WO2015036637A1 - Procédé de synthèse de kojibiose et son utilisation dans la préparation de compositions alimentaires et pharmaceutiques - Google Patents

Procédé de synthèse de kojibiose et son utilisation dans la préparation de compositions alimentaires et pharmaceutiques Download PDF

Info

Publication number
WO2015036637A1
WO2015036637A1 PCT/ES2014/070687 ES2014070687W WO2015036637A1 WO 2015036637 A1 WO2015036637 A1 WO 2015036637A1 ES 2014070687 W ES2014070687 W ES 2014070687W WO 2015036637 A1 WO2015036637 A1 WO 2015036637A1
Authority
WO
WIPO (PCT)
Prior art keywords
kojibiosa
yeast
lactose
stage
fermentation
Prior art date
Application number
PCT/ES2014/070687
Other languages
English (en)
Spanish (es)
Inventor
Marina DIEZ MUNICIO
Francisco Javier Moreno Andujar
Miguel HERRERO CALLEJA
Antonia Montilla Corredera
Original Assignee
Consejo Superior De Investigaciones Cientificas (Csic)
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 Consejo Superior De Investigaciones Cientificas (Csic) filed Critical Consejo Superior De Investigaciones Cientificas (Csic)
Publication of WO2015036637A1 publication Critical patent/WO2015036637A1/fr

Links

Classifications

    • 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
    • C07H3/04—Disaccharides
    • 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
    • A23L33/20—Reducing nutritive value; Dietetic products with reduced nutritive value
    • A23L33/21—Addition of substantially indigestible substances, e.g. dietary fibres
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7016—Disaccharides, e.g. lactose, lactulose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients

Definitions

  • the present invention relates to the field of the agri-food and pharmaceutical industry, since it specifically refers to an efficient and economical process for obtaining kojibiosa disacáhdo.
  • Said disaccharide is applicable in the elaboration of functional foods useful for the improvement of the health of the gastrointestinal system and in pharmaceutical compositions useful for the therapeutic treatment of viral diseases, as well as for the treatment of problems associated with diseases such as diabetes mellitus. , prediabetes, gastritis, tooth decay or obesity.
  • Kojibiosa is a disaccharide found naturally in koji extract (obtained after the fermentation process of steamed rice and / or a seed mixture derived from inoculation with Aspergillus oryzae), in beer, honey and in starch hydrolyzate.
  • natural levels are very low, making it difficult to obtain kojibiosa on a large scale.
  • the kojibiosa has a great prebiotic potential because the a- (1 ⁇ 2) bond has a high resistance to gastrointestinal digestion in humans and animals (Valette P, et al. J. Sci. Food Agr., 1993, 62, 121-127; Remaud-Siméon M, et al. J. Molec. Catal.
  • kojibiosa is a glucosidase inhibitor, with the ability to inhibit the elimination of terminal glucose from the ma ⁇ osa / V-oligosaccharide (Glc) 3 (Man) 9 (GlcNAc) 2 from both a free oligosaccharide and a bound oligosaccharide to the amino group of a protein.
  • Glucosidases inhibitors have been shown to have an important antiviral activity (Tan A, et al. J. Biol. Chem., 1991, 266, 14504-14510).
  • kojibiosa as an inhibitor is based on the fact that it specifically inhibits a-glucosidase I in different target tissues and organs (European Patent 0 830 365 B1, 2002). This property is of particular importance for the development of new drugs, especially of the "pseudodisaccharide" class for the treatment of human immunodeficiency virus (HIV) type 1 infections (Borges de Meló E, et al. Tetrahedron, 2006, 62 , 10277-10302).
  • HIV human immunodeficiency virus
  • the inhibition of ⁇ -glucosidase I prevents the elimination of glucose residues during normal processing of the membrane protein of the HIV gp120 virus, which results in modified structure glycoproteins involved in the breaking of the replication cycle of said virus.
  • Kojibiosa has been shown to inhibit the action of ⁇ -glucosidase I in mammary glands of bovine origin (Shailubhai K, et al. Biochem. J., 1987, 247, 555-562,) or in hepatic microsomes in rats (Takeuchi M, et al. J. Biol. Chem., 1990, 108, 42-46), among other targets.
  • kojibiosa in other types of viral diseases such as hepatitis B and C has also been suggested (Goffin D, et al. Crit. Rev. Food Sci. Nutr., 201 1, 51, 394-409).
  • kojibiosa could be used to delay the digestion of carbohydrates ingested in the diet such as starch and sucrose, in therapeutic applications of other diseases, such as obesity, diabetes mellitus, prediabetes, gastritis, duodenal and gastric ulcers, caries or cancer (European Patent 0 830 365 B1, 2002; Goffin D, et al. Crit. Rev. Food Sci. Nutr, 201 1, 51, 394-409).
  • Some methods described so far for obtaining kojibiosa include its synthesis from partially acetylated dextran (Matsuda K, et al. Nature, 1961, 191, 278); from glycosidases, such as a-glucosidase, (Takahashi M, et al. Agrie. Biol. Chem., 1969, 33, 1399-1410); from glucoamylase (Fujimoto H, et al. Agrie. Biol. Chem., 1988, 52, 1345-1351); from sucrose phosphorylase (Kitao S, et al. Biosci. Biotech.
  • This document proposes an alternative procedure for obtaining kojibiosa, which has a high yield and a reduced cost, and which makes it possible to obtain it industrially in a viable way, which will allow its subsequent use through functional or nutraceutical foods designed for Improvement of the health of the gastrointestinal system or of drugs designed for the treatment of viral infections or other pathologies.
  • the technical problem solved by the present invention is the synthesis of kojibiosa by means of a simple, economical and high-performance procedure that allows it to be obtained at a price much lower than its current market value, thus enabling the development of products that allow exploiting its potential activity in the health field.
  • a first object of the invention is a method of synthesis of kojibiosa that starts from a reaction mixture comprising as main component trisaccharide 2-aD-glucopyranosyl-lactose, ⁇ - ⁇ -D-galactopyranosyl- (1 ⁇ 4) -0 - [aD-glucopyranosyl- (1 ⁇ 2)] - pD-glucopyranoside, as well as leucrose, lactose, fructose, glucose and sucrose, obtained by a transglycosylation reaction that uses sucrose and lactose as starting substrates, and characterized by be carried out at a temperature between 20 and 60 ° C and to comprise at least the following steps:
  • step (a) fermentation of the previously diluted starting reaction mixture to a concentration of sugars between 5 and 30 g / 100 mL and preferably 20 g / 100 mL, using a yeast of the Saccharomycetaceae family, in a ratio between 5 and 50 g of yeast per 100 g of sugars, and b) hydrolysis of the fermented reaction mixture in step (a) at a pH between 5 and 8, which is preferably 7, by the addition of an enzyme with ⁇ -galactosidase activity at a concentration between 10 and 100 U / mL
  • the synthesis process uses Saccharomyces cerevisiae as a yeast in a ratio of 16 g of yeast per 100 grams of sugar, and the hydrolysis step (b) is carried out at a pH of 7 with the enzyme Kluyveromyces lactis ⁇ -galactosidase with a concentration of 65 U / mL, obtaining a production yield of kojibiosa greater than 25%, which is preferably greater than 35%, and more preferably is greater than 45%.
  • the synthesis process comprises an additional stage (c) of purification, subsequent to step (b) of hydrolysis, which is performed using a technique selected from fermentation with the remaining yeast added during stage ( a) or chromatography or nanofiltration, and in any case it obtains a kojibiosa with a percentage of wealth in weight with respect to the dry extract, between 60 and 100%.
  • an additional step (d) of lyophilization or atomization is included, subsequent to the stage (c) of purification.
  • the kojibiosa obtained according to the synthesis procedure constitutes a second object of the invention.
  • the third object of the invention constitutes the use of the kojbiosa obtained by the synthesis procedure in the preparation of a prebiotic food composition useful for preventing gastrointestinal pathologies, or in the preparation of a pharmaceutical composition useful for the treatment of viral-type diseases or to delay carbohydrate digestion.
  • the fourth object of the invention constitutes a prebiotic food composition useful for preventing gastrointestinal pathologies characterized in that it comprises the kojibiosa obtained by the synthesis procedure.
  • the fifth object of the invention constitutes a pharmaceutical composition useful for the treatment of viral diseases or for delaying carbohydrate digestion, characterized in that it comprises a therapeutically effective amount of kojibiosa obtained by the synthesis procedure, a salt, or a solvate. thereof pharmaceutically acceptable.
  • the present invention is based on the observation that a synthesis process using a starting reaction mixture comprising trisaccharide 2-aD-glucopyranosyl-lactose, 0-pD-galactopyranosyl- (1 ⁇ 4) -0- [aD - glucopyranosyl- (1 ⁇ 2)] - pD-glucopyranoside, leucose, lactose, fructose, glucose and sucrose, which is obtained from an enzymatic transglycosylation reaction of lactose using a dextransacarase, and characterized by comprising a fermentation stage with Saccharomyces cerevisiae yeast, and a hydrolysis stage using a ⁇ -galactosidase from Kluyveromyces lactis, allows obtaining a disaccharide of high added value and difficult to obtain, such as kojibiosa (see Examples 1 to 4).
  • kojibiosa means a disaccharide defined by the formula 2-OaD-glucopyranosyl-aD-glucopyranoside with bioactive and / or functional properties due to its structural characteristics.
  • mesenteroides B512-F which allows the transfer of the glucose unit from sucrose (donor substrate) to lactose (acceptor substrate) (D ⁇ ez-Municio M, et al. J. Agrie. Food. Chem. 2012, 60, 1945-1953), hereinafter starting reaction mixture.
  • the starting reaction mixture is composed of a combination of carbohydrates comprising the above trisaccharide, leucose, lactose, fructose, glucose and sucrose.
  • concentration of sugars or sugar means a mixture of carbohydrates whose composition may vary depending on the stage of the process to which it refers.
  • the first object of the invention constitutes a kojibious synthesis process that starts from the starting reaction mixture, and which is characterized by being carried out at a temperature between 20 and 60 ° C, which is preferably 30 ° C and by comprise at least the following steps: a) fermentation of the previously diluted starting reaction mixture to a concentration of sugars between 5 and 30 g / 100 mL, and preferably 20 g / 100 mL, using a yeast of the Saccharomycetaceae family, in a ratio between 5 and 50 g of yeast per 100 g of sugar, and
  • step (a) hydrolysis of the fermented reaction mixture in step (a) at a pH between 5 and 8, which is preferably 7, by the addition of an enzyme with ⁇ -galactosidase activity at a concentration between 10 and 100 U / mL
  • "2-aD-glucopyranosyl-lactose, ⁇ - ⁇ -D-galactopyranosyl- (1 ⁇ 4) -0- [aD-glucopyranosyl- (1 ⁇ 2)] - pD-glucopyranoside” means a trisaccharide kojibious galactosylate, glycosylated derivative of lactose, 4'-galactosyl-kojibose or oligosaccharide derived from kojibiosa.
  • the starting reaction mixture used in the synthesis process of the invention is obtained from abundant and low-cost substrates such as sucrose and lactose disaccharides. However, it is possible to replace it with by-products of the low-cost and easily obtainable agro-food industry such as whey permeate or by-products derived from the sugar industry.
  • transglycosylation reaction is meant the enzymatic reaction of transferring a glycosidic residue to another carbohydrate that acts as an acceptor.
  • the function of the yeast in the synthesis process of the invention is the purification of the starting reaction mixture, eliminating the fructose released in the synthesis reaction thereof, which is an enzyme inhibitor with ⁇ -galactosidase activity, which It is used in step (b) of hydrolysis.
  • Yeast also removes traces of sucrose and glucose. More specifically, the reaction produced by yeast is the removal of sugars through glycolysis in which anaerobic carbohydrates are converted to ethanol and C0 2.
  • yeasts of the Saccharomycetaceae family that are used within the scope of the invention are those of the genus Saccharomyces and Kluyveromyces, and preferably Saccharomyces cerevisiae, which is characterized by its selective ability to remove carbohydrates by fermentation (Yoon, SH, et al. ., 2003. Carbohyd. Res., 338, 1 127-1 132).
  • the yeast used in stage (a) of fermentation is Saccharomyces cerevisiae and is used in a ratio of 16 g of yeast per OO g of sugar.
  • alginate immobilized yeast is used.
  • the function of the enzyme with ⁇ -galactosidase activity is to hydrolyze lactose (P-D-galactopyranosyl- (1 ⁇ 4) -D-glucose) and trisaccharide 2-a-D-glucopyranosyllactose, which will lead to the release of kojibiosa.
  • lactose P-D-galactopyranosyl- (1 ⁇ 4) -D-glucose
  • trisaccharide 2-a-D-glucopyranosyllactose which will lead to the release of kojibiosa.
  • Examples of enzymes that can be used within the scope of the invention are ⁇ -galactosidases of microbial and food grade origin, and preferably of Kluyveromyces lactis.
  • the enzyme with ⁇ -galactosidase activity that is used in the scope of the invention, prioritizes the hydrolytic capacity over the ability to transgalactosilar, thus allowing to obtain a final product with greater purity and free of galactobioses and / or other di and trisaccharides other than kojibiosa that would not be achieved with the use of other enzymes.
  • step (b) of hydrolysis is carried out at a pH of 7, the enzyme with ⁇ -galactosidase activity is Kluyveromyces lactis and is used with a concentration of 65 U / mL
  • the synthesis process of the invention achieves the obtaining of a compound comprising kojibiosa and other products such as leucrose.
  • the synthesis process of the invention achieves a production yield of kojibiosa that is greater than 25%, preferably greater than 35%, and more preferably greater than 45%.
  • the synthesis process of the invention includes a purification step (c) subsequent to the hydrolysis stage (b) to obtain a product with a high degree of purity that facilitates its subsequent applications, and which is performed using a technique selected, by way of illustration and not limitation, between fermentation with the remaining yeast added during step (a) of fermentation or chromatography or nanofiltration.
  • the purification step (c) is carried out by allowing the remaining yeast reacted during the fermentation stage (a) followed by centrifugation or filtration and kojibiosa is obtained with a percentage of wealth in weight with respect to the dry extract, superior to 60%.
  • chromatographic techniques as a purification method results in a considerable reduction in the duration of the process, as well as an increase in the purity of the product, in relation to for example the application of the continued yeast treatment.
  • chromatographic techniques that include the scope of the present invention are molecular exclusion chromatography (SEC) and liquid chromatography with refractive index detector (LC-RID).
  • SEC Molecular exclusion chromatography
  • the step (c) of purification is carried out by means of molecular exclusion chromatography (SEC), prior to centrifugation or filtering and kojibiosa is obtained with a percentage of richness in weight with respect to the extract dry, more than 70%.
  • SEC molecular exclusion chromatography
  • LC-RID liquid chromatography with refractive index detector
  • the purification step (c) is carried out by liquid chromatography with refractive index detector (LC-RID), prior to centrifugation or filtering, and achieves kojibiosa with a percentage of wealth in weight with respect to the dry extract, greater than 95%.
  • LC-RID refractive index detector
  • the process of the invention includes an additional step (d) of lyophilization or atomization of the kojibiosa after the stage (c) of purification that allows to obtain a final powder product.
  • beneficial bacteria such as, for example, Bifidobacterium and Lactobacillus
  • kojibiosa as an inhibitor of the enzyme a-glucosidase, gives it an important antiviral activity and ability to delay the digestion of carbohydrates ingested in the diet, such as starch and sucrose, by the inhibition of intestinal glucosidases, allowing its use in therapeutic applications for different diseases, which are preferably obesity, diabetes mellitus, prediabetes, gastritis or caries.
  • the use of the kojibiosa of the invention for the preparation of a prebiotic food composition hereinafter use of the invention, which is useful for preventing pathologies related to the gastrointestinal system or for the preparation of a composition pharmaceutical that is useful for the treatment of viral diseases or to delay the digestion of starch and sucrose in diseases such as obesity, diabetes mellitus, prediabetes or caries.
  • viral disease is chosen, by way of indication and not limitation, from among human immunodeficiency virus (HIV) type 1, or hepatitis B virus or hepatitis virus C.
  • prebiotic food composition for preventing gastrointestinal pathologies, hereinafter prebiotic food composition of the invention, characterized by comprising the kojibiosa of the invention.
  • prebiotic food composition means any combination of ingredients constituting a solid or liquid food product, which in addition to its nutritional characteristics, includes specific functions of resistance to gastrointestinal digestion and selective stimulation of growth and activity of one or more strains of bacteria in the digestive system, which help improve health and reduce the risk of disease.
  • composition of the invention characterized by comprising a therapeutically effective amount of the kojibiosa of the invention, a pharmaceutically acceptable salt, solvate or pro-drug thereof, hereinafter pharmaceutical composition of the invention.
  • pharmaceutical composition of the invention is used in its broadest sense and encompasses those derivatives that are converted in vivo into kojibious derivatives suitable for the treatment of the pathologies described above.
  • the pharmaceutical composition of the invention additionally comprises at least one pharmaceutically acceptable excipient, adjuvant and / or vehicles.
  • the pharmaceutical composition of the invention additionally includes at least one other active ingredient.
  • the pharmaceutical composition of the invention can be administered by any appropriate route of administration, for example, oral, parenteral (subcutaneous, intraperitoneal, intravenous, intramuscular, etc.), rectal, etc.
  • the food composition of the invention is used in the preventive treatment of gastrointestinal pathologies.
  • the pharmaceutical composition of the invention is used for the treatment of viral type infections, which are preferably human immunodeficiency virus (HIV) type 1, hepatitis B virus or the virus of hepatitis C, or to delay the digestion of starch and sucrose in therapeutic applications of other diseases, which are preferably obesity, diabetes mellitus, prediabetes or caries.
  • viral type infections which are preferably human immunodeficiency virus (HIV) type 1, hepatitis B virus or the virus of hepatitis C
  • HIV human immunodeficiency virus
  • Figure 1 Structure of trisaccharide 2-aD-glucopyranosyl-lactose ( ⁇ - ⁇ -D-galactopyranosyl- (1 ⁇ 4) -0- [aD-glucopyranosyl- (1 ⁇ 2)] - pD-glucopyranoside) derived from galactosylated Kojibiosa present in the starting reaction mixture.
  • Figure 2 Profile obtained by gas chromatography coupled to mass spectrometry (GC-MS) of the sugar mixture obtained by transglycosylation reaction of the enzyme-catalyzed lactose Leuconostoc mesenteroides B-512F dextransacarase and subsequent treatment with the yeast Saccharomyces cerevisiae and with the ⁇ -galactosidase of Kluyveromyces lactis according to Example 1 with stage (c) of yeast purification, where the synthesis of kojibiosa is observed with high yield and percentage of purity
  • GC-MS mass spectrometry
  • Example 1 Starting from a reaction mixture comprising trisaccharide 2-a-D-glucopyranosyl-lactose, leucrose, lactose, fructose, glucose and sucrose, obtained by a transglycosylation reaction with sucrose and lactose (equimolar ratio) as starting substrates.
  • the initial transglycosylation reaction using sucrose and lactose, was carried out according to D ⁇ ez-Municio M, et al. (J. Agrie. Food. Chem. 2012, 60, 1945-1953).
  • a solution of 250 g / L of sucrose and 250 g / L of lactose in 20 mM sodium acetate buffer with pH 5.2, supplemented with 1 mM calcium chloride, to which 0.8 U was added was prepared.
  • / mL of dextransacarase enzyme from L. mesenteroides B-512F Said reaction mixture was incubated at 30 ° C temperature and stirring of 500 rpm for 24 h.
  • the resulting mixture has as main compound trisaccharide 2-a-D-glucopyranosyl lactose.
  • the synthesis process of the invention was carried out at a temperature of 30 ° C.
  • First the fermentation step was carried out, for this the mixture was diluted with water to a total sugar concentration of 200 g / L, S. cerevisiae yeast was added, at a concentration of 32 g / L and kept up under strong stirring (1000 rpm) for 4 hours until the yeast had fermented all of the sucrose and residual glucose, as well as much of the fructose (90-95%).
  • a hydrolysis step was carried out, for this purpose the pH of the reaction medium was adjusted to 7 (with potassium hydroxide) and 10 mM magnesium chloride and the enzyme ⁇ -galactosidase of K.
  • lactis (Lactozym) were added Puree 6500 L) at a concentration of 65 U / mL and kept under strong stirring (1000 rpm) for an additional 90 min. In this way, the non-transglycosylated lactose was hydrolyzed to glucose and galactose (monosaccharides that are consumed by yeast). On the other hand, the main compound of the mixture, trisaccharide 2-aD-glucopyranosyl-lactose, gives rise to kojibiosa (product of interest resistant to fermentation of yeast) and galactose (fermented).
  • a purification step was carried out, using a method selected from among, to continue with the treatment of yeast initiated prior to hydrolysis with ⁇ -galactosidase, molecular exclusion chromatography (SEC) and liquid chromatography with refractive index detector (LC-RID) obtaining different degrees of purity of the kojibiosa.
  • SEC molecular exclusion chromatography
  • LC-RID liquid chromatography with refractive index detector
  • yeast After hydrolysis with ⁇ -galactosidase, the remaining yeast was allowed to act for an additional 42 hours to eliminate the monosaccharides derived from the hydrolysis of lactose and the galactose residue of trisaccharide 2-aD-glucopyranosyl lactose. After the reaction time has elapsed, the mixture of Obtained sugars were filtered or centrifuged at 10,000 rpm for 5 minutes to remove yeast.
  • the synthesized compound has a high percentage in kojibiosa, this being around 65% of the dry extract, according to the quantification performed by gas chromatography with flame ionization detector (GC-FID).
  • GC-FID gas chromatography with flame ionization detector
  • the final product obtained has a high percentage in kojibiosa, this being around 76% (with respect to the value of the dry extract determined (96%), according to the quantification performed by gas chromatography with flame ionization detector (GC- FID)
  • GC- FID gas chromatography with flame ionization detector
  • filtration was carried out immediately after the hydrolysis stage with ⁇ -galactosidase of K. lactis. centrifugation at 10,000 rpm for 5 min to remove yeast. Subsequently, a column purification process took place for 25 min by means of liquid chromatography with refractive index detector (LC-RID) using a linked amino column and using acetronitrile as the mobile phase: water (75:25, v: v), In Socratic mode.
  • LC-RID refractive index detector
  • This type of purification of carbohydrate mixtures by LC-RID can be performed at an analytical, semi-preparative or preparative level, obtaining in all cases a kojibiosa with a degree of purity close to 100%.
  • the dimensions of the analytical column are 150 - 250 mm in length and 4.6 mm in internal diameter, the workflow of 0.5 - 1.0 mL / min and the injection volume of 50 - 100 ⁇ , obtaining 1 - 10 mg of the product of interest.
  • the semi-preparative column has dimensions of 250 mm in length and 10 mm of internal diameter, the workflow of 4.0 - 5.0 mL / min and the injection volume of 500 ⁇ (obtaining 10 - 50 mg of the product of interest).
  • a column with dimensions of 250 mm in length and 21.2 mm of internal diameter, the workflow of 21.0 mL / min and the injection volume of 2-3 mL are obtained (obtaining 50 - 100 mg of the product of interest).
  • Example 2 Starting from a reaction mixture comprising trisaccharide 2-aD-glucopyranosyl-lactose, leucose, lactose, fructose, glucose and sucrose obtained by a transglycosylation reaction with sucrose and lactose (ratio 1: 2.5) as starting substrates
  • Example 1 Exactly the same steps were followed as those indicated in Example 1 (with the stage of purification of the yeast treatment and subsequent centrifugation), with the proviso that in this case it was based on a solution of 100 g / L sucrose and 250 g / L of lactose.
  • the non-transglycosylated lactose was 20% more than when using a sucrose ratio: equimolar lactose, which resulted in a decrease in yield (28% by weight with respect to the initial amount of lactose) .
  • the synthesized compound has a high percentage in kojibiosa, being around 52% (considering the value of the dry extract determined), based on the quantification performed by gas chromatography with flame ionization detector (GC-FID).
  • Example 3 Starting from a reaction mixture comprising trisaccharide 2-aD-glucopyranosyl-lactose, leucrose, lactose, fructose, glucose and sucrose, obtained by a transglycosylation reaction with sucrose and whey permeate (sucrose ratio: equimolar lactose) as starting substrates
  • Example 2 Exactly the same steps were followed as those indicated in Example 1, with the proviso that in this case it was based on a solution of 250 g / L sucrose and 320 g / L permeate from dairy whey of bovine origin (quantity equivalent to 250 g / L lactose).
  • kojibiosa a byproduct of the dairy industry, kojibiosa is obtained with greater yield than when lactose is used as a substrate (Example 1), synthesized kojibiosa with a 48% yield by weight with respect to the initial lactose amount , which means that starting from 128 g of serum permeate, 48 g of kojibiosa are obtained.
  • the purification step was carried out using a method selected from among, continuing with the yeast treatment initiated prior to hydrolysis with ⁇ -galactosidase, molecular exclusion chromatography (SEC) and liquid chromatography with detector of refractive index (LC-RID) under the same conditions as specified in that example.
  • SEC molecular exclusion chromatography
  • LC-RID liquid chromatography with detector of refractive index
  • the final product obtained has, as in Example 1, a high and similar percentage in kojibiosa ( ⁇ 65%), accompanied by leucrose, and a low proportion of trisaccharides and metabolites produced by yeast.
  • SEC molecular exclusion chromatography
  • the final product obtained showed a high and very similar percentage in kojibiosa ( ⁇ 75%) accompanied by a low proportion of leucrose.
  • the product synthesized according to Example 1 and including a purification step with continued yeast treatment presented 65% kojibiosa, 19% leucrose disaccharide, synthesized in the reaction catalyzed by dextransacarase when the fructose acts as an acceptor, a % of trisaccharides (mainly galactosylated derivatives such as aD-Glucopyranosyl- [1 -4] -pD-Galactopyranosyl- [1-6] -pD-Galactose), and 8% of metabolites produced by yeast, such as poly-alcohols (see Figure 2).
  • the concentration in mineral salts was 2%.
  • Example 1 The product synthesized according to Example 1 and purified by SEC showed a simpler composition with 76% kojibiosa and 24% leucrose, considering the value of the determined dry extract (96%). Also, following the same Example 1 and applying LC-RID as a purification step led to a purity of ⁇ 100% in kojibiosa.
  • the product synthesized according to Example 2 consisted of 52% kojibiosa, 16% disaccharides derived from the hydrolysis of lactose and subsequent transgalactosylation (alolactose, galactobiosa, among others), 14% of trisaccharides (mainly galactosylated derivatives of lactose as aD- Glucopyranosyl- [1 -4] -pD-Galactop ⁇ ranos ⁇ l- [1 -6] -pD-Galactose), 8% of the leucrose disaccharide, synthesized in the reaction catalyzed by dextransacarase when acting Fructose as an acceptor and 8% of metabolites produced by yeast, such as poly-alcohols.
  • the composition in mineral salts was 2%. All these data refer to the dry extract content that was 93%.
  • the kojibiosa and leucrosa have been identified by gas chromatography coupled to mass spectrometry (GC-MS) by analyzing their corresponding trimethylsilyloxime (TMSO) and comparing with the previously derivatized commercial pattern.
  • GC-MS gas chromatography coupled to mass spectrometry
  • TMSO trimethylsilyloxime
  • the mass spectrum of the kojibiosa obtained according to Example 1 with stage (c) of yeast purification (see Figure 3) was characterized by presenting the ions with a mass / charge ratio (m / z) of: 319, 73, 147 , 217, 361 and 205 (in decreasing order of abundance).

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Engineering & Computer Science (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Mycology (AREA)
  • Microbiology (AREA)
  • Nutrition Science (AREA)
  • Polymers & Plastics (AREA)
  • Food Science & Technology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Coloring Foods And Improving Nutritive Qualities (AREA)

Abstract

La présente invention concerne un procédé de synthèse de kojibiose caractérisé par sa simplicité et haut rendement, qui utilise un mélange réactionnel de départ qui contient comme composé principal du trisaccharide 2-α- D-glucopyranosyl-lactose, du O-β-D-galactopyranosyl-(1→4)-O-[α-D-glucopyranosyl- (1→2)]-β-D-glucopiranose, ainsi que du leucrose, du lactose, du fructose, du glucose et du saccharose et, qui comprend des étapes de fermentation, hydrolyse et purification permettant l'obtention d'un disaccharide à haute valeur ajoutée, tel que le kojibiose, de manière économique. L'invention concerne également le kojibiose qui est obtenu au moyen du procédé de l'invention et l'utilisation de ce dernier dans la préparation de compositions prébiotiques alimentaires utiles pour prévenir des pathologies associées au système gastro-intestinal et des compositions pharmaceutiques utiles pour le traitement de maladies de type viral ou pour retarder la digestion d'hydrates de carbone.
PCT/ES2014/070687 2013-09-13 2014-09-09 Procédé de synthèse de kojibiose et son utilisation dans la préparation de compositions alimentaires et pharmaceutiques WO2015036637A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ES201331333 2013-09-13
ESP201331333 2013-09-13

Publications (1)

Publication Number Publication Date
WO2015036637A1 true WO2015036637A1 (fr) 2015-03-19

Family

ID=52665125

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/ES2014/070687 WO2015036637A1 (fr) 2013-09-13 2014-09-09 Procédé de synthèse de kojibiose et son utilisation dans la préparation de compositions alimentaires et pharmaceutiques

Country Status (1)

Country Link
WO (1) WO2015036637A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108084234A (zh) * 2018-01-23 2018-05-29 佛山科学技术学院 双重随机糖基化合成三糖的方法
WO2019166514A1 (fr) 2018-02-28 2019-09-06 C-Lecta Gmbh Enrichissement enzymatique in situ d'aliments avec des glucides fonctionnels
CN110531003A (zh) * 2019-09-29 2019-12-03 中国药科大学 一种测定复方电解质葡萄糖注射液中葡萄糖含量的方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996040702A1 (fr) * 1995-06-07 1996-12-19 Alberta Research Council Analogues kojibiosides modifies

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996040702A1 (fr) * 1995-06-07 1996-12-19 Alberta Research Council Analogues kojibiosides modifies

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BORGES DE MELO, E ET AL.: "alpha- and beta-Glucosidase inhibitors: chemical structureand biological activity", TETRAHEDRON, vol. 62, 2006, pages 10277 - 10302 *
CHAEN, H ET AL.: "Enzymatic Synthesis of Kojioligosaccharides Using Kojibiose Phosphorylase", JOURNAL OF BIOSCIENCE AND BIOENGINEERING, vol. 92, no. 2, 2001, pages 177 - 182 *
GOFFIN DOROTHEE ET AL.: "Will Isomalto-Oligosaccharides, a Well-Established Functional Food in Asia", BREAK THROUGH THE EUROPEAN AND AMERICAN MARKET? THE STATUS OF KNOWLEDGE ON THESE PREBIOTICS. CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION, vol. 51, no. 5, 2011, pages 394 - 409 *
KITAO, S ET AL.: "Formation of kojibiose and nigerose by sucrose phosphotylase", BIOSCI. BIOTECH. BIOCHEM., vol. 58, no. 4, 1994, pages 790 - 791 *
TAKAHASHI, M ET AL.: "Biochemical studies on buckwheat alpha-glucosidase. Part III. Transglucosylation action of the enzyme and isolation of the reaction products''.", AGR. BIOL. CHEM., vol. 33, no. 10, 1969, pages 1399 - 141 0 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108084234A (zh) * 2018-01-23 2018-05-29 佛山科学技术学院 双重随机糖基化合成三糖的方法
WO2019166514A1 (fr) 2018-02-28 2019-09-06 C-Lecta Gmbh Enrichissement enzymatique in situ d'aliments avec des glucides fonctionnels
US20210076724A1 (en) * 2018-02-28 2021-03-18 C-Lecta Gmbh Enzymatic in-situ fortification of food with functional carbohydrates
CN110531003A (zh) * 2019-09-29 2019-12-03 中国药科大学 一种测定复方电解质葡萄糖注射液中葡萄糖含量的方法

Similar Documents

Publication Publication Date Title
Fraenkel-Conrat THE ROLE OF THE NUCLEIC ACID IN THE RECONSTITUTION OF ACTIVE TOBACCO MOSAIC VIRUS1
Antošová et al. Fructosyltransferases: the enzymes catalyzing production of fructooligosaccharides
Matsuo et al. Retraction: D-psicose inhibits intestinal α-glucosidase and suppresses the glycemic response after ingestion of carbohydrates in rats
BRPI0709667A2 (pt) Composições de b-glicano,glicosamina e n-acetilglicosanina solúveis em água e métodos para a sua produção
WO2014086373A1 (fr) Cristallisation d'oligosaccharides de lait humain
SU562202A3 (ru) Способ получени производных аминосахаров
BR112012017670B1 (pt) Método para produzir esteviol a partir de esteviosídeo
KR20130041953A (ko) 갈락토올리고당 함유 조성물 및 이것의 제조 방법
ES2665028T3 (es) Dextrina para suprimir la elevación de la concentración de alcohol en sangre
WO2015036637A1 (fr) Procédé de synthèse de kojibiose et son utilisation dans la préparation de compositions alimentaires et pharmaceutiques
WO2020000788A1 (fr) Polysaccharide sélénié estérifié et procédé de préparation et utilisation s'y rapportant
EP3411042A1 (fr) Trans-sialidase intramoléculaire
JP2000217546A (ja) マンノース含有ヘテロオリゴ糖からなる甘味剤、病原菌感染抑制剤、整腸剤又は澱粉の老化抑制剤及び該剤からなる飲食品、医薬品又は飼料。
CN115252671A (zh) 仙人掌果花色苷在制备调控肠道菌群产品中的应用
WO2015014973A2 (fr) Utilisation de glycoside phosphorylases spécifiques pour l'implantation d'une phosphorolyse ou de réactions de phosphorolyse inverse
JPH08291192A (ja) ラクトン化オリゴ糖、その製造方法及びα−アミラーゼ阻害剤
Li et al. Effects of different sea buckthorn leaf tea processing technologies on nutrient level and fecal microflora in vitro.
JP5439671B2 (ja) 抗がん剤
JP5564205B2 (ja) 皮膚掻痒症改善剤
TWI460273B (zh) 寡醣酯之酵素合成法
RU2278164C2 (ru) Способ получения кальция фруктозодифосфата
CN106397501A (zh) 低聚果糖保存工艺
JPH04288093A (ja) 新規なステビオール配糖体及びその製造方法
JP5152474B2 (ja) オリゴ糖、その製造方法、及びその用途
ES2552403B1 (es) Procedimiento bi-enzimático de síntesis eficiente de oligosacáridos fructosilados derivados de lactosacarosa, productos obtenidos y su uso en la mejora de la salud gastrointestinal

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: 14843306

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 14843306

Country of ref document: EP

Kind code of ref document: A1