Classifications

• • 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
• • 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
  • 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
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
  • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
  • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
  • C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
• • 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
  • 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
• • 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)

Definitions

• the present invention relates to a method for preparing a mixture of poorly digestible ⁇ -glucans from a mixture of oligosaccharides and polysaccharides.
• the invention also relates to a mixture of poorly digestible ⁇ -glucans.
• the present invention also relates to the sequential use of a glucanotransferase capable of creating ⁇ (1-3) glucosidic bonds and of a glucanotransferase capable of creating ⁇ (1-6) glucosidic bonds to reduce the digestibility of a mixture of ⁇ -glucans.
• Dietary fibers play an important role in the human diet.
• soluble fibers which are soluble in water and have a gelling capacity
• insoluble fibers insoluble fibers.
• Soluble fibers, including branched maltodextrins, are particularly interesting because they are poorly digestible. As a result, their incorporation into the diet can reduce the glycemic index of a food and prolong the feeling of satiety. They also have prebiotic properties on the intestinal flora, i.e. 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 mixture of oligosaccharides and polysaccharides, to obtain fibers of interest in human and animal nutrition, by enzymatic means.
• the applicant company has thus developed a process which uses two particular enzymes, one capable of creating a(1-3) bonds and the other capable of creating a(1-6) bonds, sequentially, and vice versa. .
• the present invention relates to a method for preparing a mixture of ⁇ -glucans comprising the following steps:
• a substrate said substrate being a mixture of oligosaccharides and polysaccharides having a polydispersion index of between 5 and 10, preferably between 6 and 9.5, even more preferably between 7 and 9, between 8 and 8.5, most preferably about 8.4,
• first and second enzymes being an ⁇ -glucanotransferase capable of cleaving ⁇ (1-4) glucosidic bonds and of creating ⁇ (1-3) glucosidic bonds and/or an ⁇ -glucanotransferase capable of cleaving ⁇ (1-4) glucosidic bonds and creating ⁇ (1-6) glucosidic bonds.
• ⁇ -glucan soluble fibre
• dietary soluble fibre oligosaccharides composed of at least 3 glucose units linked together by a-glycosidic (or a-glucosidic) bonds.
• the substrate used in the method according to the present invention has a DE of between 15 and 20, preferably between 17 and 20, preferably between 18 and 19, even more preferably about 18, 4.
• a measurement of the DE is however insufficient to accurately represent the molecular distribution of the ⁇ -glucans.
• the acid hydrolysis of starch, totally random, or its enzymatic hydrolysis a little more orderly, provide mixtures of glucose and glucose polymers that the only measurement of the DE does not make it possible to define with precision, and which comprise molecules of short size, low Degree of Polymerization (DP), as well as molecules of very long size, of high DP.
• DP Degree of Polymerization
• the second enzyme is an ⁇ -glucanotransferase capable of cleaving glucosidic a(1 -4) bonds and creating glucosidic a(1 -
• the first enzyme is an ⁇ -glucanotransferase capable of cleaving ⁇ (1-4) glucosidic bonds and creating ⁇ (1-6) glucosidic bonds
• the second enzyme is an ⁇ -glucosidic bond.
• glucanotransferase capable of cleaving ⁇ (1-4) glucosidic bonds and creating ⁇ (1-3) glucosidic bonds.
• each enzyme is added at a concentration of between 0.01 and 1 mg/mL of reaction medium, preferably between 0.05 and 0.5 mg/mL, even more preferably approximately 0.1 mg / mL of reaction medium, during sequential incubations.
• the bringing together of the substrate and each enzyme is carried out for a period of between 12 and 48 hours, preferably about 24 hours.
• This mixture of ⁇ -glucans is characterized by its low digestibility according to the AOAC 2002.02 method.
• the method according to the invention makes it possible to reduce by a factor of at least 2, preferably by at least 2.5, even more preferably by at least 3, the hydrolyzable fraction, measured according to the AOAC 2002.02 method, relative to the starting substrate.
• the method according to the present invention also makes it possible to create a(1-3) bonds which were absent in the starting substrate.
• the resin was then washed 5 times with 40 mL of 20 mM Phosphate buffer pH 7.4 containing 300 mM NaCl and 20 mM imidazole. Finally, the elution was carried out with 3 mL of 20 mM Phosphate buffer pH7.4 containing 300 mM of NaCl and 250 mM of imidazole for 5 minutes in order to pick up the enzymes of interest.
• the enzyme solutions were then dialysed (10 kDa sigma membrane) against 5 L of 50 mM sodium acetate buffer, pH 5.75 containing 150 Mm of NaCl (overnight, 4°C with stirring) in order to eliminate the NaCl and the imidazole. .
• Electrophoresis under denaturing conditions made it possible to control the quality of the purified enzymatic extracts.
• samples containing 30 ⁇ L of protein extract and 10 ⁇ L of loading buffer (NuPage LDS Sample buffer 4x, Invitrogen) were denatured for 5 minutes at 95°C then loaded onto pre-cast acrylamide gels.
• Mini-Protean Tris-Glycine eXtented (Biorad) Migration was carried out for 30 min in 1 x Tris/Glycine/SDS buffer under a voltage of 150 V.
• the proteins were then revealed by incubating the gels for 1 hour in a staining solution (PageBlue Protein Staining Solution, Fermentas ) then by rinsing for 30 min in three consecutive baths of water.
• the enzymatic activity of branching enzymes can be determined by measuring the initial rate of production of reducing sugars using the dinitrosalicilic acid (DNS) method.
• DNS dinitrosalicilic acid
• An enzyme unit represents the quantity of enzyme which releases one pmol of fructose per minute, at 30°C, for an initial sucrose concentration of 100 g.L-1 under the conditions of buffer of adequate activity.
• 100 pL of reaction medium were taken and the reaction stopped by adding an equivalent volume of DNS.
• the samples were then heated for 5 min at 95°C, cooled in ice, diluted halfway in water, and the absorbance was read at 540 nm.
• a standard range of 0 to 2 g.L-1 of fructose makes it possible to establish the link between the absorbance value and the concentration of reducing sugars.
• the reactions were carried out with 0.1 mg/mL of purified enzyme either GT#11 or GT#19 and dialysed in the presence of 10%, 20% or 40% of substrate in 50 mM sodium acetate buffer, pH 5.75.
• the reactions were incubated with shaking for 24 h at 20°C or 37°C.
• the reactions were stopped by heating (95°C for 5 minutes). Samples at the initial and final times were carried out to analyze the specificity of enzymes using different analytical techniques (HPAEC-PAD, NMR and HPSEC).
• the transfer reactions were lyophilized after freezing at -80° C. for 24 hours. 25 mg of freeze-dried products were taken up in 1 mL of 100 mM sodium maleate buffer containing 30 U of pancreatic ⁇ -amylase and 3 U of amyloglucosidase (Starch resistant kit, Megazyme K-STAR 06/18, which uses 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 Ampero-metric Detection).
• HPAEC PAD High Performance Anion Exchange Chromatography with Pulsed Ampero-metric Detection
• the analyzes were performed on a Thermo ICS6000 system equipped with a CarboPacTM PA100 analytical column (2 mm x 250 mm) coupled with a CarboPacTM PA100 guard guard (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; 5-35min, 0-300mM; 35-40min, 300-450mM; 40-42 mins, 450mM.
• Detection was performed using a gold working electrode and a pH Ag/AgCI reference cell. The samples were diluted to a total dry mass of 1 g.L-1 before injection.
• the size of the reaction products was also sometimes determined by size exclusion chromatography (high performance size exclusion chromatography) on a Fisher Ultimate 3000 system equipped with a Shodex OH-Pak SB-802.5 column protected by a Shodex pre-column OH-Pak SB-G guard column, placed at 70°C in the system oven.
• the mobile phase was water at a flow rate of 0.3 mL.min-1.
• the detection was carried out by refractometry. The samples were diluted to a total dry mass of 20 g.L-1 before injection.
• Hakomori Method (1964 HAKOMORI A Rapid Permethylation of Glycolipid, and Polysaccharide Catalyzed by Methylsulfinyl Carbanion in Dimethyl Sulfoxide) makes it possible to chemically characterize osidic bonds by differentiating between free OH groups and bound groups. It is a destructive method comprising the steps of methylation, hydrolysis, reduction with NaBD4, acetylation and analysis by mass spectrometry.
• the enzymatic cascade represents a good strategy for increasing the resistance of products to hydrolytic enzymes and achieving a level of digestibility of less than 40%.
• the Glucidex 19D is placed in solution at 200 g.L-1, the first ⁇ -GT is reacted at a concentration of 0.05 mg.mL-1 for 24 hours. The reaction is stopped by heating for 5 minutes at 95°C. the second enzyme is then reacted at the same concentration of 0.05 mg.ml-1. The reaction is again stopped by heating for 5 minutes at 95°C after 24 h of incubation.

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• 2021
  • 2021-08-23 FR FR2108822A patent/FR3126229B1/fr active Active
• 2022
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  • 2022-08-22 EP EP22776967.6A patent/EP4392573A1/fr active Pending
  • 2022-08-22 CA CA3229608A patent/CA3229608A1/fr active Pending
  • 2022-08-22 CN CN202280062305.8A patent/CN117940574A/zh active Pending
  • 2022-08-22 AU AU2022332653A patent/AU2022332653A1/en active Pending
  • 2022-08-22 WO PCT/FR2022/051595 patent/WO2023026010A1/fr not_active Ceased
  • 2022-08-22 US US18/684,793 patent/US20250019733A1/en active Pending
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• 2024
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