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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K10/00—Animal feeding-stuffs
  • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
  • A23K10/14—Pretreatment of feeding-stuffs with enzymes
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K20/00—Accessory food factors for animal feeding-stuffs
  • A23K20/10—Organic substances
  • A23K20/163—Sugars; Polysaccharides
• • 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
  • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
  • A23L29/30—Foods or foodstuffs containing additives; Preparation or treatment thereof containing carbohydrate syrups; containing sugars; containing sugar alcohols, e.g. xylitol; containing starch hydrolysates, e.g. dextrin
• • 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
  • A23L33/20—Reducing nutritive value; Dietetic products with reduced nutritive value
  • A23L33/21—Addition of substantially indigestible substances, e.g. dietary fibres
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/1048—Glycosyltransferases (2.4)
  • C12N9/1051—Hexosyltransferases (2.4.1)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/1048—Glycosyltransferases (2.4)
  • C12N9/1051—Hexosyltransferases (2.4.1)
  • C12N9/107—1,4-Alpha-glucan branching enzyme (2.4.1.18)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y204/00—Glycosyltransferases (2.4)
  • C12Y204/01—Hexosyltransferases (2.4.1)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y204/00—Glycosyltransferases (2.4)
  • C12Y204/01—Hexosyltransferases (2.4.1)
  • C12Y204/01005—Dextransucrase (2.4.1.5)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y204/00—Glycosyltransferases (2.4)
  • C12Y204/01—Hexosyltransferases (2.4.1)
  • C12Y204/01018—1,4-Alpha-glucan branching enzyme (2.4.1.18), i.e. glucan branching enzyme
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y204/00—Glycosyltransferases (2.4)
  • C12Y204/01—Hexosyltransferases (2.4.1)
  • C12Y204/01025—4-Alpha-glucanotransferase (2.4.1.25)
• • C—CHEMISTRY; METALLURGY
  • C13—SUGAR INDUSTRY
  • C13K—SACCHARIDES OBTAINED FROM NATURAL SOURCES OR BY HYDROLYSIS OF NATURALLY OCCURRING DISACCHARIDES, OLIGOSACCHARIDES OR POLYSACCHARIDES
  • C13K13/00—Sugars not otherwise provided for in this class

Definitions

• Dietary fibers have an important role in human nutrition. Among dietary fibers, we distinguish between soluble fibers, which are soluble in water and have a gelling capacity, and insoluble fibers. Soluble fibers, including branched maltodextrins, are particularly interesting because they are poorly digestible. As a result, their incorporation into the diet makes it possible to reduce the glycemic index of a food and prolong the feeling of satiety. They also have prebiotic properties on the intestinal flora, that is to say they are capable of selectively promoting the growth of certain probiotic type bacteria or the activity of the microbiota, providing a benefit to the health.
• the Applicant company found that it was possible, from a syrup rich in oligosaccharides having a degree of polymerization (DP) of 4, to obtain fibers of interest in human and animal food, by enzymatic pathway.
• the Applicant company has thus developed a process which uses a particular enzyme, capable of creating a(1-6) bonds from syrup rich in DP4 oligosaccharides.
• the present invention relates to a process for preparing a mixture of a-glucans, preferably a mixture of branched maltodextrins, comprising a step of bringing a substrate and an enzyme, said substrate being a syrup rich in oligosaccharides having a degree of polymerization (DP) of 4 and said enzyme being an a-glucanotransferase capable of cleaving a(1-4) glucosidic bonds and creating a(1) glucosidic bonds -6).
• DP degree of polymerization
• a-glucan soluble fiber
• soluble dietary fiber oligosaccharides composed of at least 3 glucose units linked together by a-glycosidic (or a-glucosidic) bonds.
• Such a measurement of the DE is, however, insufficient to precisely represent the molecular distribution of ⁇ -glucans. Indeed, the acid hydrolysis of starch, completely random, or its enzymatic hydrolysis, a little more ordered, provide mixtures of glucose and glucose polymers that the sole measurement of the DE does not make it possible to define with precision, and which include molecules of short size, low DP, as well as molecules of very long size, DP. pupil.
• the measurement of the DE in fact only gives an approximate idea of the average DP of the mixture of glucose and the glucose polymers constituting the a-glucans and therefore of their number average molecular mass (Mn). To complete the characterization of the molecular mass distribution of a-glucans, the determination of another parameter is important, that of the weight average molecular mass (Mp).
• the process according to the present invention makes it possible to reduce the percentage of a(1-4) bonds in favor of a(1-6) bonds, which has the advantage of reducing the digestibility of the a-glucan mixture. obtained by the process.
• the mixture of ⁇ -glucans prepared according to the process of the invention is preferably a mixture of branched maltodextrins.
• branched maltodextrins means maltodextrins whose content of ⁇ (1-6) glucosidic bonds is greater than that of standard maltodextrins.
• the syrup rich in oligosaccharides having a DP of 4 has a dextrose equivalent (DE) greater than 20.
• the syrup rich in DP4 is a syrup having the characteristics described in Table 1 below.
• sequence SEQ ID No: 1 corresponds to the Genbank accession number WP_053069107.1.
• the bringing together of the substrate and the enzyme is carried out at a temperature between 20 and 40°C, preferably around 37°C.
• the bringing together of the substrate and the enzyme is carried out at a pH between 5 and 6.5, preferably between 5.5 and 6, even more so. preferred about 5.75.
• the present invention also relates to a mixture of aglucans, preferably a mixture of branched maltodextrins, capable of being obtained by the process described above.
• This mixture of a-glucans is characterized by its low digestibility according to the AOAC 2002.02 method.
• the process according to the invention makes it possible to reduce by a factor of at least 2, preferably at least 2.5, even more preferably by at least 3, the hydrolyzable fraction, measured according to the AOAC 2002.02 method, compared to the starting substrate.
• the AOAC 2002.02 method can in particular be implemented using the “HPAEC-PAD assay” part of the “resistant Starch, K-RSTAR 06/18” kit marketed by the company Megazyme® as described in the Example 1, part 5 below.
• the percentage of a(1 -6) bonds can be measured by the Hakomori method (1964 HAKOMORI A Rapid Permethylation of Glycolipid, and Polysaccharide Catalyzed by Methylsulfinyl Carbanion in Dimethyl Sulfoxide) as described in Example 1, part 8 below or by proton NMR as described in Example 1, part 7 below.
• the present invention relates to a mixture of a-glucans, preferably a mixture of branched maltodextrins, characterized in that it presents:
• the rate of hydrolyzable fibers is greater than 5%, preferably greater than 10%, 11%, 12%, 13%, 14%, 15%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%.
• the rate of hydrolyzable fibers is between 5% and 45%, preferably between 10% and 45%, preferably between 20% and 44%, more preferably between 30% and 45%. % and 45%.
• the percentage of a(1-3) bonds is at least 2%, preferably at least 3%.
• the percentage of a(1-3) bonds is at most 6%, preferably at most 5%.
• the percentage of ⁇ (1-3) bonds is between 2% and 8%, preferably between 3% and 5%.
• the percentage of a(1-2) bonds is at least 1%, preferably at least 2%.
• the percentage of a(1-2) bonds is at most 8%, preferably at most 6%, at most 5%, at most 4%. at most 3%.
• the percentage of a(1 -4) bonds is at most 80%, preferably at most 70%, at most 65%.
• the percentage of a(1 -4) bonds is at least 50%, preferably at least 55%, at least 60%.
• the percentage of ⁇ (1-4) bonds is between 50% and 80%, preferably between 55% and 70%, preferably between 55% and 65%.
• the mixture of ⁇ -glucans which is preferably a mixture of branched maltodextrins, is characterized in that it presents:
• a rate of hydrolyzable fibers less than 55%, preferably less than 50%, even more preferably less than 45%,
• the present invention also relates to the use of a mixture of ⁇ -glucans obtained according to the process described above and of a mixture of ⁇ -glucans having the properties described above for the preparation of food for human or animal consumption.
• the a-glucan mixture according to the invention can be used to promote intestinal health, blood sugar management, satiety and weight management, and sustained energy release.
• the present invention relates to the use of a glucanotransferase capable of cleaving ⁇ (1-4) glucosidic bonds and creating ⁇ (1-6) glucosidic bonds to reduce the digestibility of 'a mixture of ⁇ -glucans, said glucanotransferase having the sequence SEQ ID No: 1 or a protein having at least 90% identity with the protein having the sequence SEQ ID No: 1.
• the mixture of ⁇ -glucans is preferably a syrup rich in oligosaccharides, in particular a syrup rich in oligosaccharides having DP of 4 as described in the first aspect of the invention.
• Example 1 preparation of branched maltodextrins from syrup rich in DP4: materials and methods
• the transfer reactions were lyophilized after freezing at -80° C. for 24 hours. 25 mg of lyophilized products were taken up in 1 mL of 100mM sodium maleate buffer containing 30 U of pancreatic a-amylase and 3 U of amyloglucosidase (Starch resistant kit, Megazyme K-STAR 06/18, which uses the AOAC 2002.02 method). The reactions were incubated for 16 hours at 37°C. The products were diluted in water before HPAEC PAD analysis.
• the products obtained were analyzed by anion exchange chromatography coupled with a pulsed amperometric detector (HPAEC PAD - High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection).
• HPAEC PAD High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection
• the analyzes were carried out on a Thermo ICS6000 system equipped with a CarboPacTM PA100 analytical column (2 mm x 250 mm) coupled with a CarboPacTM PA100 guard pre-column (2 mm x 50 mm).
• a gradient of sodium acetate in 150 mM sodium hydroxide was applied at a flow rate of 0.250 ml.min-1 according to the following profile: 0-5 min, 0 mM; 5-35 min, 0-300 mM; 35-40 min, 300-450 mM; 40-42 min, 450mM.
• Detection was carried out using a gold working electrode and an Ag/AgCI pH reference cell. The samples were diluted to a total dry mass of 1 g.L-1 before injection.
• the Hakomori method (1964 HAKOMORI A Rapid Permethylation of Glycolipid, and Polysaccharide Catalyzed by Methylsulfinyl Carbanion in Dimethyl Sulfoxide) makes it possible to chemically characterize the saccharide bonds by differentiating the free OH groups and the bound groups. It is a destructive method including the steps of methylation, hydrolysis, reduction with NaBD4, acetylation and analysis by mass spectrometry
• Example 2 preparation of branched maltodextrins from syrup rich in DP4: results
• the GT#11 enzyme leads to a reduction in the percentage of ⁇ -1,4 bonds and the appearance of ⁇ -1,3 bonds.
• the digestibility of the product obtained by treatment with the GT#11 enzyme is also reduced (53 and 55%, for respective initial concentrations of 100 and 200 mg/mL, compared to 84 and 88% for untreated substrates).

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