069625A1 PCT/JP2013/079697 JP2013079697W WO2014069625A1 WO 2014069625 A1 WO2014069625 A1 WO 2014069625A1 JP 2013079697 W JP2013079697 W JP 2013079697W WO 2014069625 A1 WO2014069625 A1 WO 2014069625A1
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crystal
crystals
hydrate
powder
fucosyl lactose
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PCT/JP2013/079697
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French (fr)
Japanese (ja)
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宏 長野
茂樹 松宮
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協和発酵バイオ株式会社
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
    • C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives

Definitions

  • the present invention relates to an oligosaccharide crystal that is useful, for example, as a product, a raw material or an intermediate for health foods, pharmaceuticals, cosmetics, and the like, and a method for producing the crystal.
  • Oligosaccharides are useful, for example, as raw materials or intermediates for health foods, pharmaceuticals, cosmetics and the like. Synthetic methods and fermentation methods are known as oligosaccharide production methods (Non-patent Documents 1 and 2, and Patent Documents 1 and 2).
  • Synthetic methods and fermentation methods are known as oligosaccharide production methods (Non-patent Documents 1 and 2, and Patent Documents 1 and 2).
  • oligosaccharides 2'- O -fucosyl lactose [4- O- (2- O- ⁇ -L-fucosylpyranosyl- ⁇ -D-galactopyranosyl) -D-galactose] is contained in human breast milk It is one of the important oligosaccharides and has functions such as intestinal bacterial growth action, antibacterial activity, antiviral activity, immune system enhancement action and brain function enhancement action.
  • Non-Patent Document 3 and Patent Document 3 have described that crystals of 2′- O -fucosyl lactose have been obtained, but there is no description regarding the properties of the obtained crystals, and industrially useful 2′- O—. There is a need for fucosyl lactose crystals.
  • An object of the present invention is to provide crystals of 2′- O -fucosyl lactose, which is an oligosaccharide useful as, for example, products such as health foods, pharmaceuticals and cosmetics, raw materials or intermediates, and a method for producing the same.
  • the present invention relates to the following (1) to (12).
  • (1) 2′- O -fucosyl lactose n-hydrate crystal (where n represents any number from 0 to 3, and when n 0, 2′- O -fucosyl lactose-anhydride Called).
  • diffraction angles (2 ⁇ ) are 10.00 ° ⁇ 0.20 °, 18.94 ° ⁇ 0.20 °, 20.44 ° ⁇ 0.20 ° and 21.72 °.
  • a crystal of 2′- O -fucosyl lactose n-hydrate according to (1) above having a peak at ⁇ 0.20 ° [n is as defined in (1) above].
  • diffraction angles (2 ⁇ ) are further changed to 11.88 ° ⁇ 0.20 °, 14.26 ° ⁇ 0.20 °, 15.60 ° ⁇ 0.20 °, and 17.
  • a crystal of 2'- O -fucosyl lactose n-hydrate according to (6) above, having a peak at 12 ° ⁇ 0.20 ° [n is as defined in (1) above].
  • the method for producing 2'- O -fucosyl lactose n-hydrate crystals [n is synonymous with the above (1)] comprising the step of gradually adding.
  • the aqueous solution amount while maintaining the temperature of 15 ⁇ 60 ° C.
  • the 2′- O -fucosyl lactose n-hydrate crystals of the above (1) comprising a step of gradually adding 0.1 to 3 times the amount of C1-C6 alcohol to the aqueous solution little by little [n is Synonymous with (1) above].
  • the method includes the step of gradually adding the C1-C6 alcohol to the 2′- O -fucosyl lactose-containing aqueous solution little by little and then holding the resulting water / alcohol solution at 15-60 ° C. for 1 hour or more ( 9) The manufacturing method of (10). (12) The production method according to any one of (9) to (11) above, wherein the C1 to C6 alcohol is methanol or ethanol.
  • the present invention provides oligosaccharide crystals that are useful as products, raw materials, intermediates, and the like such as health foods, pharmaceuticals, and cosmetics, and a method for producing the same.
  • FIG. 1 is a diagram showing a powder X-ray diffraction peak pattern of 2′- O -fucosyl lactose 1.4 hydrate crystals.
  • the vertical axis of the graph is intensity (unit cps), and the horizontal axis is diffraction angle (2 ⁇ ) (unit °).
  • FIG. 2 is a diagram showing a powder X-ray diffraction peak pattern of 2′- O -fucosyl lactose 2.5 hydrate crystals.
  • the vertical axis of the graph is intensity (unit cps), and the horizontal axis is diffraction angle (2 ⁇ ) (unit °).
  • FIG. 1 is a diagram showing a powder X-ray diffraction peak pattern of 2′- O -fucosyl lactose 1.4 hydrate crystals.
  • the vertical axis of the graph is intensity (unit cps), and the horizontal axis is diffraction angle (2 ⁇ ) (unit °).
  • FIG. 3 is a diagram showing the results of thermogravimetric analysis (TG) of crystal A of 2′- O -fucosyl lactose.
  • the vertical axis of the graph is weight (%), and the horizontal axis is temperature.
  • the region represented by A represents the amount of water loss between room temperature and 50 ° C, and the region represented by B represents the amount of water loss between 50 and 180 ° C.
  • FIG. 4 is a diagram showing a temperature variable powder X-ray diffraction peak pattern of 2′- O -fucosyl lactose crystals.
  • the vertical axis of the graph is intensity (unit cps), and the horizontal axis is diffraction angle (2 ⁇ ) (unit °).
  • 2'O of the present invention - fucosyl sill lactose crystals present invention 2'O - fucosyllactose crystal (hereinafter sometimes abbreviated as 2'FL) ⁇ n-hydrate (where, n is 0-3 Any number, preferably n is any number greater than 0 and less than or equal to 3, more preferably n is any number from 0 to 3 in any decimal place, more preferably n is greater than 0; Any number up to the first decimal place of 3 or less.
  • 2′FL ⁇ anhydride can be cited as A and B crystals, and 2′- O -fucosyl.
  • Crystal A Crystal in which the conformation of glucose in lactose is ⁇ -type and / or ⁇ -type can be mentioned.
  • the crystal of 2′FL ⁇ 1.4 hydrate is a crystal whose powder X-ray diffraction pattern using CuK ⁇ as an X-ray source is defined by the values shown in FIG. 1 and Table 1.
  • the analyzer used for powder X-ray diffraction and the measurement conditions are the sample horizontal multi-purpose X-ray diffractometer Ultima IV manufactured by Rigaku Corporation, and the measurement conditions are a copper target sealed tube in the X-ray source. It is possible to raise conditions for irradiating a sample with X-rays that are not monochromatic and detecting diffracted X-rays derived from CuK ⁇ rays (wavelength 1.541 ⁇ ⁇ ⁇ ⁇ ⁇ ) using an energy-resolving detector.
  • the powder X-ray diffraction patterns simulated from the single crystal structures of 2′FL ⁇ 1.3 hydrate and 1.4 hydrate are as shown in Table 2.
  • a structural analysis by a single crystal X-ray diffraction method can be mentioned.
  • a single crystal of 2'FL ⁇ n hydrate is attached to a diffractometer, and a diffraction image is measured using X-rays having a predetermined wavelength in an atmosphere of room temperature or an inert gas stream at a predetermined temperature.
  • the structure is determined by the direct method and the structure is refined by the least square method to obtain a single crystal structure.
  • a method for simulating a powder X-ray diffraction pattern from a single crystal structure for example, a crystal structure display program Mercury (Cambridge Crystallographic Data Center) or PowderCell (German Federal Materials Research Laboratory) can be used.
  • a powder X-ray diffraction pattern is calculated by inputting a crystal lattice constant, atomic coordinates, and an X-ray wavelength used for calculation.
  • the diffraction angle (2 ⁇ ) was 17.00 ° ⁇ 0.20 °, 18.82 ° ⁇ 0.20 ° and 21.58 ° ⁇ 0.20 °, preferably 17.00 ° ⁇ 0.20 °, 18.82 ° ⁇ 0.20 °, 21.58 ° ⁇ 0.20 °, 9.86 ° ⁇ 0.20 °, 14.16 ° ⁇ 0.20 °, 16.12 ° ⁇ 0.20 ° and 20.30 ° ⁇ 0.20 °, more preferably 17.00 ° ⁇ 0.20 °, 18.82 ° ⁇ 0.20 °, 21.58 ° ⁇ 0.20 °, 9.86 ° ⁇ 0.20 °, 14.16 ° ⁇ 0.20 °, 16.12 ° ⁇ 0.20 °, 20.30 ° ⁇ 0.20 °, 15.52 ° ⁇ 0.20 °, 23.88 ° ⁇ 0.20 °, preferably 17.00 ° ⁇ 0.20 °, 18.82 ° ⁇ 0.20 °,
  • Crystal B Crystal B is 2′FL ⁇ p hydrate (where p is any number greater than 1.7 and less than or equal to 3, preferably p is any number from 1.8 to 3.0
  • the crystal of 2′FL ⁇ 2.5 hydrate, which is a B crystal, is a powder X-ray diffraction pattern using CuK ⁇ as an X-ray source. And crystals defined by the values shown in Table 3.
  • the powder X-ray diffraction pattern simulated from the single crystal structure of 2′FL ⁇ 2.5 hydrate crystals is as shown in Table 4.
  • the powder X-ray diffraction pattern of 2'FL ⁇ 2.5 hydrate crystals and the powder X-ray diffraction pattern simulated from the single crystal structure analysis of 2'FL ⁇ 2.5 hydrate crystals are in good agreement That is, the peak intensity changes depending on the number of water molecules contained in the crystal.
  • the crystal B also has a tendency to move to a high angle because the crystal lattice contracts as water molecules escape from the crystal.
  • the crystal of the crystal B can be defined as follows.
  • the diffraction angle (2 ⁇ ) was 10.00 ° ⁇ 0.20 °, 18.94 ° ⁇ 0.20 °, 20.44 ° ⁇ 0.20 ° and 21.72 ° ⁇ 0.20 °, preferably 10.00 ° ⁇ 0.20 °, 18.94 ° ⁇ 0.20 °, 20.44 ° ⁇ 0.20 °, 21.72 ° ⁇ 0.20 °, 11.88 ° ⁇ 0.20 °, 14.26 ° ⁇ 0.20 °, 15.60 ° ⁇ 0.20 ° and 17.12 ° ⁇ 0.20 °, more preferably 10.00 ° ⁇ 0.20 °, 18.94 ° ⁇ 0.20 °, 20.44 ° ⁇ 0.20 °, 21.72 ° ⁇ 0.20 °, 11.88 ° ⁇ 0.20 °, 14.26 ° ⁇ 0.20 °, 15.60 ° ⁇ 0.20 ° and 17.12 ° ⁇ 0.20 °, more preferably 10.00 ° ⁇ 0.20 °
  • 2′FL hydrate crystals having a peak at 8 ° ⁇ 0.20 ° and 19.86 ° ⁇ 0.20 °, most preferably 2 ⁇ in Table 3, are 2′FL ⁇ p hydration of the present invention. It is a crystal of an object.
  • the 2′FL-containing aqueous solution may be any aqueous solution containing 2′FL, for example, 2′FL
  • the purity of FL is 80% or more, more preferably 85% or more, and more preferably 90% or more
  • a 2'FL-containing aqueous solution can be given, and most of the impurities contained in the aqueous solution are sugars such as lactose. Does not hinder crystallization even if the purity of the 2′FL aqueous solution is less than 80%.
  • 2′FL-containing aqueous solution is, for example, Chem. Rev., Vol.100, p4445, 2000, Curr.Opin. In Drug Discovery & Develop., Vol. 3, p756, 2000, International Publication No. 98/12343 pamphlet.
  • 2′FL obtained by a synthesis method, an enzymatic method, a method using resting cells, or a fermentation method is also described in the above-mentioned literature. It can be obtained by purification using a certain known purification method. It can also be obtained by dissolving commercially available 2'FL powder.
  • solids such as bacterial cells can be obtained by centrifugation, filtration, ceramic filter or the like from 2′FL obtained by enzymatic method, method using resting bacterial cells, or fermentation method as necessary. After removing the substances, the resulting aqueous solution can be passed through a column packed with an ion exchange resin such as Diaion (Mitsubishi Chemical Corporation), desalted, concentrated, and crystallized. . In the case of commercially available 2'FL, it can be concentrated and crystallized as it is.
  • an ion exchange resin such as Diaion (Mitsubishi Chemical Corporation)
  • the concentration of the 2′FL-containing aqueous solution used in the method of the present invention is 500 g / L or more, preferably 600 g / L or more, more preferably 700 g / L or more, and more preferably 800 g / L or more.
  • the 2′FL-containing aqueous solution obtained above is set to 15 to 60 ° C., preferably 20 to 55 ° C., more preferably 25 to 50 ° C., still more preferably 30 to 45 ° C., and most preferably 35 to 43 ° C.
  • 2′FL crystals are 0.001 to 0.1 wt%, preferably 0.005 to 0.05 wt%, more preferably 0 Crystals are maintained at 35 to 50 ° C. for 30 minutes to 10 hours, preferably 1 to 8 hours, more preferably 3 to 6 hours after addition to the aqueous solution so as to be 0.01 to 0.03% by weight. It is also possible to promote precipitation.
  • the 2'FL crystals added above can also be obtained by adding methanol to a 2'FL-containing aqueous solution with a concentration of 700 g / L to a concentration of 50-60% and storing at 4 ° C for about 30 days. can do.
  • C1 to C6 alcohol is gradually added to the aqueous solution little by little to precipitate crystals.
  • the amount of the C1 to C6 alcohol added is 0.1 to 3 times, preferably 0.2 to 2.5 times, more preferably 0.4 to 2 times the amount of the 2′FL-containing aqueous solution. I can give you.
  • C1-C6 alcohols examples include ethanol, methanol, n-propanol, i-propanol, n-butanol, i-butanol, s-butanol, t-butanol, amyl alcohol, and n-hexanol, preferably C1-C4.
  • alcohols such as ethanol, methanol, n-propanol, i-propanol, n-butanol, i-butanol, s-butanol, and t-butanol, more preferably methanol and ethanol, and still more preferably ethanol.
  • Gradual addition of C1 to C6 alcohols means dropwise addition to the aqueous solution so that the temperature of the 2'FL-containing aqueous solution does not change abruptly.
  • the dropping speed is 2 to 20 hours, preferably 4 to 18 hours, more preferably 6 to 15 hours, and further preferably 8 to 12 hours.
  • the rate at which the total amount of ethanol is dropped over time can be increased, and can be set as appropriate according to the type of alcohol used, the 2′FL-containing aqueous solution, and the amount of alcohol.
  • the obtained water / alcohol solution is kept for 1 hour or more, preferably 1 to 30 hours, more preferably 5 to 25 hours, further preferably 8 to 20 hours, most preferably 12 to 18 hours, at 15 to 60 ° C.
  • the crystals are aged by holding. After aging the crystals, the crystals may be further precipitated by gradually cooling the water / alcohol solution to a temperature lower than the temperature of the aging step by 5 ° C. or more, preferably 10 ° C. or more, more preferably 15 ° C. or more. Good.
  • the temperature of the aging step is 40 ° C.
  • the crystal is obtained by gradually cooling the water / alcohol solution to 35 ° C. or less, more preferably 30 to 10 ° C., and further preferably 25 to 20 ° C. It may be deposited.
  • the term “gradual cooling” means to lower the temperature at a rate of, for example, 1 to 10 ° C./hour, preferably 2 to 8 ° C./hour, more preferably 4 to 6 ° C./hour.
  • the obtained crystals are separated using a basket-type centrifuge, etc., and are washed with a C1 to C6 alcohol used for crystallization, an aqueous solution of the alcohol, or without being washed, and then subjected to vacuum in a conical dryer or the like.
  • a ventilator such as a dryer or a fluidized bed dryer, the highly pure 2′FL ⁇ n hydrate crystals of the present invention having a purity of 95% or more are obtained. Can be acquired.
  • the drying temperature in the drying step is 10 to 105 ° C., preferably 20 to 90 ° C., and the drying time is 1 to 24 hours, preferably 1 to 10 hours.
  • the drying time is 10 to 90 ° C., preferably 20 to 60 ° C., and the drying time is 1 to 24 hours, preferably 1 to 10 hours.
  • the 2′FL ⁇ n hydrate crystals of the present invention having a purity of 95% or more can be obtained without drying with a dryer. Further, by repeating the above-described crystallization process of the 2′FL ⁇ n hydrate of the present invention 2 to 5 times, preferably 2 to 3 times, the purity of the 2′FL ⁇ n hydrate of the present invention having a purity of 99.5% or more is increased. Crystals of n-hydrate can be obtained.
  • reaction solution supernatant was passed through a column packed with SK1B (Mitsubishi Chemical Corporation) and then with WA30 (Mitsubishi Chemical Corporation), and 2'- O -fucosyl lactose from which the salt had been removed was removed.
  • the containing fraction was collected.
  • the fraction was concentrated under reduced pressure to obtain a 2′- O -fucosyl lactose-containing aqueous solution having a 2′FL concentration of 800 g / L.
  • the purity of 2'FL of the aqueous solution was measured by HPLC, it was 80.7% (area%).
  • the moisture content of the 2'FL crystals obtained in (2) above was measured by the following method. Using a moisture analyzer AQV-2200 (Hiranuma Sangyo Co., Ltd., Karl Fischer method), 0.5 g of sample was precisely weighed and stirred for 10 minutes in 50 mL of anhydrous methanol, then HYDRANAL-Composite 5 (1 mL-5 mg H 2 O ) To determine the water content. As a result, the moisture content of the 2′FL crystals obtained in the above (2) was 5.2%. From comparison with the theoretical moisture content, the 2′FL crystals were 2′FL ⁇ 1.4 hydrate.
  • the 2′FL single crystal obtained by the above method is attached to the R-AXIS V diffractometer (Rigaku) installed in the beam line BL26B1 of the large synchrotron radiation facility SPring-8.
  • the diffraction image was measured using synchrotron radiation (wavelength 0.711 ⁇ ) corresponding to MoK ⁇ rays while blowing a 100 K nitrogen stream on the single crystal. From the set of plane index and diffraction intensity calculated from the diffraction image, the structure was determined by the direct method and the structure was refined by the least square method using SHELX-97 (University of Göttingen) to obtain a single crystal structure.
  • the crystals used for the analysis are 2′FL ⁇ 1.3 hydrate and 1.4 hydrate crystals, and both hydrate crystals have two water molecules having different occupancy rates, that is, occupancy rates of 100 % Occupancy is 30% in the I-position water molecule and 2′FL ⁇ 1.3 hydrate crystals, and 38.1% in the 2′FL ⁇ 1.4 hydrate crystals. It was found that the water molecule at position II was included, and the water molecule at position II was more easily dehydrated than the water molecule at position I.
  • glucose in 2′FL ⁇ 1.4 hydrate is 11.6 and 88.4% of ⁇ type and ⁇ type, respectively. It was shown to contain. This is probably because the water molecule at position II collides with the oxygen atom of ⁇ -type glucose, so the ⁇ -type ratio is lower than ⁇ -type. Therefore, in the 2′FL ⁇ m hydrate crystals containing more than 1.4 water molecules, the abundance ratio of ⁇ -type glucose is lower than 11.6%, or all are ⁇ -type.
  • thermogravimetric analysis Thermogravimetric analysis (TG) The thermogravimetric analysis was performed using Q500 (manufactured by TA Instruments). Crystals of 2′FL ⁇ 1.7 hydrate obtained by the method described in Example 1 to be described later are weighed and attached to the apparatus, and at a rate of 10 ° C./min from room temperature to 200 ° C. in a dry nitrogen stream. The temperature was raised, and the change in mass with respect to temperature and time was measured. If a mass change rate of 0.20% / min or more is observed during measurement, the measurement is performed while maintaining the temperature at that time, and when the mass change rate becomes less than 0.05% / min, the temperature is increased. Resumed.
  • the obtained 2′FL crystals were recovered using a basket-type centrifuge and washed with 200 ml of ethanol. The obtained crystals were dried at 30 ° C. for 180 minutes using a vacuum dryer to obtain 2′FL ⁇ 1.3 hydrate crystals. The amount of hydrated crystals was determined by the same method as in Example 1 (4). (3) Production of 2′FL ⁇ 1.0 hydrate crystals 2′FL was produced in the same manner as in Example 1, and the crystals were precipitated and aged.
  • the obtained 2′FL crystals were recovered using a basket-type centrifuge and washed with 200 ml of ethanol. The obtained crystals were dried at 40 ° C. for 180 minutes using a vacuum dryer to obtain 2′FL ⁇ 1.0 hydrate crystals. The amount of hydrated crystals was determined by the same method as in Example 1 (4). (4) Production of 2′FL ⁇ anhydride crystals 2′FL is produced in the same manner as in Example 1, and the crystals are precipitated and aged.
  • the obtained 2′FL crystals are recovered using a basket-type centrifuge and washed with 200 ml of ethanol.
  • the obtained crystals are dried at 90 ° C. for 24 hours using a vacuum dryer to obtain 2′FL ⁇ anhydride crystals.
  • the amount of hydrated crystals can be determined by the same method as in Example 1 (4).
  • Production of crystals of 2'- O -fucosyl lactose p-hydrate 1 (1) Production of 2′FL 2′FL was produced in the same manner as in Example 1 (1). (2) Production of 2′FL ⁇ 2.5 hydrate crystals After 2′FL crystals were precipitated by the same method as in Example 1 (2), the 2′FL crystals were hydrated in a water / ethanol solution. The crystals were aged.
  • the obtained 2′FL crystals were recovered using a basket-type centrifuge and used as 2′FL crystals used for the subsequent analysis. Purity measurement by HPLC confirms that 95.2% (area%) or more of 2′FL crystals have been obtained. By dissolving the crystals once more in water and repeating the above crystallization procedure, 99 ′ is obtained. More than 4% (area%) of 2′FL crystals could be obtained.
  • (3) Powder X-ray diffraction The 2′FL crystals obtained in (2) above were subjected to powder X-ray diffraction. The analysis conditions are the same as in Example 1 (3), and the measurement results are shown in Table 7.
  • Production of 2'- O -fucosyl lactose p-hydrate crystals 2 (1) Production of 2′FL ⁇ 2.0 hydrate crystals 2′FL was produced in the same manner as in Example 1, and the crystals were precipitated and aged. The obtained 2′FL crystals were recovered using a basket-type centrifuge. The obtained crystals were dried at 30 ° C. for 30 minutes using a vacuum dryer to obtain 2′FL ⁇ 2.0 hydrate crystals. (2) Production of 2′FL ⁇ 2.2 hydrate crystals 2′FL was produced in the same manner as in Example 1, and the crystals were precipitated and aged.
  • the obtained 2′FL crystals were recovered using a basket-type centrifuge. The obtained crystals were dried at 25 ° C. for 30 minutes using a vacuum dryer to obtain 2′FL ⁇ 2.2 hydrate crystals. (3) Production of 2′FL ⁇ 2.7 hydrate crystals 2′FL was produced in the same manner as in Example 1, and the crystals were precipitated and aged.
  • the obtained 2′FL crystals were recovered using a basket-type centrifuge.
  • the obtained crystals were dried at 20 ° C. for 30 minutes using a fluidized bed dryer to obtain 2′FL ⁇ 2.7 hydrate crystals.
  • 2′- O -fucosyl lactose crystals useful as products, raw materials or intermediates for health foods, pharmaceuticals, cosmetics and the like, and a method for producing the same.

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Abstract

A crystal of 2'-O-fucosyllactose n-hydrate (wherein n represents an arbitrary numerical number of 0 to 3, wherein when n = 0, the compound is referred to as "2'-O-fucosyllactose anhydride") is produced by a method comprising a step of adding a C1-C6 alcohol to an aqueous solution containing 2'-O-fucosyllactose and having a temperature of 15 to 60˚C while keeping the temperature of the aqueous solution at the above-mentioned temperature, wherein the amount of the alcohol to be added is 0.1 to 3 folds the volume of the aqueous solution and the alcohol is added to the aqueous solution slowly in small portions.

Description

オリゴ糖の結晶およびその製造方法Oligosaccharide crystals and method for producing the same
　本発明は、例えば健康食品、医薬品、化粧品等の製品、原料もしくは中間体等として有用であるオリゴ糖の結晶、および該結晶の製造方法に関する。 The present invention relates to an oligosaccharide crystal that is useful, for example, as a product, a raw material or an intermediate for health foods, pharmaceuticals, cosmetics, and the like, and a method for producing the crystal.
　オリゴ糖は、例えば健康食品、医薬品、化粧品等の原料または中間体等として有用である。
　オリゴ糖の製造方法としては、合成法および発酵法が知られている（非特許文献１及び２、並びに特許文献１及び２）。
　オリゴ糖のうち、２’－O－フコシルラクトース［４－O－（２－O－α－L－フコシルピラノシル－β－D－ガラクトピラノシル）－D－ガラクトース］はヒト母乳に含まれる重要なオリゴ糖の１つであり、腸内細菌増殖作用、抗菌活性、抗ウィルス活性、免疫システム亢進作用及び脳機能亢進作用などの機能があるとされている。 Oligosaccharides are useful, for example, as raw materials or intermediates for health foods, pharmaceuticals, cosmetics and the like.
Synthetic methods and fermentation methods are known as oligosaccharide production methods (Non-patent Documents 1 and 2, and Patent Documents 1 and 2).
Among oligosaccharides, 2'- O -fucosyl lactose [4- O- (2- O- α-L-fucosylpyranosyl-β-D-galactopyranosyl) -D-galactose] is contained in human breast milk It is one of the important oligosaccharides and has functions such as intestinal bacterial growth action, antibacterial activity, antiviral activity, immune system enhancement action and brain function enhancement action.
　上記のような有用な作用を有する２’－O－フコシルラクトースを、健康食品等の原料として用いるためには、保存安定性が良好で、かつ高純度であることが求められている。結晶は保存安定性が高く、また結晶化は純度を上げる手段として非常に有効である。非特許文献３および特許文献３には、２’－O－フコシルラクトースの結晶が取得されたとの記載はあるが、取得された結晶の性質に関する記載はなく、産業上有用な２’－O－フコシルラクトースの結晶が求められている。 In order to use 2′- O -fucosyl lactose having the above-described useful action as a raw material for health foods or the like, it is required to have good storage stability and high purity. Crystals have high storage stability, and crystallization is very effective as a means for increasing purity. Non-Patent Document 3 and Patent Document 3 have described that crystals of 2′- O -fucosyl lactose have been obtained, but there is no description regarding the properties of the obtained crystals, and industrially useful 2′- O—. There is a need for fucosyl lactose crystals.
国際公開第98/12343号パンフレットInternational Publication No. 98/12343 Pamphlet 国際公開第99/40205号パンフレットWO99 / 40205 pamphlet 国際公開第2011/150939号パンフレットInternational Publication No. 2011/150939 Pamphlet
　本発明の目的は、例えば健康食品、医薬品、化粧品等の製品、原料もしくは中間体等として有用なオリゴ糖である２’－O－フコシルラクトースの結晶、およびその製造方法を提供することにある。 An object of the present invention is to provide crystals of 2′- O -fucosyl lactose, which is an oligosaccharide useful as, for example, products such as health foods, pharmaceuticals and cosmetics, raw materials or intermediates, and a method for producing the same.
　　本発明は、以下の（１）～（１２）に関する。
（１）２’－O－フコシルラクトース・ｎ水和物の結晶（ただし、ｎは０～３のいずれかの数字を表し、ｎ＝０の場合は、２’－O－フコシルラクトース・無水物という）。
（２）粉末Ｘ線回折において、回折角（２θ）が、１７．００°±０．２０°、１８．８２°±０．２０°および２１．５８°±０．２０°にピークを有することを特徴とする請求項１記載の２’－O－フコシルラクトース・ｎ水和物の結晶［ｎは上記（１）と同義］。
（３）粉末Ｘ線回折において、回折角（２θ）が、さらに９．８６°±０．２０°、１４．１６°±０．２０°、１６．１２°±０．２０°および２０．３０°±０．２０°にピークを有することを特徴とする上記（２）の２’－O－フコシルラクトース・ｎ水和物の結晶［ｎは上記（１）と同義］。
（４）粉末Ｘ線回折において、回折角（２θ）が、さらに１５．５２°±０．２０°、２３．８８°±０．２０°、２４．７６°±０．２０°および２７．５２°±０．２０°にピークを有することを特徴とする上記（３）の２’－O－フコシルラクトース・ｎ水和物の結晶［ｎは上記（１）と同義］。
（５）粉末Ｘ線回折において、回折角（２θ）が、さらに１２．７２°±０．２０°にピークを有することを特徴とする上記（４）の２’－O－フコシルラクトース・ｎ水和物の結晶［ｎは上記（１）と同義］。
（６）粉末Ｘ線回折において、回折角（２θ）が、１０．００°±０．２０°、１８．９４°±０．２０°、２０．４４°±０．２０°および２１．７２°±０．２０°にピークを有することを特徴とする上記（１）の２’－O－フコシルラクトース・ｎ水和物の結晶［ｎは上記（１）と同義］。
（７）粉末Ｘ線回折において、回折角（２θ）が、さらに、１１．８８°±０．２０°、１４．２６°±０．２０°、１５．６０°±０．２０°および１７．１２°±０．２０°にピークを有することを特徴とする上記（６）の２’－O－フコシルラクトース・ｎ水和物の結晶［ｎは上記（１）と同義］。
（８）粉末Ｘ線回折において、回折角（２θ）が、さらに、１６．２４°±０．２０°、１６．８８°±０．２０°、１８．５８°±０．２０°および１９．８６°±０．２０°にピークを有することを特徴とする上記（７）の２’－O－フコシルラクトース・ｎ水和物の結晶［ｎは上記（１）と同義］。
（９）１５～６０℃の２’－O－フコシルラクトース含有水溶液に、その温度を維持しながら該水溶液量に対して０．１～３倍量のＣ１～Ｃ６のアルコールを該水溶液に少量ずつ徐々に添加する工程を含む上記（１）の２’－O－フコシルラクトース・ｎ水和物の結晶［ｎは上記（１）と同義］の製造方法。
（１０）１５～６０℃の２’－O－フコシルラクトース含有水溶液に、種晶として２’－O－フコシルラクトースの結晶を添加した後、１５～６０℃の温度を維持しながら該水溶液量に対して０．１～３倍量のＣ１～Ｃ６のアルコールを該水溶液に少量ずつ徐々に添加する工程を含む上記（１）の２’－O－フコシルラクトース・ｎ水和物の結晶［ｎは上記（１）と同義］の製造方法。
（１１）Ｃ１～Ｃ６のアルコールを２’－O－フコシルラクトース含有水溶液に少量ずつ徐々に添加した後、得られた水／アルコール溶液を１５～６０℃で１時間以上保持する工程を含む上記（９）または（１０）の製造方法。
（１２）Ｃ１～Ｃ６のアルコールが、メタノールまたはエタノールである上記（９）～（１１）のいずれか１つに記載の製造方法。 The present invention relates to the following (1) to (12).
(1) 2′- O -fucosyl lactose n-hydrate crystal (where n represents any number from 0 to 3, and when n = 0, 2′- O -fucosyl lactose-anhydride Called).
(2) In powder X-ray diffraction, the diffraction angle (2θ) has peaks at 17.00 ° ± 0.20 °, 18.82 ° ± 0.20 °, and 21.58 ° ± 0.20 °. The crystal of 2'- O -fucosyl lactose n-hydrate according to claim 1, wherein n is as defined in (1) above.
(3) In powder X-ray diffraction, the diffraction angles (2θ) are further increased to 9.86 ° ± 0.20 °, 14.16 ° ± 0.20 °, 16.12 ° ± 0.20 °, and 20.30. A crystal of 2′- O -fucosyl lactose n-hydrate according to (2) above, having a peak at ° ± 0.20 ° [n is as defined in (1) above].
(4) In powder X-ray diffraction, diffraction angles (2θ) are further 15.52 ° ± 0.20 °, 23.88 ° ± 0.20 °, 24.76 ° ± 0.20 °, and 27.52. A crystal of 2′- O -fucosyl lactose n-hydrate according to (3) above having a peak at ° ± 0.20 ° [n is as defined in (1) above].
(5) 2′- O -fucosyl lactose / n water according to (4) above, wherein the diffraction angle (2θ) further has a peak at 12.72 ° ± 0.20 ° in powder X-ray diffraction Japanese crystals [n is as defined in (1) above].
(6) In powder X-ray diffraction, diffraction angles (2θ) are 10.00 ° ± 0.20 °, 18.94 ° ± 0.20 °, 20.44 ° ± 0.20 ° and 21.72 °. A crystal of 2′- O -fucosyl lactose n-hydrate according to (1) above having a peak at ± 0.20 ° [n is as defined in (1) above].
(7) In powder X-ray diffraction, diffraction angles (2θ) are further changed to 11.88 ° ± 0.20 °, 14.26 ° ± 0.20 °, 15.60 ° ± 0.20 °, and 17. A crystal of 2'- O -fucosyl lactose n-hydrate according to (6) above, having a peak at 12 ° ± 0.20 ° [n is as defined in (1) above].
(8) In powder X-ray diffraction, the diffraction angle (2θ) is further changed to 16.24 ° ± 0.20 °, 16.88 ° ± 0.20 °, 18.58 ° ± 0.20 °, and 19. A crystal of 2′- O -fucosyl lactose · n hydrate according to (7) above having a peak at 86 ° ± 0.20 ° [n is as defined in (1) above].
(9) Into the aqueous solution containing 2′- O -fucosyl lactose at 15 to 60 ° C., 0.1 to 3 times the amount of C1 to C6 alcohol is added to the aqueous solution little by little while maintaining the temperature. The method for producing 2'- O -fucosyl lactose n-hydrate crystals [n is synonymous with the above (1)] comprising the step of gradually adding.
The fucosyllactose containing aqueous solution, 2'O as seed - - (10) 2'- O of 15 ~ 60 ° C. After addition of fucosyl lactose crystals, the aqueous solution amount while maintaining the temperature of 15 ~ 60 ° C. In contrast, the 2′- O -fucosyl lactose n-hydrate crystals of the above (1) comprising a step of gradually adding 0.1 to 3 times the amount of C1-C6 alcohol to the aqueous solution little by little [n is Synonymous with (1) above].
(11) The method includes the step of gradually adding the C1-C6 alcohol to the 2′- O -fucosyl lactose-containing aqueous solution little by little and then holding the resulting water / alcohol solution at 15-60 ° C. for 1 hour or more ( 9) The manufacturing method of (10).
(12) The production method according to any one of (9) to (11) above, wherein the C1 to C6 alcohol is methanol or ethanol.
　本発明により、例えば健康食品、医薬品、化粧品等の製品、原料もしくは中間体等として有用であるオリゴ糖の結晶およびその製造方法が提供される。 The present invention provides oligosaccharide crystals that are useful as products, raw materials, intermediates, and the like such as health foods, pharmaceuticals, and cosmetics, and a method for producing the same.
図１は、２’－O－フコシルラクトース・１．４水和物の結晶の粉末Ｘ線回折のピークパターンを示す図である。グラフの縦軸は強度（単位cps）、横軸は回折角（２θ）（単位°）である。FIG. 1 is a diagram showing a powder X-ray diffraction peak pattern of 2′- O -fucosyl lactose 1.4 hydrate crystals. The vertical axis of the graph is intensity (unit cps), and the horizontal axis is diffraction angle (2θ) (unit °). 図２は、２’－O－フコシルラクトース・２．５水和物の結晶の粉末Ｘ線回折のピークパターンを示す図である。グラフの縦軸は強度（単位cps）、横軸は回折角（２θ）（単位°）である。FIG. 2 is a diagram showing a powder X-ray diffraction peak pattern of 2′- O -fucosyl lactose 2.5 hydrate crystals. The vertical axis of the graph is intensity (unit cps), and the horizontal axis is diffraction angle (2θ) (unit °). 図３は、２’－O－フコシルラクトースのＡ結晶の熱重量分析（ＴＧ）の結果を表す図である。グラフの縦軸は重量（％）、横軸は温度である。Ａで表す領域は室温～５０℃の間の水減量分、Ｂで表す領域は５０～１８０℃の間の水減量分を表す。FIG. 3 is a diagram showing the results of thermogravimetric analysis (TG) of crystal A of 2′- O -fucosyl lactose. The vertical axis of the graph is weight (%), and the horizontal axis is temperature. The region represented by A represents the amount of water loss between room temperature and 50 ° C, and the region represented by B represents the amount of water loss between 50 and 180 ° C. 図４は、２’－O－フコシルラクトースの結晶の温度可変粉末Ｘ線回折のピークパターンを示す図である。グラフの縦軸は強度（単位cps）、横軸は回折角（２θ）（単位°）である。FIG. 4 is a diagram showing a temperature variable powder X-ray diffraction peak pattern of 2′- O -fucosyl lactose crystals. The vertical axis of the graph is intensity (unit cps), and the horizontal axis is diffraction angle (2θ) (unit °).
１．本発明の２’－O－フコシルラクトースの結晶
　本発明の２’－O－フコシルラクトース（以下、２’ＦＬと略す場合もある）・ｎ水和物の結晶（ただし、ｎは０～３の任意の数字、好ましくはｎは０より大きく、３以下の任意の数字であり、より好ましくはｎは０～３の任意の小数数第１位までの数字、さらに好ましくはｎは０より大きく、３以下の任意の小数第１位までの数字である。ｎ＝０のときは２’ＦＬ・無水物という）としては、Ａ晶及びＢ晶をあげることができ、さらに２’－O－フコシルラクトース中のグルコースの立体配座がα型及び/又はβ型である結晶をあげることができる。
（１）Ａ晶
　Ａ晶は、２’ＦＬ・ｍ水和物の結晶（ただし、ｍは０～１．７の任意の数字、好ましくはｍは０より大きく、１．７以下の任意の数字であり、より好ましくはｍは０．１～１．７の任意の小数第一位までの数字である。ｍ＝０のときは２’ＦＬ・無水物という）であり、例えばＡ晶である２’ＦＬ・１．４水和物の結晶は、Ｘ線源としてCuKαを用いた粉末Ｘ線回折パターンが、図１及び表１に示す値で規定される結晶である。 1. 2'O of the present invention - fucosyl sill lactose crystals present invention 2'O - fucosyllactose crystal (hereinafter sometimes abbreviated as 2'FL) · n-hydrate (where, n is 0-3 Any number, preferably n is any number greater than 0 and less than or equal to 3, more preferably n is any number from 0 to 3 in any decimal place, more preferably n is greater than 0; Any number up to the first decimal place of 3 or less. When n = 0, 2′FL · anhydride can be cited as A and B crystals, and 2′- O -fucosyl. A crystal in which the conformation of glucose in lactose is α-type and / or β-type can be mentioned.
(1) Crystal A Crystal A is a 2′FL · m hydrate crystal (where m is any number from 0 to 1.7, preferably m is greater than 0 and any number less than 1.7) More preferably, m is a number up to the first decimal place in the range of 0.1 to 1.7. When m = 0, it is 2′FL · anhydride), for example, crystal A The crystal of 2′FL · 1.4 hydrate is a crystal whose powder X-ray diffraction pattern using CuKα as an X-ray source is defined by the values shown in FIG. 1 and Table 1.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　粉末Ｘ線回折を行う際に用いる分析装置、および測定条件としては、分析装置は株式会社リガク社製の試料水平型多目的X線回折装置UltimaIV、測定条件は、Ｘ線源に銅ターゲット封入管を用いて、単色化しないＸ線を試料に照射し、エネルギー分解型検出器を用いてCuKα線（波長1.541 Å）由来の回折X線を検出する条件をあげることができる。
　また、２’ＦＬ・１．３水和物及び１．４水和物の単結晶構造からシミュレーションされる粉末Ｘ線回折パターンは、表２に示すとおりである。 The analyzer used for powder X-ray diffraction and the measurement conditions are the sample horizontal multi-purpose X-ray diffractometer Ultima IV manufactured by Rigaku Corporation, and the measurement conditions are a copper target sealed tube in the X-ray source. It is possible to raise conditions for irradiating a sample with X-rays that are not monochromatic and detecting diffracted X-rays derived from CuKα rays (wavelength 1.541 エ ネ ル ギ ー) using an energy-resolving detector.
The powder X-ray diffraction patterns simulated from the single crystal structures of 2′FL · 1.3 hydrate and 1.4 hydrate are as shown in Table 2.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　結晶構造を決定する方法としては、単結晶Ｘ線回折法による構造解析を挙げることができる。２’ＦＬ・ｎ水和物の単結晶を回折計に取り付け、室温の大気中あるいは所定の温度の不活性ガス気流中で、所定の波長のＸ線を用いて、回折画像を測定する。回折画像から算出された面指数と回折強度の組から、直接法による構造決定と最小二乗法による構造精密化を行い、単結晶構造を得る。 As a method for determining the crystal structure, a structural analysis by a single crystal X-ray diffraction method can be mentioned. A single crystal of 2'FL · n hydrate is attached to a diffractometer, and a diffraction image is measured using X-rays having a predetermined wavelength in an atmosphere of room temperature or an inert gas stream at a predetermined temperature. From a set of plane index and diffraction intensity calculated from the diffraction image, the structure is determined by the direct method and the structure is refined by the least square method to obtain a single crystal structure.
　単結晶構造から粉末Ｘ線回折パターンのシミュレーションを行う方法としては、例えば結晶構造表示プログラムＭｅｒｃｕｒｙ（ケンブリッジ結晶学データセンター）やＰｏｗｄｅｒＣｅｌｌ（ドイツ連邦材料研究試験所）を用いることができる。結晶の格子定数と原子座標、そして計算に用いるＸ線の波長を入力することにより、粉末Ｘ線回折パターンを算出する。 As a method for simulating a powder X-ray diffraction pattern from a single crystal structure, for example, a crystal structure display program Mercury (Cambridge Crystallographic Data Center) or PowderCell (German Federal Materials Research Laboratory) can be used. A powder X-ray diffraction pattern is calculated by inputting a crystal lattice constant, atomic coordinates, and an X-ray wavelength used for calculation.
　２’ＦＬ・１．４水和物結晶の粉末Ｘ線回折パターンの実測値と、２’ＦＬ・１．４水和物および１．３水和物の単結晶構造解析からシミュレートされる粉末Ｘ線回折パターンはよく一致しており、結晶に含まれる水分子の数によってピーク強度が変化する程度である。
　Ａ晶は、結晶から水分子が抜けるに従って結晶の格子が収縮するため、ピークが高角に移動する傾向にあるが、Ａ晶の結晶は以下のように定義することができる。 Powders simulated from measured values of powder X-ray diffraction patterns of 2′FL · 1.4 hydrate crystals and single crystal structure analysis of 2′FL · 1.4 hydrate and 1.3 hydrate The X-ray diffraction patterns are in good agreement, and the peak intensity changes depending on the number of water molecules contained in the crystal.
In crystal A, the crystal lattice contracts as water molecules escape from the crystal, so the peak tends to move to a high angle, but crystal A can be defined as follows.
　すなわち、上記装置を用いて上記条件下で粉末Ｘ線回折パターンの分析を行ったとき、回折角（２θ）が、１７．００°±０．２０°、１８．８２°±０．２０°および２１．５８°±０．２０°、好ましくは１７．００°±０．２０°、１８．８２°±０．２０°、２１．５８°±０．２０°、９．８６°±０．２０°、１４．１６°±０．２０°、１６．１２°±０．２０°および２０．３０°±０．２０°、より好ましくは１７．００°±０．２０°、１８．８２°±０．２０°、２１．５８°±０．２０°、９．８６°±０．２０°、１４．１６°±０．２０°、１６．１２°±０．２０°、２０．３０°±０．２０°、１５．５２°±０．２０°、２３．８８°±０．２０°、２４．７６°±０．２０°および２７．５２°±０．２０°、さらに好ましくは１７．００°±０．２０°、１８．８２°±０．２０°、２１．５８°±０．２０°、９．８６°±０．２０°、１４．１６°±０．２０°、１６．１２°±０．２０°、２０．３０°±０．２０°、１５．５２°±０．２０°、２３．８８°±０．２０°、２４．７６°±０．２０°、２７．５２°±０．２０°および１２．７２°±０．２０°、最も好ましくは表１の２θにピークを有する２’ＦＬ・ｍ水和物の結晶、は本発明の２’ＦＬ・ｍ水和物の結晶である。 That is, when the powder X-ray diffraction pattern was analyzed using the above apparatus under the above conditions, the diffraction angle (2θ) was 17.00 ° ± 0.20 °, 18.82 ° ± 0.20 ° and 21.58 ° ± 0.20 °, preferably 17.00 ° ± 0.20 °, 18.82 ° ± 0.20 °, 21.58 ° ± 0.20 °, 9.86 ° ± 0.20 °, 14.16 ° ± 0.20 °, 16.12 ° ± 0.20 ° and 20.30 ° ± 0.20 °, more preferably 17.00 ° ± 0.20 °, 18.82 ° ± 0.20 °, 21.58 ° ± 0.20 °, 9.86 ° ± 0.20 °, 14.16 ° ± 0.20 °, 16.12 ° ± 0.20 °, 20.30 ° ± 0.20 °, 15.52 ° ± 0.20 °, 23.88 ° ± 0.20 °, 24.76 ° ± 0.20 ° and 27.52 ° ± 0.20 °, more preferably 17. 00 ° ± 0. 0 °, 18.82 ° ± 0.20 °, 21.58 ° ± 0.20 °, 9.86 ° ± 0.20 °, 14.16 ° ± 0.20 °, 16.12 ° ± 0. 20 °, 20.30 ° ± 0.20 °, 15.52 ° ± 0.20 °, 23.88 ° ± 0.20 °, 24.76 ° ± 0.20 °, 27.52 ° ± 0. 2′FL · m hydrate crystals having a peak at 20 ° and 12.72 ° ± 0.20 °, most preferably 2θ in Table 1, are the 2′FL · m hydrate crystals of the present invention. is there.
（２）Ｂ晶
　Ｂ晶は、２’ＦＬ・ｐ水和物（ただし、ｐは１．７より大きく３以下の任意の数字であり、好ましくはｐは１．８～３．０の任意の小数第一位までの数字である）の結晶であり、Ｂ晶である２’ＦＬ・２.５水和物の結晶は、Ｘ線源としてCuKαを用いた粉末Ｘ線回折パターンが、図２及び表３に示す値で規定される結晶である。 (2) Crystal B Crystal B is 2′FL · p hydrate (where p is any number greater than 1.7 and less than or equal to 3, preferably p is any number from 1.8 to 3.0 The crystal of 2′FL · 2.5 hydrate, which is a B crystal, is a powder X-ray diffraction pattern using CuKα as an X-ray source. And crystals defined by the values shown in Table 3.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
　２’ＦＬ・２.５水和物の結晶の単結晶構造からシミュレーションされる粉末Ｘ線回折パターンは、表４に示すとおりである。 The powder X-ray diffraction pattern simulated from the single crystal structure of 2′FL · 2.5 hydrate crystals is as shown in Table 4.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　２’ＦＬ・２．５水和物結晶の粉末Ｘ線回折パターンと、２’ＦＬ・２．５水和物結晶の単結晶構造解析からシミュレートされる粉末Ｘ線回折パターンはよく一致しており、結晶に含まれる水分子の数によってピーク強度が変化する程度である。
　Ｂ晶も、その結晶から水分子が抜けるに従って結晶の格子が収縮するため、ピークが高角に移動する傾向にあるが、Ｂ晶の結晶は以下のように定義することができる。 The powder X-ray diffraction pattern of 2'FL · 2.5 hydrate crystals and the powder X-ray diffraction pattern simulated from the single crystal structure analysis of 2'FL · 2.5 hydrate crystals are in good agreement That is, the peak intensity changes depending on the number of water molecules contained in the crystal.
The crystal B also has a tendency to move to a high angle because the crystal lattice contracts as water molecules escape from the crystal. The crystal of the crystal B can be defined as follows.
　すなわち、上記装置を用いて上記条件下で粉末Ｘ線回折パターンの分析を行ったとき、回折角（２θ）が、１０．００°±０．２０°、１８．９４°±０．２０°、２０．４４°±０．２０°および２１．７２°±０．２０°、好ましくは１０．００°±０．２０°、１８．９４°±０．２０°、２０．４４°±０．２０°、２１．７２°±０．２０°、１１．８８°±０．２０°、１４．２６°±０．２０°、１５．６０°±０．２０°および１７．１２°±０．２０°、さらに好ましくは１０．００°±０．２０°、１８．９４°±０．２０°、２０．４４°±０．２０°、２１．７２°±０．２０°、１１．８８°±０．２０°、１４．２６°±０．２０°、１５．６０°±０．２０°、１７．１２°±０．２０°、１６．２４°±０．２０°、１６．８８°±０．２０°、１８．５８°±０．２０°および１９．８６°±０．２０°、最も好ましくは表３の２θにピークを有する２’ＦＬの水和物の結晶、は本発明の２’ＦＬ・ｐ水和物の結晶である。 That is, when the powder X-ray diffraction pattern was analyzed using the above apparatus under the above conditions, the diffraction angle (2θ) was 10.00 ° ± 0.20 °, 18.94 ° ± 0.20 °, 20.44 ° ± 0.20 ° and 21.72 ° ± 0.20 °, preferably 10.00 ° ± 0.20 °, 18.94 ° ± 0.20 °, 20.44 ° ± 0.20 °, 21.72 ° ± 0.20 °, 11.88 ° ± 0.20 °, 14.26 ° ± 0.20 °, 15.60 ° ± 0.20 ° and 17.12 ° ± 0.20 °, more preferably 10.00 ° ± 0.20 °, 18.94 ° ± 0.20 °, 20.44 ° ± 0.20 °, 21.72 ° ± 0.20 °, 11.88 ° ± 0.20 °, 14.26 ° ± 0.20 °, 15.60 ° ± 0.20 °, 17.12 ° ± 0.20 °, 16.24 ° ± 0.20 °, 16.88 ° ± 0.20 °, 18. 2′FL hydrate crystals having a peak at 8 ° ± 0.20 ° and 19.86 ° ± 0.20 °, most preferably 2θ in Table 3, are 2′FL · p hydration of the present invention. It is a crystal of an object.
２．本発明の２’－O－フコシルラクトース・ｎ水和物の結晶の製造方法
　本発明において、２’ＦＬ含有水溶液としては、２’ＦＬを含有する水溶液であればいずれでもよいが、例えば２’ＦＬの純度が８０％以上、より好ましくは８５％以上、さらに好ましくは９０％以上の２’ＦＬ含有水溶液をあげることができ、該水溶液に含まれる不純物のほとんどがラクトースのような糖類である場合は、２’ＦＬ水溶液の純度が８０％未満であっても結晶化には支障がない。 2. 2. Method for Producing Crystal of 2′- O- Fucosyl Lactose n Hydrate of the Present Invention In the present invention, the 2′FL-containing aqueous solution may be any aqueous solution containing 2′FL, for example, 2′FL In the case where the purity of FL is 80% or more, more preferably 85% or more, and more preferably 90% or more, a 2'FL-containing aqueous solution can be given, and most of the impurities contained in the aqueous solution are sugars such as lactose. Does not hinder crystallization even if the purity of the 2′FL aqueous solution is less than 80%.
　上記の２’ＦＬ含有水溶液は、例えばChem. Rev., Vol.100, p4445, 2000、Curr.Opin. in Drug Discovery & Develop., Vol. 3,p756,　2000、国際公開第98/12343号パンフレット、または国際公開第99/40205号パンフレットに記載の方法に準じて、合成法、酵素法、休止菌体を用いた方法、または発酵法により得られる２’ＦＬを、上記した文献にも記載がある公知の精製方法を用いて精製することによって取得することができる。また市販されている２’ＦＬの粉末を溶解することによっても取得できる。 The above 2′FL-containing aqueous solution is, for example, Chem. Rev., Vol.100, p4445, 2000, Curr.Opin. In Drug Discovery & Develop., Vol. 3, p756, 2000, International Publication No. 98/12343 pamphlet. According to the method described in International Publication No. 99/40205 pamphlet, 2′FL obtained by a synthesis method, an enzymatic method, a method using resting cells, or a fermentation method is also described in the above-mentioned literature. It can be obtained by purification using a certain known purification method. It can also be obtained by dissolving commercially available 2'FL powder.
　より具体的な精製方法としては、酵素法、休止菌体を用いた方法、または発酵法により取得した２’ＦＬから必要に応じて遠心分離、濾過またはセラミックフィルタ等を用いて菌体等の固形物を除去した後、得られた水溶液をダイヤイオン（三菱化学株式会社）等のイオン交換樹脂を充填したカラムに通塔して脱塩してから濃縮し、結晶化する方法をあげることができる。市販されている２’ＦＬの場合は、そのまま濃縮して結晶化することができる。 As a more specific purification method, solids such as bacterial cells can be obtained by centrifugation, filtration, ceramic filter or the like from 2′FL obtained by enzymatic method, method using resting bacterial cells, or fermentation method as necessary. After removing the substances, the resulting aqueous solution can be passed through a column packed with an ion exchange resin such as Diaion (Mitsubishi Chemical Corporation), desalted, concentrated, and crystallized. . In the case of commercially available 2'FL, it can be concentrated and crystallized as it is.
　本発明の方法に用いられる２’ＦＬ含有水溶液の濃度としては、５００ｇ/Ｌ以上、好ましくは６００ｇ/Ｌ以上、さらに好ましくは７００ｇ/Ｌ以上、より好ましくは８００ｇ/Ｌ以上をあげることができる。
　上記で取得される２’ＦＬ含有水溶液を１５～６０℃、好ましくは２０～５５℃、より好ましくは２５～５０℃、さらに好ましくは３０～４５℃、最も好ましくは３５～４３℃にする。 The concentration of the 2′FL-containing aqueous solution used in the method of the present invention is 500 g / L or more, preferably 600 g / L or more, more preferably 700 g / L or more, and more preferably 800 g / L or more.
The 2′FL-containing aqueous solution obtained above is set to 15 to 60 ° C., preferably 20 to 55 ° C., more preferably 25 to 50 ° C., still more preferably 30 to 45 ° C., and most preferably 35 to 43 ° C.
　濃度が７００ｇ/Ｌ以上の２’ＦＬ含有水溶液を用いる場合には、２’ＦＬの結晶を０．００１～０．１重量％、好ましくは０．００５～０．０５重量％、より好ましくは０．０１～０．０３重量％になるように該水溶液に添加してから３０分～１０時間、好ましくは１～８時間、より好ましくは３～６時間、３５～５０℃に維持することにより結晶の析出を促すこともできる。
　上記で添加する２’ＦＬの結晶は、濃度が７００ｇ/Ｌの２’ＦＬ含有水溶液に、５０～６０％になるようにメタノールを加えたのち、４℃で３０日程度保存することによっても取得することができる。 When a 2′FL-containing aqueous solution having a concentration of 700 g / L or more is used, 2′FL crystals are 0.001 to 0.1 wt%, preferably 0.005 to 0.05 wt%, more preferably 0 Crystals are maintained at 35 to 50 ° C. for 30 minutes to 10 hours, preferably 1 to 8 hours, more preferably 3 to 6 hours after addition to the aqueous solution so as to be 0.01 to 0.03% by weight. It is also possible to promote precipitation.
The 2'FL crystals added above can also be obtained by adding methanol to a 2'FL-containing aqueous solution with a concentration of 700 g / L to a concentration of 50-60% and storing at 4 ° C for about 30 days. can do.
　次に、２’ＦＬ含有水溶液の温度を１５～６０℃に維持した状態で、Ｃ１～Ｃ６のアルコールを少量ずつ徐々に該水溶液に添加することで、結晶を析出させる。
　Ｃ１～Ｃ６のアルコールの添加量としては、２’ＦＬ含有水溶液に対して０．１～３倍量、好ましくは０．２～２．５倍量、より好ましくは０．４～２倍量をあげることができる。 Next, while maintaining the temperature of the 2′FL-containing aqueous solution at 15 to 60 ° C., C1 to C6 alcohol is gradually added to the aqueous solution little by little to precipitate crystals.
The amount of the C1 to C6 alcohol added is 0.1 to 3 times, preferably 0.2 to 2.5 times, more preferably 0.4 to 2 times the amount of the 2′FL-containing aqueous solution. I can give you.
　Ｃ１～Ｃ６のアルコールとしては、例えばエタノール、メタノール、ｎ-プロパノール、i-プロパノール、ｎ-ブタノール、i-ブタノール、s-ブタノール、t-ブタノール、アミルアルコール、およびn-ヘキサノール、好ましくはＣ１～Ｃ４のアルコール、例えばエタノール、メタノール、ｎ-プロパノール、i-プロパノール、ｎ-ブタノール、i-ブタノール、s-ブタノール、およびt-ブタノール、より好ましくはメタノールおよびエタノール、さらに好ましくはエタノールをあげることができる。 Examples of C1-C6 alcohols include ethanol, methanol, n-propanol, i-propanol, n-butanol, i-butanol, s-butanol, t-butanol, amyl alcohol, and n-hexanol, preferably C1-C4. And alcohols such as ethanol, methanol, n-propanol, i-propanol, n-butanol, i-butanol, s-butanol, and t-butanol, more preferably methanol and ethanol, and still more preferably ethanol.
　Ｃ１～Ｃ６のアルコールを少量ずつ徐々に添加するとは、２’ＦＬ含有水溶液の温度が急激に変化しないように、該水溶液に滴下することを意味する。滴下する速度としては、例えば５００ｍｌの２’ＦＬ含有水溶液に２５０ｍｌのエタノールを滴下する場合は、２～２０時間、好ましくは４～１８時間、より好ましくは６～１５時間、さらに好ましくは８～１２時間かけてエタノール全量を滴下する速度をあげることができ、用いるアルコールの種類、２’ＦＬ含有水溶液およびアルコールの量に応じて適宜設定できる。 Gradual addition of C1 to C6 alcohols means dropwise addition to the aqueous solution so that the temperature of the 2'FL-containing aqueous solution does not change abruptly. For example, when 250 ml of ethanol is dropped into 500 ml of 2′FL-containing aqueous solution, the dropping speed is 2 to 20 hours, preferably 4 to 18 hours, more preferably 6 to 15 hours, and further preferably 8 to 12 hours. The rate at which the total amount of ethanol is dropped over time can be increased, and can be set as appropriate according to the type of alcohol used, the 2′FL-containing aqueous solution, and the amount of alcohol.
　その後、得られた水／アルコール溶液を１時間以上、好ましくは１～３０時間、より好ましくは５～２５時間、さらに好ましくは８～２０時間、最も好ましくは１２～１８時間、１５～６０℃で保持することによって結晶を熟成させる。
　結晶を熟成させた後、熟成工程の温度より５℃以上、好ましくは１０℃以上、より好ましくは１５℃以上低い温度に徐々に水／アルコール溶液を冷却することにより、さらに結晶を析出させてもよい。例えば、上記熟成工程の温度が４０℃であった場合、好ましくは３５℃以下、より好ましくは３０～１０℃、さらに好ましくは２５～２０℃に水／アルコール溶液を徐々に冷却することによって結晶を析出させてもよい。 Thereafter, the obtained water / alcohol solution is kept for 1 hour or more, preferably 1 to 30 hours, more preferably 5 to 25 hours, further preferably 8 to 20 hours, most preferably 12 to 18 hours, at 15 to 60 ° C. The crystals are aged by holding.
After aging the crystals, the crystals may be further precipitated by gradually cooling the water / alcohol solution to a temperature lower than the temperature of the aging step by 5 ° C. or more, preferably 10 ° C. or more, more preferably 15 ° C. or more. Good. For example, when the temperature of the aging step is 40 ° C., the crystal is obtained by gradually cooling the water / alcohol solution to 35 ° C. or less, more preferably 30 to 10 ° C., and further preferably 25 to 20 ° C. It may be deposited.
　徐々に冷却するとは、例えば１～１０℃／時間、好ましくは２～８℃／時間、より好ましくは４～６℃／時間の割合で温度を下げることを意味する。
　得られた結晶は、バスケット型遠心分離機等を用いて分離し、結晶化に用いたＣ１～Ｃ６のアルコール、または該アルコールの水溶液で洗浄後、または洗浄せずにそのままコニカル乾燥機等の真空乾燥機または流動層乾燥機等の通風乾燥機を用いて適宜温度および乾燥時間を調整することにより、純度が９５％以上である高純度の本発明の２’ＦＬ・ｎ水和物の結晶を取得することができる。当該乾燥工程の乾燥温度は真空乾燥の場合、１０～１０５℃、好ましくは２０～９０℃、乾燥時間は１～２４時間、好ましくは１～１０時間である。通風乾燥の場合、１０～９０℃、好ましくは２０～６０℃、乾燥時間は１～２４時間、好ましくは１～１０時間である。 The term “gradual cooling” means to lower the temperature at a rate of, for example, 1 to 10 ° C./hour, preferably 2 to 8 ° C./hour, more preferably 4 to 6 ° C./hour.
The obtained crystals are separated using a basket-type centrifuge, etc., and are washed with a C1 to C6 alcohol used for crystallization, an aqueous solution of the alcohol, or without being washed, and then subjected to vacuum in a conical dryer or the like. By adjusting the temperature and drying time as appropriate using a ventilator such as a dryer or a fluidized bed dryer, the highly pure 2′FL · n hydrate crystals of the present invention having a purity of 95% or more are obtained. Can be acquired. In the case of vacuum drying, the drying temperature in the drying step is 10 to 105 ° C., preferably 20 to 90 ° C., and the drying time is 1 to 24 hours, preferably 1 to 10 hours. In the case of ventilation drying, the drying time is 10 to 90 ° C., preferably 20 to 60 ° C., and the drying time is 1 to 24 hours, preferably 1 to 10 hours.
　また、乾燥機による乾燥も行わなくとも純度が９５％以上である高純度の本発明の２’ＦＬ・ｎ水和物の結晶を取得することもできる。
　また上記した本発明の２’ＦＬ・ｎ水和物の結晶化の工程を２～５回、好ましくは２～３回繰り返すことにより、純度が９９．５％以上の本発明の２’ＦＬ・ｎ水和物の結晶を取得することができる。 Further, the 2′FL · n hydrate crystals of the present invention having a purity of 95% or more can be obtained without drying with a dryer.
Further, by repeating the above-described crystallization process of the 2′FL · n hydrate of the present invention 2 to 5 times, preferably 2 to 3 times, the purity of the 2′FL · n hydrate of the present invention having a purity of 99.5% or more is increased. Crystals of n-hydrate can be obtained.
　以下に本発明の実施例を記載するが、本発明はこれに限定されるものではない。 Examples of the present invention will be described below, but the present invention is not limited thereto.
２’－O－フコシルラクトース・ｍ水和物の結晶の製造１
（１）２’ＦＬの製造
　国際公開第98/12343号パンフレットに記載の方法に従って取得した２’－O－フコシルラクトースを含有する１０Ｌの休止菌体反応液に塩酸を加えてpH３に合わせてから、遠心分離により菌体を除去した。 Production of crystals of 2′- O -fucosyl lactose m hydrate 1
(1) Production of 2′FL After adding hydrochloric acid to 10 L resting cell reaction solution containing 2′- O -fucosyl lactose obtained according to the method described in the pamphlet of WO 98/12343, the pH is adjusted to 3. The cells were removed by centrifugation.
　次に、得られた反応液上清をＳＫ１Ｂ（三菱化学株式会社）、続いてＷＡ３０（三菱化学株式会社）を充填したカラムに通塔し、塩が除去された２’－O－フコシルラクトースを含有する画分を回収した。
　続いて当該画分を減圧濃縮することで２’ＦＬの濃度が８００ｇ／Ｌである２’－O－フコシルラクトース含有水溶液を取得した。当該水溶液の２’ＦＬの純度をＨＰＬＣで測定したところ、８０．７％（面積％）であった。
（２）２’ＦＬ・１．４水和物の結晶の製造
　上記で取得した８００ｇ／Ｌの２’ＦＬ含有水溶液５００ｍｌを４０℃に加温した。次に該水溶液に２’ＦＬの結晶を０．１ｇ添加した後、４０℃の温度を保持したままで５時間放置することにより２’ＦＬの結晶を析出させた。 Next, the obtained reaction solution supernatant was passed through a column packed with SK1B (Mitsubishi Chemical Corporation) and then with WA30 (Mitsubishi Chemical Corporation), and 2'- O -fucosyl lactose from which the salt had been removed was removed. The containing fraction was collected.
Subsequently, the fraction was concentrated under reduced pressure to obtain a 2′- O -fucosyl lactose-containing aqueous solution having a 2′FL concentration of 800 g / L. When the purity of 2'FL of the aqueous solution was measured by HPLC, it was 80.7% (area%).
(2) Production of 2′FL · 1.4 hydrate crystals 500 ml of the 800 g / L 2′FL-containing aqueous solution obtained above was heated to 40 ° C. Next, 0.1 g of 2′FL crystals was added to the aqueous solution, and then left for 5 hours while maintaining a temperature of 40 ° C., thereby precipitating 2′FL crystals.
　次に、結晶が析出している該水溶液の温度を４０℃に維持させながら、２５０ｍｌのエタノールを１０時間かけて該溶液に滴下添加することで、さらに結晶を析出させた。
　エタノールの添加が終了した後、得られた水／エタノール溶液を４０℃で１６時間保持することにより結晶を熟成させた。
　得られた２’ＦＬの結晶は、バスケット型遠心分離機を用いて回収し、２００ｍｌのエタノールで洗浄した。 Next, while maintaining the temperature of the aqueous solution in which crystals were precipitated at 40 ° C., 250 ml of ethanol was added dropwise over 10 hours to further precipitate crystals.
After the addition of ethanol was completed, the resulting water / ethanol solution was kept at 40 ° C. for 16 hours to age the crystals.
The obtained 2′FL crystals were recovered using a basket-type centrifuge and washed with 200 ml of ethanol.
　得られた結晶を、真空乾燥機を用いて３０℃で３０分間乾燥することにより２’ＦＬの結晶を取得した。
　ＨＰＬＣによる純度測定では９５.２％（面積％）以上の２’ＦＬの結晶が得られていることが確認され、当該結晶をもう一度水に溶解して上記の晶析操作を繰り返すことにより、９９.８％（面積％）以上の２’－O－フコシルラクトースの結晶を取得することができた。
（３）粉末Ｘ線回折
　上記（２）で得られた２’ＦＬの結晶を粉末Ｘ線回折に供した。結果を表５に示す。 The obtained crystals were dried at 30 ° C. for 30 minutes using a vacuum dryer to obtain 2′FL crystals.
Purity measurement by HPLC confirms that 95.2% (area%) or more of 2′FL crystals have been obtained. By dissolving the crystals once more in water and repeating the above crystallization procedure, 99 ′ is obtained. It was possible to obtain crystals of 2′- O -fucosyl lactose in an amount of 0.8% (area%) or more.
(3) Powder X-ray diffraction The 2′FL crystals obtained in (2) above were subjected to powder X-ray diffraction. The results are shown in Table 5.
　分析条件および測定結果は以下のとおりである。
分析装置：リガク電機社製　試料水平型多目的Ｘ線回折装置UltimaIV
測定条件：Ｘ線原はCuKα、測定波長1.541Å、モノクロメーターを使用した単色化は未実施のKα線を使用 Analysis conditions and measurement results are as follows.
Analyzer: Rigaku Electric Co., Ltd. Sample horizontal multipurpose X-ray diffractometer UltimaIV
Measurement conditions: X-ray source is CuKα, measurement wavelength is 1.541 mm, and monochromatization using a monochromator uses unimplemented Kα rays
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
（４）水分量測定
　上記（２）で得られた２’ＦＬの結晶の水分量を以下の方法で測定した。
　水分測定装置AQV-2200型（平沼産業社製、カールフィッシャー法）を用い、試料0.5gを精秤して無水メタノール50ｍL中で10分間撹拌した後、HYDRANAL-Composite 5（1mL-5mg H2O）で滴定を行うことで水分量を求めた。その結果、上記（２）で得られた２’ＦＬの結晶の水分量は5.2％であり、理論水分量との比較から、当該２’ＦＬの結晶は２’ＦＬ・１．４水和物であることがわかった。
（５）単結晶構造解析
　上記（１）と同様に２’ＦＬの結晶を熟成させた後、遠心分離により結晶を分離し、水：エタノール＝１：２の水溶液を用いて再度結晶化を行い、風乾した後、単結晶構造解析に供した。
　単結晶Ｘ線構造解析は、以下の手順により行った。室温（２９８　Ｋ）での測定では、上記の方法で取得した２’ＦＬの単結晶を、Ｒ－ＡＸＩＳ　ＲＡＰＩＤ－Ｆ回折計（リガク）に取り付け、大気中で、ＣｕＫα線を用いて、回折画像を測定した。低温（１００　Ｋ）での測定では、上記の方法で取得した２’ＦＬの単結晶を、大型放射光施設ＳＰｒｉｎｇ－８のビームラインＢＬ２６Ｂ１に設置されたＲ－ＡＸＩＳ　Ｖ回折計（リガク）に取り付け、１００　Ｋの窒素気流を単結晶に吹き付けながら、ＭｏＫα線相当のシンクロトロン放射光（波長０．７１1　Å）を用いて、回折画像を測定した。回折画像から算出された面指数と回折強度の組から、ＳＨＥLＸ－９７（ゲッチンゲン大学）を用いて直接法による構造決定と最小二乗法による構造精密化を行い、単結晶構造を得た。　 (4) Moisture content measurement The moisture content of the 2'FL crystals obtained in (2) above was measured by the following method.
Using a moisture analyzer AQV-2200 (Hiranuma Sangyo Co., Ltd., Karl Fischer method), 0.5 g of sample was precisely weighed and stirred for 10 minutes in 50 mL of anhydrous methanol, then HYDRANAL-Composite 5 (1 mL-5 mg H 2 O ) To determine the water content. As a result, the moisture content of the 2′FL crystals obtained in the above (2) was 5.2%. From comparison with the theoretical moisture content, the 2′FL crystals were 2′FL · 1.4 hydrate. I found out that
(5) Single crystal structure analysis As in (1) above, after ripening 2'FL crystals, the crystals are separated by centrifugation and recrystallized using an aqueous solution of water: ethanol = 1: 2. After air drying, it was subjected to single crystal structure analysis.
Single crystal X-ray structural analysis was performed according to the following procedure. In the measurement at room temperature (298 K), the 2′FL single crystal obtained by the above method was attached to an R-AXIS RAPID-F diffractometer (Rigaku), and a diffraction image was obtained using CuKα rays in the atmosphere. Was measured. For measurement at low temperature (100 K), the 2′FL single crystal obtained by the above method is attached to the R-AXIS V diffractometer (Rigaku) installed in the beam line BL26B1 of the large synchrotron radiation facility SPring-8. The diffraction image was measured using synchrotron radiation (wavelength 0.711 Å) corresponding to MoKα rays while blowing a 100 K nitrogen stream on the single crystal. From the set of plane index and diffraction intensity calculated from the diffraction image, the structure was determined by the direct method and the structure was refined by the least square method using SHELX-97 (University of Göttingen) to obtain a single crystal structure.
　解析に供した結晶は、２’ＦＬ・１．３水和物及び１．４水和物の結晶であり、両水和物結晶には占有率が異なる２つの水分子、すなわち占有率が１００％であるI位の水分子、及び２’ＦＬ・１．３水和物の結晶では占有率が３０％、２’ＦＬ・１．４水和物の結晶では占有率が３８．１％であるII位の水分子が包含されており、II位の水分子はI位の水分子に比べ、より脱水されやすい状態にあることがわかった。 The crystals used for the analysis are 2′FL · 1.3 hydrate and 1.4 hydrate crystals, and both hydrate crystals have two water molecules having different occupancy rates, that is, occupancy rates of 100 % Occupancy is 30% in the I-position water molecule and 2′FL · 1.3 hydrate crystals, and 38.1% in the 2′FL · 1.4 hydrate crystals. It was found that the water molecule at position II was included, and the water molecule at position II was more easily dehydrated than the water molecule at position I.
　また、２’ＦＬ・１．４水和物の結晶構造解析によれば、２’ＦＬ・１．４水和物中のグルコースは、α型及びβ型をそれぞれ１１．６及び８８．４％で含むことが示された。これはII位の水分子がα型のグルコースの酸素原子と衝突するため、β型よりα型の比率が低いと考えられる。よって、水分子を１．４分子より多く含む２’ＦＬ・ｍ水和物の結晶では、α型のグルコースの存在比率は１１．６％より低いか、すべてβ型であることになる。
（６）単結晶構造解析からの粉末Ｘ線回折パターンのシミュレーション
　上記（５）の解析結果に基づき、２’ＦＬ・１．３水和物及び１．４水和物の粉末Ｘ線結晶回折パターンのシミュレーションを行った。単結晶構造からの粉末Ｘ線回折パターンのシミュレーションには、Ｍｅｒｃｕｒｙを用いた。上記の単結晶Ｘ線構造解析によって得られた結晶の格子定数と原子座標から、ＣｕＫα線に対する粉末Ｘ線回折パターンを算出した。 Further, according to the crystal structure analysis of 2′FL · 1.4 hydrate, glucose in 2′FL · 1.4 hydrate is 11.6 and 88.4% of α type and β type, respectively. It was shown to contain. This is probably because the water molecule at position II collides with the oxygen atom of α-type glucose, so the α-type ratio is lower than β-type. Therefore, in the 2′FL · m hydrate crystals containing more than 1.4 water molecules, the abundance ratio of α-type glucose is lower than 11.6%, or all are β-type.
(6) Simulation of powder X-ray diffraction pattern from single crystal structure analysis Based on the analysis result of (5) above, powder X-ray crystal diffraction pattern of 2′FL · 1.3 hydrate and 1.4 hydrate A simulation was performed. Mercury was used for the simulation of the powder X-ray diffraction pattern from the single crystal structure. A powder X-ray diffraction pattern for CuKα rays was calculated from the lattice constant and atomic coordinates of the crystal obtained by the single crystal X-ray structural analysis.
　表６に示すとおり、２’ＦＬ・１．４水和物結晶の粉末Ｘ線回折パターンの実測値と、２’ＦＬ・１．３水和物及び１．４水和物の単結晶構造からシミュレートした粉末Ｘ線回折パターンはよく一致しており、これらは新規な２’ＦＬ・水和物の結晶であった。 As shown in Table 6, from the measured value of the powder X-ray diffraction pattern of 2′FL · 1.4 hydrate crystals and the single crystal structures of 2′FL · 1.3 hydrate and 1.4 hydrate The simulated powder X-ray diffraction patterns were in good agreement, and these were new 2′FL hydrate crystals.
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
（７）熱重量分析（ＴＧ）
　熱重量分析は、Ｑ５００（ティー・エイ・インスツルメント社製）を用いて行った。後述の実施例１に記載の方法で取得した２’ＦＬ・１．７水和物の結晶を秤量して装置に取り付け、乾燥窒素気流中で室温から２００　℃まで、１０　℃／ｍｉｎの速度で昇温し、温度並びに時間に対する質量の変化を測定した。測定中に０．２０％／ｍｉｎ以上の質量変化速度が観測された場合は、その時点の温度を保持して測定を行い、質量変化速度が０．０５％／ｍｉｎ未満になったら、昇温を再開した。 (7) Thermogravimetric analysis (TG)
The thermogravimetric analysis was performed using Q500 (manufactured by TA Instruments). Crystals of 2′FL · 1.7 hydrate obtained by the method described in Example 1 to be described later are weighed and attached to the apparatus, and at a rate of 10 ° C./min from room temperature to 200 ° C. in a dry nitrogen stream. The temperature was raised, and the change in mass with respect to temperature and time was measured. If a mass change rate of 0.20% / min or more is observed during measurement, the measurement is performed while maintaining the temperature at that time, and when the mass change rate becomes less than 0.05% / min, the temperature is increased. Resumed.
　熱重量分析結果から温度上昇にしたがって２’ＦＬ・１．７水和物の２つの水分子は徐々に脱水されて結晶から抜けてゆくこと（図３、Ａの領域はII位の水分子の脱水、Ｂの領域はI位の水分子の脱水と考えられる）、及び温度可変粉末Ｘ線回折の結果から脱水されても結晶構造を維持している（図４、１６０℃までＡ晶のピークを示している）ことから、２’ＦＬ・水和物の結晶には、無水物から約１．７水和物までの連続したｍ水和物の結晶状態が存在することが明らかとなり、これは新規な２’ＦＬ・ｍ水和物の結晶（Ａ晶。ｍは０～１．７の任意の数字であり、好ましくは０～１．７の任意の小数第一位までの数字である。ｍが０の場合は２’ＦＬ・無水和物の結晶という）である。 From the thermogravimetric analysis results, the two water molecules of 2'FL · 1.7 hydrate are gradually dehydrated and escape from the crystal as the temperature rises (Fig. 3, area A is the water molecule at position II) Dehydration, region B is considered to be dehydration of water molecule at position I), and maintains the crystal structure even after dehydration from the results of temperature variable powder X-ray diffraction (FIG. 4, peak of crystal A up to 160 ° C. From the above, it is clear that 2′FL · hydrate crystals have a continuous m-hydrate crystal state from anhydride to about 1.7 hydrate. Is a novel 2′FL · m hydrate crystal (crystal A. m is an arbitrary number from 0 to 1.7, preferably an arbitrary number from 0 to 1.7 up to the first decimal place. When m is 0, it is called 2'FL.anhydrous crystal).
２’－O－フコシルラクトース・ｍ水和物の結晶の製造２
（１）２’ＦＬ・１．７水和物の結晶の製造
　実施例１と同様の方法により２’ＦＬを製造し、その結晶を析出、熟成させた。
　得られた２’ＦＬの結晶は、バスケット型遠心分離機を用いて回収し、２００ｍｌのエタノールで洗浄した。 Production of crystals of 2'- O -fucosyl lactose m hydrate 2
(1) Production of 2′FL · 1.7 hydrate crystals 2′FL was produced in the same manner as in Example 1, and the crystals were precipitated and aged.
The obtained 2′FL crystals were recovered using a basket-type centrifuge and washed with 200 ml of ethanol.
　得られた結晶を、真空乾燥機を用いて３０℃で３０分間乾燥することにより２’ＦＬ・１．７水和物の結晶を取得した。
　結晶の水和物量は、実施例１の（４）と同様の方法により決定した。
（２）２’ＦＬ・１．３水和物の結晶の製造
　実施例１と同様の方法により２’ＦＬを製造し、その結晶を析出、熟成させた。 The obtained crystals were dried at 30 ° C. for 30 minutes using a vacuum dryer to obtain crystals of 2′FL · 1.7 hydrate.
The amount of hydrated crystals was determined by the same method as in Example 1 (4).
(2) Production of 2′FL · 1.3 hydrate crystals 2′FL was produced in the same manner as in Example 1, and the crystals were precipitated and aged.
　得られた２’ＦＬの結晶は、バスケット型遠心分離機を用いて回収し、２００ｍｌのエタノールで洗浄した。
　得られた結晶を、真空乾燥機を用いて３０℃で１８０分間乾燥することにより２’ＦＬ・１．３水和物の結晶を取得した。
　結晶の水和物量は、実施例１の（４）と同様の方法により決定した。
（３）２’ＦＬ・１．０水和物の結晶の製造
　実施例１と同様の方法により２’ＦＬを製造し、その結晶を析出、熟成させた。 The obtained 2′FL crystals were recovered using a basket-type centrifuge and washed with 200 ml of ethanol.
The obtained crystals were dried at 30 ° C. for 180 minutes using a vacuum dryer to obtain 2′FL · 1.3 hydrate crystals.
The amount of hydrated crystals was determined by the same method as in Example 1 (4).
(3) Production of 2′FL · 1.0 hydrate crystals 2′FL was produced in the same manner as in Example 1, and the crystals were precipitated and aged.
　得られた２’ＦＬの結晶は、バスケット型遠心分離機を用いて回収し、２００ｍｌのエタノールで洗浄した。
　得られた結晶を、真空乾燥機を用いて４０℃で１８０分間乾燥することにより２’ＦＬ・１．０水和物の結晶を取得した。
　結晶の水和物量は、実施例１の（４）と同様の方法により決定した。
（４）２’ＦＬ・無水物の結晶の製造
　実施例１と同様の方法により２’ＦＬを製造し、その結晶を析出、熟成させる。 The obtained 2′FL crystals were recovered using a basket-type centrifuge and washed with 200 ml of ethanol.
The obtained crystals were dried at 40 ° C. for 180 minutes using a vacuum dryer to obtain 2′FL · 1.0 hydrate crystals.
The amount of hydrated crystals was determined by the same method as in Example 1 (4).
(4) Production of 2′FL · anhydride crystals 2′FL is produced in the same manner as in Example 1, and the crystals are precipitated and aged.
　得られた２’ＦＬの結晶は、バスケット型遠心分離機を用いて回収し、２００ｍｌのエタノールで洗浄する。
　得られた結晶を、真空乾燥機を用いて９０℃で２４時間乾燥することにより２’ＦＬ・無水物の結晶を取得できる。
　結晶の水和物量は、実施例１の（４）と同様の方法により決定することができる。 The obtained 2′FL crystals are recovered using a basket-type centrifuge and washed with 200 ml of ethanol.
The obtained crystals are dried at 90 ° C. for 24 hours using a vacuum dryer to obtain 2′FL · anhydride crystals.
The amount of hydrated crystals can be determined by the same method as in Example 1 (4).
２’－O－フコシルラクトース・ｐ水和物の結晶の製造１
（１）２’ＦＬの製造
　実施例１の（１）と同様の方法により２’ＦＬを製造した。
（２）２’ＦＬ・２．５水和物の結晶の製造
　実施例１の（２）と同様の方法により２’ＦＬの結晶を析出させた後、水／エタノール溶液内で２’ＦＬの結晶を熟成させた。 Production of crystals of 2'- O -fucosyl lactose p-hydrate 1
(1) Production of 2′FL 2′FL was produced in the same manner as in Example 1 (1).
(2) Production of 2′FL · 2.5 hydrate crystals After 2′FL crystals were precipitated by the same method as in Example 1 (2), the 2′FL crystals were hydrated in a water / ethanol solution. The crystals were aged.
　得られた２’ＦＬの結晶は、バスケット型遠心分離機を用いて回収し、その後の解析に用いる２’ＦＬの結晶とした。
　ＨＰＬＣによる純度測定では９５.２％（面積％）以上の２’ＦＬの結晶が得られていることが確認され、当該結晶をもう一度水に溶解して上記の晶析操作を繰り返すことにより、９９.４％（面積％）以上の２’ＦＬの結晶を取得することができた。
（３）粉末Ｘ線回折
　上記（２）で得られた２’ＦＬの結晶を粉末Ｘ線回折に供した。分析条件は実施例１の（３）と同様であり、測定結果は表７のとおりである。 The obtained 2′FL crystals were recovered using a basket-type centrifuge and used as 2′FL crystals used for the subsequent analysis.
Purity measurement by HPLC confirms that 95.2% (area%) or more of 2′FL crystals have been obtained. By dissolving the crystals once more in water and repeating the above crystallization procedure, 99 ′ is obtained. More than 4% (area%) of 2′FL crystals could be obtained.
(3) Powder X-ray diffraction The 2′FL crystals obtained in (2) above were subjected to powder X-ray diffraction. The analysis conditions are the same as in Example 1 (3), and the measurement results are shown in Table 7.
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
（４）水分量測定
　実施例１の（４）と同様の方法により、上記（２）で取得した２’ＦＬの結晶の水分量を測定したところ、該結晶は２’ＦＬ・２．５水和物であった。
（５）単結晶構造解析
　上記（２）で取得した２’ＦＬの結晶を試料として、実施例１の（５）と同様の方法で単結晶構造解析を行った。ただし、室温での測定に際しては、結晶からの脱水による単結晶の崩壊を防ぐために、結晶を少量の晶析溶媒とともに毛細管に封入した試料を使用した。 (4) Moisture content measurement The moisture content of the 2′FL crystal obtained in (2) above was measured by the same method as in (4) of Example 1, and the crystal was found to be 2′FL · 2.5 water. It was a Japanese product.
(5) Single crystal structure analysis Single crystal structure analysis was performed in the same manner as (5) of Example 1 using the 2′FL crystal obtained in (2) above as a sample. However, in the measurement at room temperature, a sample in which the crystal was sealed in a capillary together with a small amount of a crystallization solvent was used in order to prevent the single crystal from collapsing due to dehydration from the crystal.
　その結果、２’ＦＬ・２．５水和物の結晶は、２’ＦＬ・２水和物：２’ＦＬ・３水和物＝１：１からなる結晶であると考えられ、グルコース分子の配座によって結晶から押し出されやすい水分子が存在することが明らかとなった。よって、Ａ晶と同様にＢ晶もまた１．７水和物より大きく３水和物までの連続した水和物の結晶状態が存在することが明らかとなった。またＢ晶のグルコースはβ型であった。
（６）単結晶構造解析に基づく粉末Ｘ線結晶回折パターンのシミュレーション
　実施例１の（６）と同様の方法により、単結晶構造解析の結果に基づく２’ＦＬ・２．５水和物の結晶について粉末Ｘ線回折パターン分析を行った。結果は表８のとおりである。 As a result, the 2′FL · 2.5 hydrate crystal is considered to be a crystal consisting of 2′FL · 2 hydrate: 2′FL · 3 hydrate = 1: 1, It was found that there are water molecules that are easily pushed out of the crystal by conformation. Therefore, it was clarified that the crystal state of continuous hydrate of B crystal as well as A crystal is larger than 1.7 hydrate up to trihydrate. In addition, the glucose of crystal B was β-type.
(6) Simulation of powder X-ray crystal diffraction pattern based on single crystal structure analysis Crystal of 2′FL · 2.5 hydrate based on the result of single crystal structure analysis by the same method as (6) of Example 1 A powder X-ray diffraction pattern analysis was performed on. The results are shown in Table 8.
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
　上記の２’ＦＬ・２．５水和物の結晶の粉末Ｘ線回折パターンの実測値と、その単結晶構造解析からシミュレーションした粉末Ｘ線回折パターンはよく一致しており、Ａ晶は粉末Ｘ線回折パターンが明らかに異なる新規な２’ＦＬ・水和物の結晶（Ｂ晶）であった。
　単結晶構造解析の結果を合わせると、Ａ晶と同様、Ｂ晶もその結晶に含有される水含量により、１．７より多い水分を含有する水和物から３水和物までの連続したｐ水和物の結晶状態が存在することは明らかであり、これは新規な２’ＦＬ・ｐ水和物の結晶（Ｂ晶。ｐは１．７より大きく３以下の任意の数字であり、好ましくは１．７より大きく３．０以下の任意の少数第一位までの数字である）である。 The measured value of the powder X-ray diffraction pattern of the above 2′FL · 2.5 hydrate crystal agrees well with the powder X-ray diffraction pattern simulated from the single crystal structure analysis. It was a novel 2'FL hydrate crystal (crystal B) with a clearly different line diffraction pattern.
Combining the results of the single crystal structure analysis, like crystal A, crystal B is a continuous p from hydrate containing more than 1.7 to trihydrate depending on the water content contained in the crystal. It is clear that there is a crystalline state of hydrate, which is a novel 2′FL · p hydrate crystal (crystal B. p is any number greater than 1.7 and less than or equal to 3, preferably Is a number up to a first decimal place of any number greater than 1.7 and less than or equal to 3.0).
２’－O－フコシルラクトース・ｐ水和物の結晶の製造２
（１）２’ＦＬ・２．０水和物の結晶の製造
　実施例１と同様の方法により２’ＦＬを製造し、その結晶を析出、熟成させた。
　得られた２’ＦＬの結晶は、バスケット型遠心分離機を用いて回収した。
　得られた結晶を、真空乾燥機を用いて３０℃で３０分間乾燥することにより２’ＦＬ・２．０水和物の結晶を取得した。
（２）２’ＦＬ・２．２水和物の結晶の製造
　実施例１と同様の方法により２’ＦＬを製造し、その結晶を析出、熟成させた。 Production of 2'- O -fucosyl lactose p-hydrate crystals 2
(1) Production of 2′FL · 2.0 hydrate crystals 2′FL was produced in the same manner as in Example 1, and the crystals were precipitated and aged.
The obtained 2′FL crystals were recovered using a basket-type centrifuge.
The obtained crystals were dried at 30 ° C. for 30 minutes using a vacuum dryer to obtain 2′FL · 2.0 hydrate crystals.
(2) Production of 2′FL · 2.2 hydrate crystals 2′FL was produced in the same manner as in Example 1, and the crystals were precipitated and aged.
　得られた２’ＦＬの結晶は、バスケット型遠心分離機を用いて回収した。
　得られた結晶を、真空乾燥機を用いて２５℃で３０分間乾燥することにより２’ＦＬ・２．２水和物の結晶を取得した。
（３）２’ＦＬ・２．７水和物の結晶の製造
　実施例１と同様の方法により２’ＦＬを製造し、その結晶を析出、熟成させた。 The obtained 2′FL crystals were recovered using a basket-type centrifuge.
The obtained crystals were dried at 25 ° C. for 30 minutes using a vacuum dryer to obtain 2′FL · 2.2 hydrate crystals.
(3) Production of 2′FL · 2.7 hydrate crystals 2′FL was produced in the same manner as in Example 1, and the crystals were precipitated and aged.
　得られた２’ＦＬの結晶は、バスケット型遠心分離機を用いて回収した。
　得られた結晶を、流動層乾燥機を用いて２０℃で３０分間乾燥することにより２’ＦＬ・２．７水和物の結晶を取得した。 The obtained 2′FL crystals were recovered using a basket-type centrifuge.
The obtained crystals were dried at 20 ° C. for 30 minutes using a fluidized bed dryer to obtain 2′FL · 2.7 hydrate crystals.
　本発明により、例えば健康食品、医薬品、化粧品等の製品、原料もしくは中間体等として有用である２’－O－フコシルラクトースの結晶およびその製造方法が提供される。 According to the present invention, there are provided 2′- O -fucosyl lactose crystals useful as products, raw materials or intermediates for health foods, pharmaceuticals, cosmetics and the like, and a method for producing the same.

Claims (12)

  1. ２’－O－フコシルラクトース・ｎ水和物の結晶（ただし、ｎは０～３のいずれかの数字を表し、ｎ＝０の場合は、２’－O－フコシルラクトース・無水物という）。 Crystal of 2'- O -fucosyl lactose n-hydrate (where n represents any number from 0 to 3, and when n = 0, it is referred to as 2'- O -fucosyl lactose-anhydride).
  2. 粉末Ｘ線回折において、回折角（２θ）が、１７．００°±０．２０°、１８．８２°±０．２０°および２１．５８°±０．２０°にピークを有することを特徴とする請求項１記載の２’－O－フコシルラクトース・ｎ水和物の結晶（ｎは請求項１と同義）。 In powder X-ray diffraction, the diffraction angle (2θ) has peaks at 17.00 ° ± 0.20 °, 18.82 ° ± 0.20 ° and 21.58 ° ± 0.20 °. A crystal of 2′- O -fucosyl lactose n-hydrate according to claim 1, wherein n is as defined in claim 1.
  3. 粉末Ｘ線回折において、回折角（２θ）が、さらに９．８６°±０．２０°、１４．１６°±０．２０°、１６．１２°±０．２０°および２０．３０°±０．２０°にピークを有することを特徴とする請求項２記載の２’－O－フコシルラクトース・ｎ水和物の結晶（ｎは請求項１と同義）。 In powder X-ray diffraction, diffraction angles (2θ) are further increased to 9.86 ° ± 0.20 °, 14.16 ° ± 0.20 °, 16.12 ° ± 0.20 °, and 20.30 ° ± 0. The 2'- O -fucosyl lactose n-hydrate crystal according to claim 2, which has a peak at 20 ° (n is as defined in claim 1).
  4. 粉末Ｘ線回折において、回折角（２θ）が、さらに１５．５２°±０．２０°、２３．８８°±０．２０°、２４．７６°±０．２０°および２７．５２°±０．２０°にピークを有することを特徴とする請求項３記載の２’－O－フコシルラクトース・ｎ水和物の結晶（ｎは請求項１と同義）。 In powder X-ray diffraction, diffraction angles (2θ) are further 15.52 ° ± 0.20 °, 23.88 ° ± 0.20 °, 24.76 ° ± 0.20 ° and 27.52 ° ± 0. The 2'- O -fucosyl lactose n-hydrate crystal according to claim 3, which has a peak at 20 ° (n is as defined in claim 1).
  5. 粉末Ｘ線回折において、回折角（２θ）が、さらに１２．７２°±０．２０°にピークを有することを特徴とする請求項４記載の２’－O－フコシルラクトース・ｎ水和物の結晶（ｎは請求項１と同義）。 The powder of 2'- O -fucosyl lactose n-hydrate according to claim 4, characterized in that, in powder X-ray diffraction, the diffraction angle (2θ) further has a peak at 12.72 ° ± 0.20 °. Crystal (n is as defined in claim 1).
  6. 粉末Ｘ線回折において、回折角（２θ）が、１０．００°±０．２０°、１８．９４°±０．２０°、２０．４４°±０．２０°および２１．７２°±０．２０°にピークを有することを特徴とする請求項１記載の２’－O－フコシルラクトース・ｎ水和物の結晶（ｎは請求項１と同義）。 In powder X-ray diffraction, diffraction angles (2θ) are 10.000 ° ± 0.20 °, 18.94 ° ± 0.20 °, 20.44 ° ± 0.20 ° and 21.72 ° ± 0. The 2'- O -fucosyl lactose n-hydrate crystal according to claim 1, which has a peak at 20 ° (n is as defined in claim 1).
  7. 粉末Ｘ線回折において、回折角（２θ）が、さらに１１．８８°±０．２０°、１４．２６°±０．２０°、１５．６０°±０．２０°および１７．１２°±０．２０°にピークを有することを特徴とする請求項６の２’－O－フコシルラクトース・ｎ水和物の結晶（ｎは請求項１と同義）。 In powder X-ray diffraction, the diffraction angles (2θ) are further 11.88 ° ± 0.20 °, 14.26 ° ± 0.20 °, 15.60 ° ± 0.20 ° and 17.12 ° ± 0. A crystal of 2'- O -fucosyl lactose n-hydrate according to claim 6, which has a peak at 20 ° (n is as defined in claim 1).
  8. 粉末Ｘ線回折において、回折角（２θ）が、さらに１６．２４°±０．２０°、１６．８８°±０．２０°、１８．５８°±０．２０°および１９．８６°±０．２０°にピークを有することを特徴とする請求項７の２’－O－フコシルラクトース・ｎ水和物の結晶（ｎは請求項１と同義）。 In powder X-ray diffraction, diffraction angles (2θ) are further increased to 16.24 ° ± 0.20 °, 16.88 ° ± 0.20 °, 18.58 ° ± 0.20 ° and 19.86 ° ± 0. A crystal of 2'- O -fucosyl lactose n-hydrate according to claim 7, which has a peak at 20 ° (n is as defined in claim 1).
  9. １５～６０℃の２’－O－フコシルラクトース含有水溶液に、その温度を維持しながら該水溶液量に対して０．１～３倍量のＣ１～Ｃ６のアルコールを該水溶液に少量ずつ徐々に添加する工程を含む請求項１記載の２’－O－フコシルラクトース・ｎ水和物の結晶（ｎは請求項１と同義）の製造方法。 To a 2'- O -fucosyl lactose-containing aqueous solution at 15 to 60 ° C, 0.1 to 3 times the amount of C1-C6 alcohol is gradually added to the aqueous solution little by little while maintaining the temperature. A method for producing a crystal of 2'- O -fucosyl lactose n-hydrate according to claim 1, wherein n is as defined in claim 1.
  10. １５～６０℃の２’－O－フコシルラクトース含有水溶液に、種晶として２’－O－フコシルラクトースの結晶を添加した後、１５～６０℃の温度を維持しながら該水溶液量に対して０．１～３倍量のＣ１～Ｃ６のアルコールを該水溶液に少量ずつ徐々に添加する工程を含む請求項１記載の２’－O－フコシルラクトース・ｎ水和物の結晶（ｎは請求項１と同義）の製造方法。 Of 15 ~ 60 ℃ 2'- O - in fucosyllactose containing aqueous solution, 2'O as seed - After addition of fucosyl lactose crystals, 0 for the aqueous solution amount while maintaining the temperature of 15 ~ 60 ° C. 2. The crystal of 2′- O -fucosyl lactose n-hydrate according to claim 1, comprising the step of gradually adding 1 to 3 times the amount of C1-C6 alcohol to the aqueous solution little by little. The same method).
  11. Ｃ１～Ｃ６のアルコールを２’－O－フコシルラクトース含有水溶液に少量ずつ徐々に添加した後、得られた水／アルコール溶液を１５～６０℃で１時間以上保持する工程を含む請求項９または１０記載の製造方法。 The method further comprises the step of gradually adding the C1-C6 alcohol to the 2'- O -fucosyl lactose-containing aqueous solution little by little and then maintaining the resulting water / alcohol solution at 15-60 ° C for 1 hour or longer. The manufacturing method as described.
  12. Ｃ１～Ｃ６のアルコールが、メタノールまたはエタノールである請求項９～１１のいずれか１項に記載の製造方法。 The production method according to any one of claims 9 to 11, wherein the C1-C6 alcohol is methanol or ethanol.
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WO2015188834A1 (en) * 2014-06-11 2015-12-17 Glycom A/S Separation of 2'-o-fucosyllactose from fermentation broth
WO2016086947A1 (en) * 2014-12-05 2016-06-09 Glycom A/S Crystalline difucosyllactose
US10500221B2 (en) 2014-12-05 2019-12-10 Glycom A/S Crystalline difucosyllactose
WO2016095924A1 (en) 2014-12-16 2016-06-23 Glycom A/S Separation of 2'-fl from a fermentation broth
US12060594B2 (en) 2014-12-16 2024-08-13 Glycom A/S Separation of 2′-FL from a fermentation broth
EP4151645A3 (en) * 2014-12-16 2023-06-21 Glycom A/S Separation of 2'-fl from a fermentation broth
US10676770B2 (en) 2014-12-16 2020-06-09 Glycom A/S Separation of 2′-FL from a fermentation broth
WO2017195743A1 (en) 2016-05-09 2017-11-16 協和発酵バイオ株式会社 Crystal of 3'-sialyllactose sodium salt n-hydrate, and method for producing same
US11384110B2 (en) 2016-10-31 2022-07-12 Glycom A/S Method for removing residual organic solvent from a crystalline oligosaccharide
WO2018077368A1 (en) 2016-10-31 2018-05-03 Glycom A/S Method for removing residual organic solvent from a crystalline oligosaccharide
US11505567B2 (en) 2017-07-12 2022-11-22 Glycom A/S Amorphous mixture comprising a neutral mono- or oligosaccharide and an acidic non-carbohydrate component
US11939351B2 (en) 2017-07-12 2024-03-26 Glycom A/S Amorphous mixture comprising a neutral mono- or oligosaccharide and an acidic non-carbohydrate component
US12054513B2 (en) 2017-07-12 2024-08-06 Glycom A/S Amorphous mixture comprising a neutral mono- or oligosaccharide and an acidic non-carbohydrate component
WO2020079146A1 (en) 2018-10-18 2020-04-23 Basf Se Crystalline form ii of 2'-o-fucosyllactose, process for its preparation, nutritional, cosmetic or pharmaceutical formulation containing the same
CN115873051A (en) * 2022-05-17 2023-03-31 山东恒鲁生物科技有限公司 Novel crystal form of trisaccharide
CN116425810A (en) * 2023-06-14 2023-07-14 山东合成远景生物科技有限公司 Purification method of 3-fucosyllactose in mixed solution
CN116425810B (en) * 2023-06-14 2023-08-11 山东合成远景生物科技有限公司 Purification method of 3-fucosyllactose in mixed solution

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