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WO2025012361A1 - Cristallisation de lacto-n-triose ii - Google Patents

Cristallisation de lacto-n-triose ii Download PDF

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Publication number
WO2025012361A1
WO2025012361A1 PCT/EP2024/069607 EP2024069607W WO2025012361A1 WO 2025012361 A1 WO2025012361 A1 WO 2025012361A1 EP 2024069607 W EP2024069607 W EP 2024069607W WO 2025012361 A1 WO2025012361 A1 WO 2025012361A1
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Prior art keywords
lnt
lacto
triose
polymorph
crystalline
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Tobias SEITZ
Mischa BAIER
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Chr Hansen AS
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Chr Hansen AS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/40—Complete food formulations for specific consumer groups or specific purposes, e.g. infant formula
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H5/00—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium
    • C07H5/04—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium to nitrogen
    • C07H5/06—Aminosugars

Definitions

  • the present disclosure concerns a method for providing crystalline lacto-N-triose II (LNT-II), a novel polymorph of crystalline lacto-N-triose II (LNT-II), and uses thereof.
  • HMOs human milk oligosaccharides
  • HMOs further contain one or more fucose moieties, at least one /V-acetylglucosamine moiety and/or one or more /V-acetylneuraminic acid moieties. Furthermore, there are linear as well as branched representative molecules within the family of HMOs. Generally, the monosaccharide residues of HMOs are selected from the group consisting of D-glucose, D-galactose, /V-acetylglucosamine, L-fucose and /V-acetylneuraminic acid.
  • HMOs for infant nutrition, health and development are directly linked to their biological activities which include protecting the neonate from pathogens, supporting development of the infant’s immune system and cognitive abilities.
  • HMOs serve as a substrate for beneficial bacteria in the infant’s evolving microbiome such as Bifidobacteria and Lactobacilli.
  • HMOs The health benefits associated with HMOs render them a valued ingredient for infant formula.
  • this requires an industrial scale production for at least some HMOs. Due to challenges and drawbacks that are associated with the chemical synthesis of oligosaccharides, industrial scale production of HMOs currently focuses on biochemical and especially fermentative approaches utilizing metabolically engineered microbial cells for intracellular biosynthesis of an HMO of interest.
  • the industrial scale production of certain HMOs for infant formula requires the recovery of the HMO of interest from a complex composition, i.e. a cell lysate and/or a fermentation broth. Typically, the HMO of interest is dried at the end of the recovery process, either by means of spray-drying or by means of crystallization.
  • a member of the HMO family is the trisaccharide lacto- N-triose II (LNT-II; 2- Acetamido-2-deoxy-b-D-glucopyranosyl-(1->3)-b-D-galactopyranosyl-(1->4)-D- glucose; GlcNAc(pi,3)Gal(pi,4)Glc, CAS No. 75645-27-1).
  • Lacto- N-triose II is able to modulate the growth of Bifidobacterium longum subsp. infantis, may increase intestinal immunity, may increase adhesion of the commensal bacterium L plantarum WCFS1 to intestinal epithelial cells, and appears to support the crosstalk between commensal bacteria and epithelial cells.
  • LNT-II polymorph A a white microcrystalline powder
  • LNT-II crystals were obtained in that 100 ml of an aqueous LNT-II solution (cone. 200 g/L) was gradually added to 500 ml methanol heated to 60°C in about 30 minutes, and the resulting mixture was refluxed at 60°C for about 3 hours, then cooled to 20°C and stirred for 1 hour. LNT-II crystals were obtained and washed with methanol, then dried by airflow. A Powder X-ray diffraction spectrum was taken and the data presented in table 3 of the document describes a crystal form of LNT-II (herein referred to as “LNT-II polymorph B”).
  • LNT-II polymorph A of Kuhn’s disclosure and LNT-II polymorph B of WO 2002/100875 are the same crystalline polymorph form of LNT-II.
  • Crystallization or recrystallization is one of the simplest and cheapest methods to separate a product from contaminants and obtain a pure substance.
  • providing one or more crystalline modifications (polymorphs) of a solid compound is an important factor in product development, because the different crystalline forms affect the compound's properties — for example, solubility, dissolution rate, thermodynamic stability, density, hygroscopicity, electrical properties (such as dielectric constant, conductivity), mechanical properties (such as friability, hardness, breaking strength, elasticity), optical properties (such as colour, transparency, refraction), etc. — diversely. Polymorphs enlarge the repertoire of materials that are available for improving the product's characteristics.
  • the present invention provides a method for producing a crystalline form of LNT-II.
  • the problem is further solved by providing a novel crystalline LNT-II polymorph as described herein below.
  • the method described herein below resulted in crystalline LNT-II possessing a crystal structure that differs from the structures of LNT-II crystals that were obtained by crystallization utilizing an organic solvent, i.e. utilizing a crystallization from a methanol solution.
  • a method for producing crystalline LNT-II is provided.
  • a crystalline form of LNT-II as novel polymorph.
  • polymorph C of LNT-II for manufacturing pharmaceutical and/or nutritional compositions.
  • compositions containing polymorph C LNT-II are provided.
  • FIG. 1 displays the X-ray powder diffraction pattern of the crystalline LNT-II polymorph C obtained in example 1.
  • FIG. 2 displays the X-ray powder diffraction pattern of the crystalline LNT-II polymorph C obtained in example 2.
  • FIG. 3 displays the X-ray powder diffraction pattern of the crystalline LNT-II polymorph C obtained in example 3.
  • FIG. 4 is a schematic representation which illustrates the method for producing crystalline LNT-II pursuant to the present invention. Detailed description
  • a method for producing a crystalline LNT-II is provided.
  • the method comprises the following steps: i) crude lacto-N-triose II (LNT-II) is dissolved in water, or an aqueous solution of lacto-N-triose II (LNT-II) is provided, ii) the aqueous solution of lacto-N-triose II (LNT-II) is adjusted to a concentration of at least about 40 wt.-%, iii) the adjusted aqueous solution is cooled to a temperature of about 2 °C to about 15 °C, iv) the cooled aqueous solution is kept at a temperature in the range of from about 2°C to about 15°C for at least 2 days to allow formation of crystals, v) optionally washing the crystals with a water/ethanol mixture; and vi) drying the crystals at ambient pressure.
  • an aqueous solution of lacto-N-triose II (LNT-II) is provided.
  • the aqueous solution can be a crude aqueous solution from a work-up process of a fermentation method to produce LNT-II.
  • the aqueous solution can as well be a solution of an amorphous or crystalline crude form of LNT-II in water.
  • the water used to prepare the solution is preferably a kind of purified, for instance HPLC grade, water.
  • the concentration of the aqueous LNT-II solution is adjusted to at least 40 wt.-%, in particular to about 45 wt.-%, 50 wt.-%, 55 wt.-%, 60 wt.-%, or 65wt.-%.
  • the solution from which lacto-N- triose II is crystallized does not contain methanol, and preferably does not contain an organic solvent.
  • the concentration of the aqueous solution of lacto-N-triose II is adjusted to a concentration of more than 50 wt.-%, preferably more than 60 wt.-%, most preferably to a concentration of about 65 wt.- %.
  • solid LNT-II can be dissolved in an appropriate amount of water by rotating and moderate heating to a temperature of about 30°C to about 40°C, preferably to about 35°C, until completely dissolved.
  • the aqueous solution of LNT-II is cooled to a temperature of about 2 °C to about 15 °C, and the cooled aqueous solution is kept at a temperature in the range of from about 2°C to about 15°C for at least 2 days to allow formation of crystals.
  • the aqueous solution of lacto-N- triose II (LNT-II) is cooled to a temperature of between 3°C and 10°C, preferably to about 4°C to 6°C.
  • This temperature range is advantageous in terms of optimizing the speed of crystal growth, overall yield and purity of the resulting crystal structure, which can often be considered to be interdepentend features of the crystallization process.
  • the crystallization time is from about 5 hours to about 50 days, or from about 1 day to about 40 days, preferably from about 2 days to about 30 days, or from about 5 days to about 25 days.
  • the resulting crystal mass is dried at ambient pressure.
  • the drying is carried out at a temperature below 50°C, preferably below 40°C, more preferably at about 35°C - 37°C.
  • the drying of the crystals is continued until a constant weight of the crystal mass is observed.
  • the preferred drying temperature range is advantageous in terms of speed of drying, overall yield of crystals with constant weight, and preserving the crystal structure without risking a part of the material to become amorphous.
  • the inventive method can be distinguished from the previous known methods of crystallizing LNT-II especially in that the crystallization is carried out from an aqueous crystallization solution rather than from a methanol solution.
  • water is not considered to be a good choice as a crystallization medium for oligosaccharides because the crystallization tends to be slow and sluggish, often ending in more honey-like outcomes or hydrogels. Further drying of such outcomes often requires harsher conditions, and in the end then leads to amorphous solid forms. Therefore, all previously known crystallization methods of LNT-II started from a solution having methanol as solvent. However, with the present invention, it was surprisingly found that LNT-II can be nicely crystallized from an aqueous solution resulting in obtaining crystals in a good yield.
  • the inventive method for producing crystalline LNT-II is thus advantageous in that the use of an organic solvent and/or an antisolvent for crystallizing the LNT-II is not necessary. Neither methanol as toxic and harmful solvent nor ethanol or another organic solvent is required as medium from which the crystallization is carried out. This reduces costs in manufacturing crystalline LNT-II, makes the production process safer and more environment friendly, and also makes the product itself safer because no traces of any solvent other than water can be left, for instance as part of the crystal structure.
  • the obtained crystals were formed in a novel polymorph form (polymorph C) of LNT-II.
  • polymorph C polymorph form
  • the novel crystal structure of the crystalline LNT-II obtained by the method of the present invention was determined by X-Ray diffraction analysis and compared to the details of the X-Ray diffraction pattern of the LNT-II polymorph B as described in WO 2002/100875, and thus found to be novel.
  • Different polymorphs of a crystalline material are known often to possess different physical behavior, for instance in respect to solubility, dissolution rate, thermodynamic stability, density, hygroscopicity, electrical properties (such as dielectric constant, conductivity), mechanical properties (such as friability, hardness, breaking strength, elasticity), optical properties (such as colour, transparency, refraction), etc.
  • electrical properties such as dielectric constant, conductivity
  • mechanical properties such as friability, hardness, breaking strength, elasticity
  • optical properties such as colour, transparency, refraction
  • the LNT-II polymorph C is a polymorph that exhibits an X-ray powder diffraction reflections, based on a measurement using Cu Ka (1 ,54056 A) radiation, at 8,19°, 19,29°, and 19,60° ⁇ 0.2 20 angle, preferably at 8,19°, 19,29°, 19,60°, and 19,86° ⁇ 0.2 20 angles, more preferably at 8,19°, 19,29°, 19,60°, 19,86°, and 9,92° ⁇ 0.2 20 angles, yet more preferably at 8,19°, 19,29°, 19,60°, 19,86°, 9,92°, and 21 ,37° ⁇ 0.2 20 angles, even more preferably at 8,19°, 19,29°, 19,60°, 19,86°, 9,92°, 21 ,37°, and 23,05° ⁇ 0.2 20 angles, even more preferably at 8,19°, 19,29°, 19,60°, 19,86°, 9,92°, 21 ,37°, and 23,05° ⁇ 0.2 20 angles, even more preferably at 8,19°, 19,29°,
  • the LNT-II polymorph C is a polymorph that exhibits an X-ray powder diffraction reflections, based on a measurement using Cu Ka (1 ,54056 A) radiation, at at 8,19°, 19,29°, and 19,60° ⁇ 0.1 20 angle, preferably at 8,19°, 19,29°, 19,60°, and 19,86° ⁇ 0.1 20 angles, more preferably at 8,19°, 19,29°, 19,60°, 19,86°, and 9,92° ⁇ 0.1 20 angles, yet more preferably at 8,19°, 19,29°, 19,60°, 19,86°, 9,92°, and 21 ,37° ⁇ 0.1 20 angles, even more preferably at 8,19°, 19,29°, 19,60°, 19,86°, 9,92°, 21 ,37°, and 23,05° ⁇ 0.1 20 angles, even more preferably at 8,19°, 19,29°, 19,60°, 19,86°, 9,92°, 21 ,37°, and 23,05° ⁇ 0.1 20 angles, even more preferably at 8,19°, 19,29°
  • the measurement has a range of measurements from 2° to 40° 20 with a step size of 0.013° 20.
  • the LNT-II polymorph C has an X-ray powder diffraction diagram as shown in FIG. 1 or FIG. 2 or FIG. 3.
  • the powder X-ray diffraction pattern of the crystalline LNT-II polymorph C of this invention is different from the known X-ray diffraction pattern of LNT-II polymorph B as there are notable differences which show in their respective diffractogram.
  • the prior art LNT-II polymorph B has peak positions of the three peaks with the highest relative intensity at 4.78°, 4.49°, and 9.25° 20 angles, which are all not present in the X-ray pattern of the polymorph C of the present invention.
  • the most characteristic peaks at 8,19°, 19,29°, and 19,60° 20 angles of the LNT-II polymorph C in the present invention are all absent in the X-ray pattern of the prior art polymorph B.
  • the crystalline LNT-II polymorph C can be considered as an anomeric mixture of a- and p-anomers or even a pure form of one of the anomers.
  • the crystalline LNT-II polymorph C of this invention is substantially pure.
  • substantially pure preferably means that the crystalline LNT-II polymorph contains less than 10 w/w % of impurity, preferably less than 5 w/w % of impurity, more preferably less than 2 w/w % or less than 1 w/w% of impurity, most preferably less than 0.5 w/w % of impurity, wherein “impurity” refers to any physical entity different from the crystalline LNT-II polymorph, such as an amorphous LNT-II, different LNT-II polymorph(s) or form(s), unreacted intermediate(s) remained from the synthesis of LNT-II, by-product(s), degradation product(s), inorganic salt(s) and/or other contaminants different from water.
  • impurity refers to any physical entity different from the crystalline LNT-II polymorph, such as an amorphous LNT-II, different LNT-II polymorph(s) or form(s), unreacted intermediate(s)
  • the crystalline LNT-II polymorph C of this invention is free from an organic solvent.
  • the crystalline LNT-II polymorph C of this invention is free from methanol.
  • the novel LNT-II polymorph C does not contain any traces of an organic solvent, and especially no traces of methanol. Thus, no further washing steps are needed and a high product safety can be quite easily achieved. Especially in view of the field of its main use as part of a nutritional formula for infants or other vulnerable humans in need of a special nutrition this is very beneficial as even very small traces of methanol are regarded as being harmful.
  • polymorph C as crystalline LNT-II obtainable by a process for its production comprising the following consecutive steps: a) Providing an aqueous solution of lacto-N-triose II (LNT-II), b) Increasing the concentration of the aqueous solution of step a) to a concentration of more than 40 wt.-%, preferably more than 50 wt.-%, most preferably more than 60 wt.-%, in particular to a concentration of about 65 wt.-%, thereby obtaining a concentrated solution or slurry, c) Setting the resulting concentrated solution or slurry to crystallization at a temperature between 2°C and 15°C, preferably between 3°C and 10°C, more preferably at about 4°C to 6°C, for a period of time, thereby obtaining a white crystal mass, and d) Drying the resulting crystal mass at ambient pressure at a temperature below 50°C, preferably below 40°C, more
  • an aqueous solution of lacto-N-triose II (LNT-II) is provided.
  • the aqueous solution can be a crude aqueous solution from a work-up process of a fermentation method to produce LNT-II.
  • the aqueous solution can as well be a solution of an amorphous or crystalline form of LNT-II in water.
  • the water used to prepare the solution is preferably a kind of purified, for instance HPLC grade, water.
  • the concentration of the aqueous LNT-II solution in step a) is not critical and can range from about 0.5 wt.-% to about 50.0 wt.-%, preferably from about 1.0 wt.-% to about 45.0 wt.-%, and most preferred from about 30 wt.-% to about 45 wt.-%, in particular about 40 wt.-%.
  • solid LNT-II can be dissolved in an appropriate amount of water by rotating and moderate heating to a temperature of about 30°C to about 40°C, preferably to about 35°C, until completely dissolved.
  • step b) the concentration of the aqueous solution of LNT-II is increased to a concentration of more than 40 wt.-%, preferably more than 50 wt.-%, most preferably more than 60 wt.-%, in particular to a concentration of about 65 wt.-%. At such a concentration, it is observed that this is a point where a slurry is obtained.
  • the increase in concentration can be achieved by any means known to the skilled person.
  • the increase in concentration is achieved by applying reduced pressure while moderately heating and rotating.
  • step c) the resulting concentrated solution or slurry is set to crystallization at a temperature between 2°C and 15°C, preferably between 3°C and 10°C, more preferably at about 4°C to 6°C, for a period of time, thereby obtaining a white crystal mass.
  • the crystallization time is from about 5 hours to about 50 days, or from about 1 day to about 40 days, preferably from about 2 days to about 30 days, or from about 5 days to about 25 days.
  • step d) the resulting crystal mass is dried at ambient pressure at a temperature below 50°C, preferably below 40°C, more preferably at about 35°C - 37°C.
  • the method is not only clearly easier and safer than the methods for producing crystal LNT-II because no harmful organic solvent is used, and especially no methanol, but also a novel polymorph C can be produced which shows a combination of different physical behaviors that can be beneficial in regard to its use in nutritional and pharmaceutical applications.
  • step c the crystal mass is washed with a water/ethanol mixture before the drying of step d).
  • a water/ethanol mixture before the drying of step d.
  • crystalline LNT-II polymorph C as described herein before and/or as obtained by a method as described herein before for the manufacturing of a nutritional and/or pharmaceutical composition.
  • the crystalline LNT-II polymorph C according to this invention is suitable for nutritional use.
  • LNT-II alone or in combination with other N- acetyllactosamine and/or fucose and/or sialic acid containing human milk oligosaccharides, is particularly effective in the education and/or maturation of the immune system of neonatal infants, and have preventive effect against secondary infections following viral infections such as influenza.
  • LNT-II as prebiotic enhances the beneficial effects and efficiency of probiotics, such as Lactobacillus and Bifidobacterium species, in promoting the development of an early bifidogenic intestinal microbiota in infants, in reducing the risk of development or allergy and/or asthma in infants, in preventing and treating pathogenic infections in such as diarrhoea in infants.
  • probiotics such as Lactobacillus and Bifidobacterium species
  • the crystalline LNT-II polymorph C of the invention is used for the preparation of nutritional formulations, such as foods, drinks and feeds, preferably infant formulas, food supplements and digestive health functional food.
  • the nutritional formulations can be prepared in a conventional manner. For example, it can be prepared by admixing micronutrient components in appropriate proportions, and then adding vitamins and minerals while avoiding thermal degradation or decomposition of heat sensitive vitamins by adding them after homogenization. Lipophilic vitamins can be dissolved in a fat source before mixing. A liquid mixture is formed using water, whose temperature is preferably about 50- 80° C. to help dissolution or dispersal of the ingredients.
  • the crystalline LNT-II polymorph C can be suitably added at this stage.
  • the resulting mixture is then homogenized by flash heating to about 80-150° C. by means of steam injection, heat exchanger or autoclave. This thermal treatment also reduces significantly the bacterial load.
  • the hot mixture is then cooled rapidly to about 60-80° C. If needed, further homogenization can be carried out at this temperature under high pressure of about 2-30 MPa. After cooling, heat sensitive constituents can be added, and the pH and the solids content can be conveniently adjusted.
  • the resulting mixture is then dried in a conventional manner, such as by spray drying or freeze drying to powder. Probiotics can be added at this point by dry-mixing.
  • LNT-II polymorph C is suitable for pharmaceutical use.
  • LNT-II alone or in combination with other N- acetyllactosamine and/or fucose and/or sialic acid containing human milk oligosaccharides, is particularly effective in the education and/or maturation of the immune system of neonatal infants, and have preventive effect against secondary infections following viral infections such as influenza.
  • LNT-II as prebiotic enhances the beneficial effects and efficiency of probiotics, such as Lactobacillus and Bifidobacterium species, in promoting the development of an early bifidogenic intestinal microbiota in infants, in reducing the risk of development or allergy and/or asthma in infants, in preventing and treating pathogenic infections in such as diarrhoea in infants.
  • probiotics such as Lactobacillus and Bifidobacterium species
  • the crystalline LNT-II polymorph C of the invention can be used for the preparation of pharmaceutical compositions.
  • Pharmaceutical compositions can be manufactured in a conventional manner, e.g. as described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field. 5.
  • a nutritional formulation (such as food, drink or feed) comprising the crystalline LNT-II polymorph C of this invention.
  • the nutritional formulation can also contain edible micronutrients, vitamins and minerals as well. The amounts of such ingredient can vary depending on whether the formulation is intended for use with normal, healthy infants, children, adults or subjects having specialized needs (e.g. suffering from metabolic disorders).
  • Micronutrients include for example edible oils, fats or fatty acids (such as coconut oil, soy-bean oil, monoglycerides, diglycerides, palm olein, sunflower oil, fish oil, linoleic acid, linolenic acid etc.), carbohydrates (such as glucose, fructose, sucrose, maltodextrin, starch, hydrolysed cornstarch, etc.) and proteins from casein, soy-bean, whey or skim milk, or hydrolysates of these proteins, but protein from other source (either intact or hydrolysed) may be used as well.
  • edible oils such as coconut oil, soy-bean oil, monoglycerides, diglycerides, palm olein, sunflower oil, fish oil, linoleic acid, linolenic acid etc.
  • carbohydrates such as glucose, fructose, sucrose, maltodextrin, starch, hydrolysed cornstarch, etc.
  • Vitamins may be chosen from the group consisting of vitamin A, B1 , B2, B5, B6, B12, C, D, E, H, K, folic acid, inositol and nicotinic acid.
  • the nutritional formulation can also contain the following minerals and trace elements: Ca, P, K, Na, Cl, Mg, Mn, Fe, Cu, Zn, Se, Cr or I.
  • the nutritional formulation is an infant formula, i.e. a foodstuff intended for use by infants during the first 4-6 months of life and satisfying, by itself, the nutritional requirements of infants.
  • It can contain one or more probiotic Bifidobacterium species, prebiotics such as fructooligosaccharides and galactooligosaccharides, proteins from casein, soy-bean, whey or skim milk, carbohydrates such as lactose, saccharose, maltodextrin, starch or mixtures thereof, lipids (e.g. palm olein, sunflower oil, safflower oil) and vitamins and minerals essential in a daily diet.
  • prebiotics such as fructooligosaccharides and galactooligosaccharides, proteins from casein, soy-bean, whey or skim milk
  • carbohydrates such as lactose, saccharose, maltodextrin, starch or mixtures thereof
  • lipids e.g. palm olein
  • the infant formula preferably contains the crystalline LNT-II polymorph C of the invention in a concentration mimicking the natural concentration in breast milk.
  • the concentration of LNT-II in human breast milk is generally around 2 - 3 mg/L in the colostrum and sinks to around 0.05 mg/L during lactation.
  • the infant formula preferably contains the novel crystalline LNT-II polymorph C in an amount which results in a concentration of between 2 - 3 mg/L and 0.05 mg/L in the final liquid foodstuff.
  • the nutritional formulation can be a food supplement containing the crystalline LNT-II polymorph C.
  • the food supplement can also contain one or more probiotics in an amount sufficient to achieve the desired effect in an individual, preferably in children and adults.
  • the food supplement can also contain vitamins, minerals, trace elements and other micronutrients as well.
  • the food supplement may be for example in the form of tablets, capsules, pastilles or a liquid.
  • the supplement can also contain conventional additives, such as binders, coatings, emulsifiers, solubilising agents, encapsulating agents, film forming agents, adsorbents, carriers, fillers, dispersing agents, wetting agents, jellifying agents, gel forming agents, etc.
  • the daily dose of LNT-II typically ranges from 0.01 to 3.0 g.
  • the food supplement is a digestive health functional food as the administration of LNT-II provides a beneficial effect on digestive health.
  • Digestive health functional food is a processed food used with intention enhance and preserve digestive health by crystalline LNT according to the present invention as physiologically functional ingredient or component in forms of tablet, capsule, powder, etc.
  • Different terms such as dietary supplement, nutraceutical, designed food, health product may also be used to refer to functional food.
  • a pharmaceutical composition comprising the crystalline LNT-II polymorph C of the invention as an active ingredient and one or more pharmaceutically acceptable carriers such as additives, adjuvants, excipients and diluents (water, gelatine, talc, sugars, starch, gum arabic, vegetable gums, vegetable oils, polyalkylene glycols, flavouring agents, preservatives, stabilizers, emulsifying agents, lubricants, colorants, fillers, wetting agents, etc.).
  • suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field.
  • the dosage form for administration includes, for example, tablets, powders, granules, pills, suspensions, emulsions, infusions, capsules, syrups, injections, liquids, elixirs, extracts and tincture.
  • Instrument PANalytical X'Pert Pro, QS-Nr. 02204, geometry: transmission, detector: Pixcel (3D), radiation: Cu Ka (1 ,54056 A), tube settings: 40 kV, 40 mA, range: 2-40° 2Theta, step width: 0,013° 2Theta, measuring time per step: 25 s, preparation: sample carrier with acetate foils, software: PANalytical HighScorePlus v.4, reference databank: ICDD PDF-4 (2021) / external standards, evaluation according to: DIN EN 13925:2003.
  • aqueous solution having a dry solid content of 31.09 % (w/v) and containing LNT-II at a purity of 74 % based on the peak areas of the HPLC chromatogram was concentrated by using a rotary evaporator at 60 °C and a reduced pressure of 42 mbar.
  • the dry solid content of the concentrate was adjusted to a solids content of 61.48 weight-% and the resulting solution was cooled to about 4 to 6 °C.
  • a crystal mass was obtained over time and washed with two volumes of 80 vol.% ethanolic solution (80 vol.% EtOH 120 vol.% H2O) at a temperature in the range of between 4 and 6 °C.

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Abstract

Est divulgué un procédé de production de LNT-II cristallin, un nouveau polymorphe d'un LNT-II cristallin et ses utilisations.
PCT/EP2024/069607 2023-07-11 2024-07-11 Cristallisation de lacto-n-triose ii Pending WO2025012361A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP23184691 2023-07-11
EP23184691.6 2023-07-11

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Citations (3)

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Publication number Priority date Publication date Assignee Title
WO2002100875A1 (fr) 2001-06-11 2002-12-19 Kyowa Hakko Kogyo Co., Ltd. Cristaux d'oligosaccharides et procedes de preparation correspondants
WO2023117673A1 (fr) * 2021-12-21 2023-06-29 Dsm Ip Assets B.V. Cristallisation de lnnt
WO2023175130A1 (fr) * 2022-03-17 2023-09-21 Dsm Ip Assets B.V. Lacto-n-triose ii cristallin

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002100875A1 (fr) 2001-06-11 2002-12-19 Kyowa Hakko Kogyo Co., Ltd. Cristaux d'oligosaccharides et procedes de preparation correspondants
CA2449736A1 (fr) * 2001-06-11 2002-12-19 Kyowa Hakko Kogyo Co., Ltd. Cristaux d'oligosaccharides et procedes de preparation correspondants
WO2023117673A1 (fr) * 2021-12-21 2023-06-29 Dsm Ip Assets B.V. Cristallisation de lnnt
WO2023175130A1 (fr) * 2022-03-17 2023-09-21 Dsm Ip Assets B.V. Lacto-n-triose ii cristallin

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Title
KUHN ET AL., CHEM. BER., vol. 89, no. 75645-27-1, 1956, pages 1027 - 1033
URASHIMA T. ET AL.: "Milk Oligosaccharides", 2011, NOVA BIOMEDICAL BOOKS

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