WO2023175130A1 - Lacto-n-triose ii cristallin - Google Patents

Lacto-n-triose ii cristallin Download PDF

Info

Publication number
WO2023175130A1
WO2023175130A1 PCT/EP2023/056863 EP2023056863W WO2023175130A1 WO 2023175130 A1 WO2023175130 A1 WO 2023175130A1 EP 2023056863 W EP2023056863 W EP 2023056863W WO 2023175130 A1 WO2023175130 A1 WO 2023175130A1
Authority
WO
WIPO (PCT)
Prior art keywords
lnt2
crystalline
powder
ray diffraction
water
Prior art date
Application number
PCT/EP2023/056863
Other languages
English (en)
Inventor
Imre Kovács
Benjámin PODÁNYI
Original Assignee
Dsm Ip Assets B.V.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Dsm Ip Assets B.V. filed Critical Dsm Ip Assets B.V.
Publication of WO2023175130A1 publication Critical patent/WO2023175130A1/fr

Links

Classifications

    • 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    • C07H5/00—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium
    • C07H5/04—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium to nitrogen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
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • 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
    • 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

Definitions

  • This invention relates to a crystalline human milk oligosaccharide (HMO), notably the trisaccharide lacto-N-triose II (LNT2, 2-acetamido-2-deoxy-p-D-glucopyranosyl-(1— >3)-p-D-galactopyranosyl- (1 ⁇ 4)-D-glucose, GlcNAcpi-3Gaipi-4Glc) in crystalline form and a method of obtaining it.
  • HMO crystalline human milk oligosaccharide
  • Lacto-N-triose II is a metabolic intermediate of lacto-N-tetraose (LNT) and lacto-N- neotetraose (LNnT) biosynthesis, and as such occurs also naturally in breast milk (Urashima et aL: Milk oligosaccharides, Nova Science, 2011 ; Chen Adv. Carbohydr. Chem. Biochem. 72, 113 (2015)).
  • LNT2 has previously been crystallized. Kuhn et al. (Chem. Ber. 89, 1027 (1956)) isolated LNT from natural source, obtained LNT2 after partial acidic hydrolysis and crystallized it from moist hot methanol and ethanol. EP-A-1405856 disclosed that crystals of LNT2 were obtained from aqueous methanol which was proved by X-ray powder diffraction data.
  • Crystalline HMOs produced in industrial amounts, are highly desirable for nutritional and medical applications. 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, thermodynamic stability, solubility, 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 a scientist has available for improving the product’s characteristics.
  • the first aspect of the invention relates to a crystalline modification of lacto-N-triose II, referred to as form 2.
  • the second aspect of the invention relates to a method for producing the crystalline lacto-N-triose II form 2.
  • the third aspect of the invention relates to a nutritional composition containing a crystalline lacto- N-triose II form 2 according to the present invention.
  • the fourth aspect of the invention relates to the use of the crystalline lacto-N-triose II form 2 according to the present invention in preparing a nutritional composition.
  • the fifth aspect of the invention relates to the crystalline lacto-N-triose II form 2 according to the present invention for use as a pharmaceutically active ingredient.
  • the sixth aspect of the invention relates to a pharmaceutical composition containing the crystalline lacto-N-triose II form 2 according to the present invention.
  • Figure 1 shows the powder X-ray diffraction pattern of the crystalline LNT2 form 1 obtained according to Example 1 ;
  • Figure 2 shows the comparison of the powder X-ray diffraction patterns of the crystalline LNT2 form 1 obtained according to Example 1 and crystalline LNT2 disclosed in EP-A-1405856 (values with *);
  • Figure 3 shows the powder X-ray diffraction pattern of the crystalline LNT2 form 2 obtained according to Example 2;
  • Figure 4 shows the comparison of the powder X-ray diffraction patterns of the crystalline LNT2 form 1 and form 2;
  • Figure 5 shows a chemical synthesis pathway to LNT2.
  • lacto-N-triose II (LNT2) can be crystallized in at least two different hydrated crystalline forms.
  • LNT2 When LNT2 was crystallized from aqueous methanol, its powder X-ray diffraction (PXRD) scan can be indexed on a trigonal unit cell, the calculated density is in agreement of a sesquihydrate and its melting (decomposition point) is 202-205 °C.
  • This crystalline form is referred to as form 1 .
  • LNT2 form 1 thus corresponds to the LNT2 crystal disclosed by Kuhn et al. (Chem. Ber. 89, 1027 (1956)) who reported the crystal to be a sesquihydrate and having a melting (decomposition point) of 201 - 202 °C.
  • the PXRD data are in good agreement with those disclosed in EP-A- 1405856 (see Fig. 2).
  • this invention relates to the novel crystalline lacto-N-triose II form 2.
  • the crystalline LNT2 of this invention designated as form 2, has powder X-ray diffraction reflections, based on a measurement using CuKa radiation, at 8.19 ⁇ 0.20, 19.36 ⁇ 0.2 and 19.66 ⁇ 0.20, preferably at 8.19 ⁇ 0.20, 19.36 ⁇ 0.2, 19.66 ⁇ 0.20 and 9.94 ⁇ 0.2, more preferably at 8.19 ⁇ 0.20, 19.36 ⁇ 0.2, 19.66 ⁇ 0.20, 9.94 ⁇ 0.2 and 19.94 ⁇ 0.2, even more preferably at 8.19 ⁇ 0.20, 19.36 ⁇ 0.2, 19.66 ⁇ 0.20, 9.94 ⁇ 0.2, 19.94 ⁇ 0.2 and 23.12 ⁇ 0.2, particularly at 8.19 ⁇ 0.20, 19.36 ⁇ 0.2, 19.66 ⁇ 0.20, 9.94 ⁇ 0.2, 19.94 ⁇ 0.2, 23.12 ⁇ 0.2 and 23.66 ⁇ 0.2 20 angles.
  • the crystalline LNT2 of this invention designated as form 2
  • the crystalline LNT2 of this invention designated as form 2
  • the crystalline LNT2 form 2 of this invention preferably, contains around 6 mols of water per 1 mol of LNT2 in the crystal structure based on the calculated density from its PXRD scan.
  • the crystalline LNT2 form 2 of this invention preferably, has a water content of about 15-16.5 w/w%, such as 15.5-16 w/w %, as determined by Karl Fischer titration, after the crystals are conventionally dried, for example in a vacuum oven at 50-100 mbar in room temperature and/or above, such as drying the crystals at 80 mbar in room temperature for 17 hours then in 50 °C for 4.5 hours.
  • the crystalline LNT2 form 2 defined above is substantially pure.
  • substantially pure preferably means herein that the crystalline LNT2 contains less than 10 w/w%, such as less than 5 w/w%, less than 2.5 w/w%, less than 1 w/w%, or less than 0.5 w/w%, of impurities.
  • impurities preferably means herein any physical entities different from the crystalline LNT2 and its water of hydration, such as an amorphous LNT2, by-products, e.g. carbohydrates different than LNT2, degradation products, inorganic salts and/or other contaminants.
  • the crystalline LNT2 form 2 according to the present invention is substantially free from organic solvent with the aid of which LNT2 form 2 was crystallized, in this case acetone.
  • the expression “substantially free from organic solvent” intends to mean that the acetone content is at most 200 ppm, preferably at most 150 ppm, more preferably at most 100 ppm, most preferably at most 50 ppm.
  • the crystalline LNT2 form 2 defined above can be an anomeric mixture of a- and p-anomers or a pure form of one of the anomers.
  • HMOs human milk oligosaccharides
  • the gut bacteria can act directly and indirectly on the immune system by stimulating innate immunity and controlling inflammatory reactions and by inducing an adaptive immune response and a tolerogenic environment.
  • HMOs directly strengthen the intestinal epithelial barrier, protecting the host against pathogens.
  • LNT2 is part of the HMO components found in breast milk, it can be putatively used for nutritional and/or therapeutic purposes.
  • the crystalline LNT2 form 2 of this invention defined above is suitable for nutritional use.
  • Nutritional compositions containing the crystalline LNT2 form 2 defined above can be for example a food composition, a medical food or a food for special medical purposes, a nutritional/dietary supplement and the like.
  • the nutritional composition can contain sources of protein, lipids and/or digestible carbohydrates and can be in powdered or liquid forms.
  • the composition can be designed to be the sole source of nutrition or as a nutritional supplement.
  • Suitable protein sources include milk proteins, soy protein, rice protein, pea protein and oat protein, or mixtures thereof.
  • Milk proteins can be in the form of milk protein concentrates, milk protein isolates, whey protein or casein, or mixtures of both.
  • the protein can be whole protein or hydrolysed protein, either partially hydrolysed or extensively hydrolysed. Hydrolysed protein offers the advantage of easier digestion which can be important for pregnant women.
  • the protein can also be provided in the form of free amino acids.
  • the protein can comprise about 5 % to about 30 % of the energy of the nutritional composition, normally about 10 % to 20 %. Ideally the source of protein does not include excessive amounts of lactose.
  • the protein source can be a source of glutamine, threonine, cysteine, serine, proline, or a combination of these amino acids.
  • the glutamine source can be a glutamine dipeptide and/or a glutamine enriched protein.
  • Glutamine can be included due to the use of glutamine by enterocytes as an energy source.
  • Threonine, serine and proline are important amino acids for the production of mucin. Mucin coats the Gl tract and can improve intestinal barrier function and promote mucosal healing.
  • Cysteine is a major precursor of glutathione, which is key for the antioxidant defences of the body.
  • Suitable digestible carbohydrates include maltodextrin, hydrolysed or modified starch or corn starch, glucose polymers, corn syrup, corn syrup solids, high fructose corn syrup, rice-derived carbohydrates, pea-derived carbohydrates, potato-derived carbohydrates, tapioca, sucrose, glucose, fructose, sucrose, honey, sugar alcohols (e.g. maltitol, erythritol, sorbitol), or mixtures thereof.
  • the composition is reduced in or free from added lactose or other FODMAP carbohydrates.
  • Generally digestible carbohydrates provide about 35 % to about 55 % of the energy of the nutritional composition.
  • a suitable digestible carbohydrate is a low dextrose equivalent (DE) maltodextrin.
  • Suitable lipids include medium chain triglycerides (MCT) and long chain triglycerides (LCT).
  • MCT medium chain triglycerides
  • LCT long chain triglycerides
  • MCTs can comprise about 30 % to about 70 % by weight of the lipids, more specifically about 50 % to about 60 % by weight.
  • MCTs offer the advantage of easier digestion which can be important for pregnant women.
  • the lipids provide about 35 % to about 50 % of the energy of the nutritional composition.
  • the lipids can contain essential fatty acids (omega-3 and omega-6 fatty acids). Preferably these polyunsaturated fatty acids provide less than about 30 % of total energy of the lipid source.
  • Suitable sources of long chain triglycerides are rapeseed oil, sunflower seed oil, palm oil, soy oil, milk fat, corn oil, high oleic oils, and soy lecithin.
  • Fractionated coconut oils are a suitable source of medium chain triglycerides.
  • the lipid profile of the nutritional composition is preferably designed to have a polyunsaturated fatty acid omega-6 (n-6) to omega-3 (n-3) ratio of about 4:1 to about 10:1 .
  • the n-6 to n-3 fatty acid ratio can be about 6:1 to about 9:1 .
  • the nutritional composition may also include vitamins and minerals. If the nutritional composition is intended to be a sole source of nutrition, it preferably includes a complete vitamin and mineral profile.
  • vitamins include vitamins A, B-complex (such as B1 , B2, B6, B9 and B12), C, D, E and K, niacin and acid vitamins such as pantothenic acid, folic acid and biotin.
  • minerals include calcium, iron, zinc, magnesium, iodine, copper, phosphorus, manganese, potassium, chromium, molybdenum, selenium, nickel, tin, silicon, vanadium and boron.
  • the nutritional composition can also include a carotenoid such as lutein, lycopene, zeaxanthin, and beta-carotene.
  • a carotenoid such as lutein, lycopene, zeaxanthin, and beta-carotene.
  • the total amount of carotenoid included can vary from about 0.001 pg/ml to about 10 pg/ml.
  • Lutein can be included in an amount of from about 0.001 pg/ml to about 10 pg/ml, preferably from about 0.044 pg/ml to about 5 pg/ml of lutein.
  • Lycopene can be included in an amount from about 0.001 pg/ml to about 10 pg/ml, preferably about 0.0185 pg/ml to about 5 pg/ml of lycopene.
  • Beta-carotene can comprise from about 0.001 pg/ml to about 10 mg
  • the nutritional composition preferably also contains reduced concentrations of sodium; for example, from about 300 mg/l to about 400 mg/l.
  • the remaining electrolytes can be present in concentrations set to meet needs without providing an undue renal solute burden on kidney function.
  • potassium is preferably present in a range of about 1180 to about 1300 mg/l; and chloride is preferably present in a range of about 680 to about 800 mg/l.
  • the nutritional composition can also contain various other conventional ingredients such as preservatives, emulsifying agents, thickening agents, buffers, fibres and prebiotics (e.g. fructooligosaccharides, galactooligosaccharides), probiotics (e.g. B. animalis subsp. lactis BB-12, B. lactis HN019, B. lactis Bi07, B. infantis ATCC 15697, L. rhamnosus GG, rhamnosus HNOOI, L. acidophilus LA-5, L. acidophilus NCFM, L. fermentum CECT5716, B. longum BB536, B. longum AH1205, B. longum AH1206, B.
  • prebiotics e.g. fructooligosaccharides, galactooligosaccharides
  • probiotics e.g. B. animalis subsp. lactis BB-12, B. lactis HN019, B. lactis Bi07
  • antioxidant/anti-inflammatory compounds including tocopherols, carotenoids, ascorbate/vitamin C, ascorbyl palmitate, polyphenols, glutathione, and superoxide dismutase (melon), other bioactive factors (e.g. growth hormones, cytokines, TFG-P), colorants, flavours, and stabilisers, lubricants, and so forth.
  • the nutritional composition can be formulated as a soluble powder.
  • the composition can be fed to a human in need via a nasogastric tube or orally.
  • Various flavours and other additives can also be present.
  • the nutritional compositions can be prepared by any commonly used manufacturing techniques for preparing nutritional compositions in solid form, for example by dry-mixing.
  • An example of nutritional compositions containing the crystalline LNT2 form 2 is an infant formula, i.e. a foodstuff intended for use by infants during their first 4-6 months of life and satisfying by itself their nutritional requirements.
  • the infant formula 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.
  • the infant formula preferably contains 0.1 -3.0 g of the crystalline LNT2 form 2 /100 g of the infant formula.
  • the nutritional composition is in a unit dosage form.
  • the unit dosage form can contain an acceptable food-grade carrier, e.g. phosphate buffered saline solution, mixtures of ethanol in water, water and emulsions such as an oil/water or water/oil emulsion, as well as various wetting agents or excipients.
  • the unit dosage form can also contain other materials that do not produce an adverse, allergic or otherwise unwanted reaction when administered to a human.
  • the carriers and other materials can include solvents, dispersants, coatings, absorption promoting agents, controlled release agents, and one or more inert excipients, such as starches, polyols, granulating agents, microcrystalline cellulose, diluents, lubricants, binders, and disintegrating agents.
  • the unit dosage form comprises the crystalline LNT2 form 2 defined above with a minimum amount of binders and/or excipients.
  • the unit dosage form may include additional nutrients such as long-chain polyunsaturated fatty acids and vitamins and minerals as described above.
  • the unit dosage form may also include prebiotics and probiotics as described above. Unit dosage forms are particularly suitable when nutritionally incomplete or not intended as a sole source of nutrition.
  • a unit dosage form can be administered orally, e.g. as a tablet, capsule, or pellet containing a predetermined amount of the mixture, or as a powder or granules containing a predetermined concentration of the mixture or a gel, paste, solution, suspension, emulsion, syrup, bolus, electuary, or slurry, in an aqueous or non-aqueous liquid, containing a predetermined concentration of the mixture.
  • An orally administered composition can include one or more binders, lubricants, inert diluents, flavouring agents, and humectants.
  • An orally administered composition such as a tablet can optionally be coated and can be formulated to provide sustained, delayed or controlled release of the crystalline LNT2 form 2 defined above.
  • a unit dosage form can also be administered by naso-gastric tube or direct infusion into the Gl tract or stomach.
  • a unit dosage form can also include therapeutic agents such as antibiotics, probiotics, analgesics, and anti-inflammatory agents.
  • the proper dosage of a nutritional composition for a human can be determined in a conventional manner, based upon factors such as the concentration of the LNT2, the patients’ condition, immune status, body weight and age.
  • the required amount of LNT2 would generally be in the range from about 1 g to about 15 g per day, in certain embodiments from about 2 g to about 10 g per day, for example about 3 g to about 7 g per day. Appropriate dose regimes can be determined by methods known to those skilled in the art.
  • the crystalline LNT2 form 2 defined above can be formulated as a pharmaceutical composition.
  • the pharmaceutical composition can contain a pharmaceutically acceptable carrier, e.g.
  • the pharmaceutical composition can also contain other materials that do not produce an adverse, allergic or otherwise unwanted reaction when administered to a human.
  • the carriers and other materials can include solvents, dispersants, coatings, absorption promoting agents, controlled release agents, and one or more inert excipients, such as starches, polyols, granulating agents, microcrystalline cellulose, diluents, lubricants, binders, and disintegrating agents.
  • compositions can be administered orally, e.g. as a tablet, capsule, or pellet containing a predetermined amount, or as a powder or granules containing a predetermined concentration or a gel, paste, solution, suspension, emulsion, syrup, bolus, electuary, or slurry, in an aqueous or non-aqueous liquid, containing a predetermined concentration.
  • Orally administered compositions can include binders, lubricants, inert diluents, flavouring agents, and humectants.
  • Orally administered compositions such as tablets can optionally be coated and can be formulated to provide sustained, delayed or controlled release of the mixture therein.
  • compositions can also be administered by rectal suppository, aerosol tube, naso-gastric tube or direct infusion into the Gl tract or stomach.
  • the pharmaceutical compositions can also include therapeutic agents such as antibiotics, probiotics, analgesics, and anti-inflammatory agents.
  • the proper dosage of a pharmaceutical composition can be determined in a conventional manner, based upon factors such as the concentration of the LNT2, the patients’ condition, immune status, body weight and age.
  • the required amount of LNT2 would generally be in the range from about 1 g to about 15 g per day, in certain embodiments from about 2 g to about 10 g per day, for example about 3 g to about 7 g per day. Appropriate dose regimes can be determined by methods known to those skilled in the art.
  • One aspect of this invention relates to a process for obtaining the crystalline LNT2 form 2 by crystallizing it from a mixture of water and acetone.
  • the crystallization process comprises the steps of: a) providing a solution of LNT2 in water, b) adding acetone to the aqueous solution of LNT2 obtained in step a) to provide a suspension, c) stirring the suspension obtained in step c) for a couple of hours, d) collecting and drying LNT2 form 2 crystals which precipitate from the mixture during step c).
  • the process is conducted at room temperature.
  • One embodiment of this process comprises the steps of: i) providing a 45-55 m/m% solution of LNT2 in water, ii) to the aqueous solution of LNT2 obtained in step i), adding 9-11 ml of acetone per 1 g of LNT2 in the aqueous solution, to provide a suspension, iii) stirring the suspension obtained in step iii) for 2-6 hours, iv) collecting and drying LNT2 form 2 crystals which precipitate from the mixture during step iii).
  • addition of seed crystals during or after step b) or ii) may assist crystallization.
  • LNT2 to be crystallized can be readily obtained by a process, which involves culturing or fermenting a genetically modified cell in an aqueous culture medium or fermentation medium containing lactose and one or more carbon-based substrates followed by separating it from the culture medium.
  • culture medium is meant the aqueous environment of the fermentation process in a fermenter outside of the genetically modified cell.
  • genetically modified cell is preferably meant a cell in which at least one DNA sequence has been added to, deleted from or changed in the cell’s genome, so that the cell has a changed phenotype. This change in phenotype alters the characteristics of the genetically modified cell from that of the wild type cell.
  • the genetically modified cell can perform at least an additional chemical transformation, when cultured or fermented, due to the added or changed DNA that encodes the expression of at least one enzyme not found in the wild type cell, or the genetically modified cell cannot perform a chemical transformation due to the deleted, added or changed DNA that encodes the expression of an enzyme found in the wild type cell.
  • the genetically modified cell can be produced by conventional genetic engineering techniques.
  • the genetically modified cell can be a bacteria or yeast but preferably is a bacterium.
  • Preferred bacteria include Escherichia coli, Bacillus spp. (e.g. B. subtilis), Campylobacter pylori, Helicobacter pylori, Agrobacterium tumefaciens, Staphylococcus aureus, Thermophilus aquaticus, Azorhizobium caulinodans, Rhizobium leguminosarum, Neisseria gonorrhoeae, N.
  • Lactobacillus spp. Lactococcus spp., Enterococcus spp., Bifidobacterium spp., Sporolactobacillus spp., Micromomospora spp., Micrococcus spp., Rhodococcus spp., Pseudomonas, particularly E. coli.
  • the genetically modified cell capable of producing LNT2 contains a recombinant gene encoding a pi,3-N-acetyl-glucosaminyl transferase which is able to transfer a GIcNAc of a UDP- GIcNAc to lactose and thereby to form LNTri II in the cell.
  • the genetically modified cell above when cultured in an aqueous culture medium containing lactose, can internalize the lactose and then transfer a GIcNAc residue of an activated sugar nucleotide in the cell to the internalized lactose to form LNTri II in the cell.
  • the recombinant gene or the equivalent DNA sequence responsible for the transfers can be introduced into the cell in a well-known manner, using conventional expression vectors.
  • the origin of the heterologous nucleic acid sequences can be any bacteria, e.g. Neisseria meningitidis.
  • An example of pi,3-N-acetyl-glucosaminyl transferase is LgtA from N. meningitidis.
  • the genetically modified cell is cultured in the presence of a carbonbased substrate such as glycerol, glucose, sucrose, glycogen, fructose, maltose, starch, cellulose, pectin, chitin, etc.
  • a carbonbased substrate such as glycerol, glucose, sucrose, glycogen, fructose, maltose, starch, cellulose, pectin, chitin, etc.
  • the cell is cultured with glycerol, glucose, sucrose and/or fructose.
  • This process also involves initially transporting the exogenous lactose from the culture medium into the genetically modified cell.
  • Lactose can be added exogenously in a conventional manner to the culture medium, from which it can then be transported into the cell.
  • the internalization of lactose should not, of course, affect the basic and vital functions or destroy the integrity of the cell.
  • the internalization can take place via a passive transport mechanism during which lactose diffuses passively across the plasma membrane of the cell. The flow is directed by the concentration difference in the extra- and intracellular space with respect to lactose to be internalized, so that lactose passes from the place of higher concentration to the place of lower concentration.
  • lactose is preferably internalized in the cell with the aid of an active transport mechanism, by which lactose diffuses across the plasma membrane of the cell under the influence of a transporter protein or lactose permease (LacY) of the cell.
  • lactose is preferably internalized in the cell with the aid of an active transport mechanism, by which lactose diffuses across the plasma membrane of the cell under the influence of a transporter protein or lactose permease (LacY) of the cell.
  • the genetically modified cell used in this process lacks enzymatic activity which would significantly degrade lactose and LNT2 in the cell.
  • the native p-galactosidase of the culturing cell encoded by the LacZ gene in E. coli, for example), which hydrolyses lactose to galactose and glucose, is preferably deleted or inactivated (LacZ- genotype).
  • a preferred genetically modified cell has a LacZ", particularly a LacZ", LacY + , more particularly a LacZ", LacY + , Lach genotype.
  • a possible way to carry out the fermentation to produce LNT2 is when a genetically modified LacZ- Y + E. coli strain as disclosed above is cultured comprising: (1) a first phase of exponential cell growth that is ensured by a carbon-based substrate, preferably glucose, provided in the culture medium and that preferably lasts until the glucose has all been consumed which is preferably at least 12 hours, more preferably at least 18 hours, still more preferably 20-25 hours, up to about 48 hours; and
  • a second phase of cell growth that is limited by a carbon-based substrate, preferably glycerol, and lactose which are provided, preferably continuously, in the culture medium after the first phase and that lasts until the glucose and preferably most (e.g. at least 60 %) of the lactose have been consumed which is preferably at least 35 hours, more preferably at least 45 hours, still more preferably 50 to 70 hours, up to about 130 hours.
  • a carbon-based substrate preferably glycerol
  • lactose which are provided, preferably continuously, in the culture medium after the first phase and that lasts until the glucose and preferably most (e.g. at least 60 %) of the lactose have been consumed which is preferably at least 35 hours, more preferably at least 45 hours, still more preferably 50 to 70 hours, up to about 130 hours.
  • the LNT2 product can be separated from the cells and impurities in the culture medium by a method comprising at least one of the following separation steps:
  • an ultrafiltration step is always comprised, preferably as the first separation step (that is applied on the fermentation broth), followed by nanofiltration, ion exchange treatment and/or active charcoal treatment.
  • LNT2 may be prepared chemically.
  • Fig. 5 depicts a chemical total synthesis of LNT2.
  • GIcNAc oxazoline donor 2 can be prepared from N-acetyl-glucosamine (GIcNAc) 1 .
  • the hydrogenolysis of benzyl glycoside 5 results in LNT2 6.
  • LNT2 may be prepared enzymatically, for example a p-galactosidase removes galactose from LNnT to produce LNT2.
  • LNT2 in crude, syrupy or amorphous form, it can be crystallized according to the above described methods of the invention.
  • LNT2 form 2 according to the present invention can be characterized by more beneficial properties compared to form 1 .
  • LNT2 form 2 has at least one of the following: higher thermodynamical stability, higher thermal stability. Other features of the invention will become apparent from the following examples which illustrate the invention but do not limit it.
  • Fig. 4 shows the comparison of the PXRD patterns of samples obtained in Example 1 and 2.
  • the two crystalline material represent different polymorphs.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Mycology (AREA)
  • Nutrition Science (AREA)
  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La divulgation concerne une nouvelle forme cristalline de lacto-N-triose II, un procédé d'obtention de celle-ci et des compositions nutritionnelles ou pharmaceutiques les comprenant.
PCT/EP2023/056863 2022-03-17 2023-03-17 Lacto-n-triose ii cristallin WO2023175130A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP22162658.3 2022-03-17
EP22162658 2022-03-17

Publications (1)

Publication Number Publication Date
WO2023175130A1 true WO2023175130A1 (fr) 2023-09-21

Family

ID=81328131

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2023/056863 WO2023175130A1 (fr) 2022-03-17 2023-03-17 Lacto-n-triose ii cristallin

Country Status (1)

Country Link
WO (1) WO2023175130A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1405856A1 (fr) 2001-06-11 2004-04-07 Kyowa Hakko Kogyo Co., Ltd Cristaux d'oligosaccharides et procedes de preparation correspondants
WO2018077368A1 (fr) * 2016-10-31 2018-05-03 Glycom A/S Procédé d'élimination de solvant organique résiduel d'un oligosaccharide cristallin

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1405856A1 (fr) 2001-06-11 2004-04-07 Kyowa Hakko Kogyo Co., Ltd Cristaux d'oligosaccharides et procedes de preparation correspondants
WO2018077368A1 (fr) * 2016-10-31 2018-05-03 Glycom A/S Procédé d'élimination de solvant organique résiduel d'un oligosaccharide cristallin

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CHEN, ADV. CARBOHYDR. CHEM. BIOCHEM., vol. 72, 2015, pages 113
KUHN ET AL., CHEM. BER., vol. 89, 1956, pages 1027
KUHN RICHARD ET AL: "Lacto-N-triose I und Lacto-N-triose II", CHEMISCHE BERICHTE, vol. 89, no. 4, 21 April 1956 (1956-04-21), DE, pages 1027 - 1033, XP093053455, ISSN: 0009-2940, Retrieved from the Internet <URL:https://onlinelibrary.wiley.com/doi/pdf/10.1002/cber.19560890429> DOI: 10.1002/cber.19560890429 *
URASHIMA ET AL.: "Milk oligosaccharides", NOVA SCIENCE, 2011

Similar Documents

Publication Publication Date Title
US20220008444A1 (en) Compositions for use in the prevention or treatment of necrotizing enterocolitis in infants and young children
EP3071208B1 (fr) Compositions pour utilisation dans la prévention ou le traitement de l&#39;entérocolite nécrosante chez des bébés et des enfants en bas âge
EP2576578B1 (fr) Formes polymorphiques du 2&#39;-o-fucosyllactose et leurs procédés de production
EP2405918B2 (fr) Oligosaccharides prébiotiques
RU2697650C1 (ru) Композиции для профилактики или лечения аллергий у младенцев, рожденных или вскармливаемых матерями несекреторного типа, путем обеспечения фукозилированными олигосахаридами, в частности среди младенцев, подверженных риску или рожденных с помощью кесарева сечения
US11419884B2 (en) Compositions comprising HMOS, their production and use for the prevention and/or treatment of viral and/or bacterial infections
ES2824679T3 (es) Mezclas de HMO
CN110621168A (zh) 与用于健康目的的用于在婴儿或幼儿中使用的hmo共混物的复杂性相关联的丁酸的协同产生
RU2720933C2 (ru) Композиция, содержащая fut2-зависимые олигосахариды и лакто-n-неотетраозу, для применения с целью активации развития головного мозга и познавательной способности
CN114468302A (zh) 用于预防或治疗有风险婴儿或幼儿的urt感染的组合物
CN107404931A (zh) 用于改善婴儿或幼儿粪便稠度或频率的组合物
CN111903766A (zh) 一种婴幼儿配方奶粉及其制备方法
WO2020166527A1 (fr) Aliment ou boisson pour bébés et nourrissons, procédé pour améliorer l&#39;environnement intestinal d&#39;un bébé ou d&#39;un nourrisson, et procédé pour renforcer l&#39;immunité du bébé ou du nourrisson
CN113163832A (zh) 用于增强胰腺成熟和胰岛素生物合成的婴儿营养组合物
AU2017324759B2 (en) Nutritional compositions for infants and/or young children comprising oligosaccharides
WO2023175130A1 (fr) Lacto-n-triose ii cristallin
CN104780788A (zh) 多糖用于护理的婴儿和/或婴儿的肠道健康的用途
US10407516B2 (en) Crystalline HMO
WO2024126677A1 (fr) Lacto-n-biosidase mutée
WO2023247483A1 (fr) Mélange de hmo fucosylés
EP4313064A1 (fr) Oligosaccharides de lait humain
TW201316910A (zh) 用於腦成長及／或認知及／或心理動作發展之組合物
Motherway et al. Bifidobacterial galacto‐oligosaccharide metabolism

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23710384

Country of ref document: EP

Kind code of ref document: A1