Enzymatic methods for obtaining soluble fiber

Abstract

The present invention relates to a method for preparing a mixture of resistant α-glucans from a substrate rich in oligosaccharides having a degree of polymerization (DP) of four.

Description

[Technical field]

[0001] The present invention relates to a method for preparing a mixture of resistant α-glucans from a mixture of oligosaccharides and polysaccharides.

[0002] The present invention also relates to a mixture of resistant α-glucans.

[0003] The present invention also relates to the sequential use of a glucanotransferase capable of producing an α(1,3) glycosidic bond and a glucanotransferase capable of producing an α(1,6) glycosidic bond to reduce digestibility of a mixture of α-glucans. [Background technology]

[0004] Dietary fibres have an important role in human nutrition. Among dietary fibres, a distinction is made between soluble fibres, which are soluble in water and have gelling capacity, and insoluble fibres. Soluble fibres, including branched maltodextrins, are particularly advantageous, as they are indigestible. For this reason, their incorporation into the diet makes it possible to lower the glycemic index of food and to prolong satiety. They are also endowed with biotic properties for the intestinal microflora. In other words, they can selectively promote the growth of certain bacteria of probiotic type or the activity of the microflora, by providing health benefits.

[0005] To date, soluble fibres, including branched maltodextrins, have been obtained primarily through physicochemical means.

[0006] This is in particular the case of the maltodextrin sold by the Applicant Company under the brand name NUTRIOSE® FM10 as a soluble fibre.

[0007] There are other soluble fibres obtained physicochemically, such as PROMITOR® sold by the company Tate and Lyl, FIBERSOL® or LITESSE® sold by the company Dupont Nutrition and Biosciences.

[0008] Many studies have demonstrated that digestibility is directly related to the proportion of different glycosidic bonds within soluble fiber.

[0009] In fact, standard maltodextrins are defined as rapidly digestible, purified and concentrated mixtures of glucose and glucose polymers essentially linked via α1→4 (hereinafter 1→4 or α(1,4)), with only 4-5% α1→6 glycosidic bonds (hereinafter 1→6 or α(1,6)), of very different molecular weights, completely soluble in water and with low reducing power.

[0010] By increasing the proportion of α1→6 or α1→3 linkages, the degree of branching of maltodextrins is increased, which makes them more resistant to digestion.

[0011] Enzymatic approaches using enzymes capable of promoting the creation of "branched" type bonds have many advantages in terms of safety and environmental protection, and also offer better specificity.

[0012] Originally, most enzymatic methods for producing soluble fiber are carried out using sucrose as a substrate for the enzyme to create new bonds. For example, WO2015183714 describes an enzymatic reaction from a mixture of sucrose and an α-glucan type substrate.

[0013] Currently, most enzymatic processes use amylomaltase to produce soluble fiber from starch.

[0014] It is desirable to obtain enzymatically soluble fiber from a substrate in the absence of sucrose. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The Applicant Company has found that it is possible to enzymatically obtain fibres of interest in human and animal nutrition from mixtures of oligosaccharides and polysaccharides, and has therefore developed a method using two specific enzymes, one capable of generating α(1,3) bonds and the other capable of generating α(1,6) bonds, in sequence (and vice versa).

[0016] In a first aspect, the present invention relates to a method for preparing a mixture of α-glucans, the method comprising the steps of: providing a substrate which is a mixture of oligosaccharides and polysaccharides having a polydispersity index of -5 to 10, preferably 6 to 9.5, even more preferably 7 to 9, 8 to 8.5, and most preferably about 8.4; - a first incubation in the presence of a first enzyme; - a second incubation with a second enzyme, wherein the first and second enzymes are α-glucanotransferases capable of cleaving α(1,4) glycosidic linkages to produce α(1,3) glycosidic linkages and / or α-glucanotransferases capable of cleaving α(1,4) glycosidic linkages to produce α(1,6) glycosidic linkages.

[0017] According to the present invention, the terms "α-glucan", "soluble fiber", and "edible soluble fiber" are used interchangeably. They define oligosaccharides composed of at least three glucose units linked together by α-glycosidic (or α-glucosidic) bonds.

[0018] The classification of α-glucans is mainly based on the measurement of their reducing power, conventionally expressed by the idea of ​​"Dextrose Equivalent (DE)". In this particular respect, the definition of maltodextrin given in the Monograph Specifications of the Food Chemical Codex specifies that the DE value of maltodextrins must not exceed 20. Above 20, they are glucose syrups.

[0019] Preferably, the substrate used in the method according to the 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.

[0020] However, such DE measurements are insufficient to accurately represent the molecular distribution of α-glucan. Indeed, acid hydrolysis of starch, which is completely random, or its enzymatic hydrolysis, which is slightly more ordered, cannot be accurately defined by a single measurement of DE, and provides a mixture of glucose and glucose polymers containing short-sized molecules with low degree of polymerization (DP), as well as very long-sized molecules with high DP.

[0021] The measurement of DE actually only gives a rough idea of ​​the average DP of the mixture of glucose and the constituent glucose polymers of α-glucan and therefore of their number-average molecular weight (Mn). To complete the characterization of the molecular weight distribution of α-glucan, it is important to determine another parameter, that of the weight-average molecular weight (Mw).

[0022] In practice, the values ​​of Mn and Mw are not calculated but are measured by different techniques, for example a measurement method suitable for glucose polymers is used, which is based on gel permeation chromatography on a chromatography column calibrated with pullulan of known molecular weight.

[0023] The Mw / Mn ratio, called the polymolecular index or polydispersity index (PI), makes it possible to characterize the overall molecular weight distribution of a polymer blend. As a rule, the molecular weight distribution of standard maltodextrins results in an IP value of 5 to 10.

[0024] These various parameters are also a reflection of the α-glycosidic bond profile of the α-glucan: indeed, a standard α-glucan mixture has a very high proportion of "linear" α(1,4) bonds (more than 90%) and a low proportion of so-called "branched" (α(1,2), α(1,3) and α(1,6) bonds).

[0025] The process according to the invention makes it possible to reduce the proportion of α(1,4) bonds in favor of α(1,3) and α(1,6) bonds, which has the advantage of reducing the digestibility of the mixture of α-glucans obtained by this process.

[0026] According to one embodiment of the invention, the substrate comprises: - 40-50% oligosaccharides with a degree of polymerization (DP) of 1-9; - 15-20% polysaccharides with a DP of 10-20, -35-40% polysaccharides with a DP greater than 20. Here, percentages are expressed as relative molar percentages that add up to 100%.

[0027] Preferably, the substrate comprises: - 90 to 97%, preferably 92 to 95%, of α(1,4) bonds; - 3 to 7%, preferably 4 to 6%, of α(1,6) bonds; - 0 to 3%, preferably 1 to 2% α(1,3) bonds; The proportion of α(1,6) linkages is the molar percentage of the respective α(1,6) linkages relative to the total number of glycosidic linkages as determined by the Hakomori method.

[0028] According to a preferred embodiment of the present invention, the substrate has a dextrose equivalent (DE) of 17-20, preferably 18-19, even more preferably about 18.4.

[0029] According to a preferred embodiment of the invention, the substrate has the characteristics set out in Table 1 below. It may for example be Glucidex 19D® sold by the Applicant Company.

[0030] In a preferred embodiment of the invention, the substrate is present in the reaction medium at a concentration of 50 g / g / L to 500 g / L, preferably 100 g / L to 200 g / L.

[0031] The two enzymes are used in series.

[0032] In one embodiment, the first enzyme is an α-glucanotransferase that can cleave α(1,4) glycosidic linkages but can also cleave α(1,4) glycosidic linkages to generate α(1,3) glycosidic linkages, and the second enzyme is an α-glucanotransferase that can cleave α(1,4) glycosidic linkages to generate α(1,6) glycosidic linkages.

[0033] In another embodiment, the first enzyme is an α-glucanotransferase capable of cleaving an α(1,4) glycosidic bond to produce an α(1,6) glycosidic bond, and the second enzyme is an α-glucanotransferase capable of cleaving an α(1,4) glycosidic bond to produce an α(1,3) glycosidic bond.

[0034] In a preferred embodiment of the invention, the α-glucanotransferase capable of hydrolyzing α(1,4) glycosidic bonds and generating α(1,6) glycosidic bonds is a protein having the sequence SEQ ID NO: 1 or a protein having at least 90% identity to the protein having the sequence SEQ ID NO: 1. Preferably, it is a protein having at least 91%, even more preferably at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% identity to the protein having the sequence SEQ ID NO: 1. SEQ ID NO: 1 corresponds to Genbank accession number WP_053069107.1.

[0035] In a preferred embodiment of the invention, the α-glucanotransferase capable of cleaving an α(1,4) glycosidic bond and generating an α(1,3) glycosidic bond is a protein having the sequence SEQ ID NO: 2 or a protein having at least 90% identity to a protein having the sequence SEQ ID NO: 2. Preferably, it is a protein having at least 91%, even more preferably at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% identity to a protein having the sequence SEQ ID NO: 2. SEQ ID NO: 2 corresponds to Genbank accession number AOR73699.1.

[0036] According to one embodiment of the present invention, each enzyme is added during successive incubations at a concentration of 0.01-1 mg / mL of reaction medium, preferably 0.05-0.5 mg / mL, even more preferably about 0.1 mg / mL of reaction medium.

[0037] According to one embodiment of the present invention, the substrate is contacted with each enzyme for 12 to 48 hours, preferably about 24 hours.

[0038] According to one embodiment of the present invention, the substrate and each enzyme are combined at a temperature between 20 and 40°C, preferably at about 37°C.

[0039] According to one embodiment of the present invention, the substrate and each enzyme are combined at a pH of 5 to 6.5, preferably 5.5 to 6, and even more preferably about 5.75.

[0040] According to one aspect, the present invention also relates to a mixture of α-glucans obtainable by the above-mentioned method.

[0041] This mixture of α-glucans is characterized by a low digestibility according to the AOAC 2002.02 method. Advantageously, the method according to the invention makes it possible to reduce the hydrolyzable fraction, measured according to the AOAC 2002.02 method, by at least 2-fold, preferably by at least 2.5-fold and even more preferably by at least 3-fold relative to the starting substrate.

[0042] The AOAC 2002.02 method can be specifically performed using the "HPAEC-PAD Assay" portion of the "Starch Resistance, K-RSTAR06 / 18" kit sold by Megazyme® Inc., as described in Example 1, Part 6 below.

[0043] The method according to the invention makes it possible to increase the proportion of α(1,6) bonds by at least 3-fold, preferably at least 4-fold, even more preferably at least 5-, 6-, 7- or 8-fold relative to the starting substrate.

[0044] The method according to the invention also makes it possible to generate an α(1,3) bond that was not present in the starting substrate.

[0045] The percentages of α(1,4), α(1,6), α(1,2), and α(1,3) linkages are measured by the Hakomori method (1964 HAKOMORI A Rapid Permethylation of Glycolipid, and Polysaccharide Catalysed by Methylsulfinyl Carbanion in Dimethyl Sulfoxide), as described in Example 1, Part 9 below.

[0046] According to one aspect, the present invention relates to an α-glucan mixture characterized by having the following: - Hydrolyzable fiber content less than 45%; and / or at least 20% (1,6) bonds, - and / or at least 3% α(1,3) bonds. Here, the fiber content corresponds to the hydrolyzable (i.e. non-resistant) fraction according to the AOAC 2002.02 method, and the proportion of α(1,6) and α(1,3) bonds represents the respective molar percentages of α(1,6) and α(1,3) bonds relative to the total number of glycosidic bonds, as determined by the Hakomori method.

[0047] Preferably, the content of hydrolysable fibres is less than 44% by weight, preferably less than 43% by weight, even more preferably less than 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30% by weight relative to the total weight of dry matter.

[0048] Preferably, the percentage of α(1,6) linkages is at least 21%, preferably at least 22%, even more preferably at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, the percentage of α(1,6) linkages being the molar percentage of α(1,6) linkages relative to the total number of glycosidic linkages as determined by the Hakomori method.

[0049] Preferably, the percentage of α(1,3) linkages is at least 4%, preferably at least 4%, at least 5%, at least 6%, at least 7% and at least 8%, the percentage of α(1,3) linkages being the respective molar percentage of α(1,3) linkages relative to the total number of glycosidic linkages as determined by the Hakomori method.

[0050] The present invention also relates to the use of a mixture of α-glucans obtained according to the above-mentioned method and having the above-mentioned properties for the preparation of a food product for human or animal nutrition.

[0051] Typically, the mixture of α-glucans of the present invention can be used to promote gut health, glycemic control, satiety and weight control, and sustained energy release.

[0052] Finally, in another aspect, the present invention relates to the sequential use of a glucanotransferase capable of cleaving α(1,4) glycosidic bonds to generate α(1,6) glycosidic bonds and a glucanotransferase capable of cleaving α(1,4) glycosidic bonds to generate α(1,3) glycosidic bonds to reduce the digestibility of a mixture of α-glucans. Preferably, the glucanotransferase has the sequence of SEQ ID NO: 1 or has at least 90% identity with a protein having the sequence of SEQ ID NO: 1. Preferably, the glucanotransferase capable of cleaving α(1,4) glycosidic bonds to generate α(1,6) glycosidic bonds has the sequence of SEQ ID NO: 1 or has at least 90% identity with a protein having the sequence of SEQ ID NO: 1. Preferably, the glucanotransferase capable of cleaving an α(1,4) glycosidic bond and generating an α(1,3) glycosidic bond has the sequence of SEQ ID NO:2 or has at least 90% identity to a protein having the sequence of SEQ ID NO:2.

[0053] Preferably, the reduction in digestibility is at least a 2-fold, preferably at least a 2.5-fold, even more preferably at least a 3-fold reduction in the hydrolyzable fraction measured according to the AOAC 2002.02 method.

[0054] The invention will be better understood with the aid of the following examples, which are intended to be illustrative and non-limiting.

[0055] Example 1: Preparation of branched maltodextrins from a mixture of oligosaccharides and polysaccharides: Materials and Methods 1 Preparation of a substrate solution containing a mixture of oligosaccharides and polysaccharides The starting substrate used was a mixture of oligo- and polysaccharides with the characteristics listed in Table 1.

[0056] [Table 1]

[0057] Solutions of the various substrates in 50 mM sodium acetate buffer (pH 5.75) were prepared at concentrations of 100 g / L, 200 g / L or 400 g / L.

[0058] 2. Production of recombinant enzymes. The following enzymes were recombinantly produced: - Enzyme GT#11: α-4,3 glucanotransferase from Lactobacillus fermentum NC2970, of the glycoside hydrolase family GH70, having as amino acid sequence the sequence listed in Genbank under the reference number AOR73699.1. - Enzyme GT#19: α-4,6 glucanotransferase from Lactobacillus mucosa, of the family of glycoside hydrolases GH70, having as amino acid sequence the sequence listed in Genbank under the reference number WP-053069107.1.

[0059] E. coli BL21 star (DE3) cells containing the plasmid pET-21 a-enzyme (to produce various enzymes including GT#11 and GT#19) were cultivated in ZYM-50524 medium containing 1% glycerol and 1% lactose. At the end of the cultivation, the cells were centrifuged at 6500g for 10 min, the cell pellet was resuspended in 20 mM phosphate buffer (pH 7.4) containing 300 mM NaCl and 20 mM imidazole at a DO of 80, and the cells were lysed by cold sonication using 4 cycles of 20 s at 30% amplitude followed by a 4 min pause. Cell debris was separated from the solubilized proteins by centrifugation at 10,000g for 30 min.

[0060] 3. Purified enzyme Purification of the protein of interest is performed using the divalent cobalt ion (CO) for which the polyhistidine tag has affinity. 2+) loaded on cobalt resin (Invitrogen). Elution was performed by creating a competition between the polyhistidine tag and increasing concentrations of imidazole. Briefly, 10-35 mL of cell extract of E. coli was contacted for 1 h with 1 mL of cobalt resin pre-equilibrated with 25 mL of 20 mM phosphate buffer (pH 7.4) containing 300 mM NaCl and 20 mM imidazole. All unbound proteins could be removed by filtering the resin on sintered glass. The resin was then washed five times with 40 mL of 20 mM phosphate buffer (pH 7.4) containing 300 mM NaCl and 20 mM imidazole. Finally, elution was performed with 3 mL of 20 mM phosphate buffer (pH 7.4) containing 300 mM NaCl and 250 mM imidazole for 5 min to isolate the enzyme of interest. The enzyme solution was then dialyzed (Sigma 10 kDa membrane) against 5 L of 50 mM sodium acetate buffer (pH 5.75) containing 150 mM NaCl (overnight at 4° C. with stirring) to remove NaCl and imidazole. The various protein solutions were assayed by measuring their absorbance at 280 nm using a 2000 spectrophotometer Nanodrop (Thermofisher). The molar extinction coefficient ε was determined using the ProtParam tool application of the ExPASy bioinformatics resource portal site.

[0061] Electrophoresis under denaturing conditions made it possible to control the quality of the purified enzyme extract. For this purpose, samples containing 30 μl of protein extract and 10 μl of loading buffer (NuPAGE LDS sample buffer 4x, Invitrogen) were denatured at 95 °C for 5 min and then deposited on a precast acrylamide gel (Mini-Protean Tris-Glycine exTender (Biorad)). The run was carried out for 30 min in 1x Tris / glycine / SDS buffer under a voltage of 150 V. The proteins were then revealed by incubating the gel in staining solution (PageBlue protein staining solution, Fermentas) for 1 h and then rinsing in three successive water baths for 30 min.

[0062] 4. Measurement of Branching Enzyme Activity The enzymatic activity of branching enzymes can be determined by measuring the initial rate of production of reducing sugars using the dinitrosalicylic acid (DNS) method. One enzyme unit represents the amount of enzyme that releases 1 pmol of fructose per minute at 30 °C for an initial sucrose concentration of 100 g.L-1 under buffer conditions of appropriate activity. During the kinetics of a volume of 1 mL, the reaction was stopped by removing 100 μL of reaction medium and adding an equal volume of DNS. The samples were then heated at 95 °C for 5 min, cooled in ice, diluted in half in water and the absorbance was read at 540 nm. A standard range of 0 to 2 g.L-1 of fructose allows to establish the relationship between the absorbance values ​​and the concentration of reducing sugars.

[0063] 5. Enzyme reaction Reactions were performed with 0.1 mg / mL of purified enzyme, either GT#11 or GT#19, and dialyzed in the presence of 10%, 20% or 40% substrate in 50 mM sodium acetate buffer (pH 5.75). Reactions were incubated at 20° C. or 37° C. for 24 h under stirring. Reactions were stopped by heating (95° C. for 5 min). Samples were taken at the beginning and end to analyze the specificity of the enzyme using different analytical techniques (HPAEC-PAD, NMR and HPSEC).

[0064] 6. Digestibility test The transposition reaction was frozen at -80°C for 24 hours and then lyophilized. 25 mg of the lyophilized product was dissolved in 1 mL of 100 mM sodium maleate buffer containing 30 U of pancreatic α-amylase and 3 U of amyloglucosidase (starch resistance kit, Megazyme K-STAR 06 / 18, which implements the AOAC 2002.02 method). The reaction was incubated at 37°C for 16 hours. Before HPAEC PAD analysis, the product was diluted with water.

[0065] 7. Chromatographic Analysis The obtained products were analyzed by anion exchange chromatography coupled to a pulsed amperometric detector (HPAEC PAD-HIGH Performance Anion Exchange Chromatography with Pulsed Amperometric Detection). The analysis was performed on a Thermo ICS6000 system equipped with a CarboPac™ PA100 analytical column (2 mm x 250 mm) coupled with a CarboPac™ PA100 guard precolumn (2 mm x 50 mm). A gradient of sodium acetate in 150 mM sodium 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, 450 mM. Detection was performed with a gold working electrode and a pHAg / AgCl reference cell. Samples were diluted to a total dry mass of 1 g.L-1 before injection. The size of the reaction products was also determined at intervals by 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 OH-Pak SB-G guard column precolumn, placed in the system's oven at 70 °C. The mobile phase was water at a flow rate of 0.3 mL.min-1. Detection was by refractive index measurement. Samples were diluted to a total dry mass of 20 g.L-1 before injection.

[0066] 8.NMR. Spectrum 1 H, 13 C and HSQC were recorded on a Bruker Avance 500 MHz instrument with a 5 mm Z-gradient H-BB-D BBI probe at 298 K. Data were acquired and processed using TopSpin 3 software.

[0067] 9.Hakomori method The Hakomori method (1964 HAKOMORI A Rapid Permethylation of Glycolipid,and Polysaccharide Catalysed by Methylsulfinyl Carbanion in Dimethyl Sulfoxide) allows the chemical characterization of glycosidic bonds by distinguishing between free OH groups and bond groups. It is a destructive method that includes the steps of methylation, hydrolysis, reduction with NaBD4, acetylation and analysis by mass spectrometry.

[0068] Example 2: Separate use of GT#11 and GT#19 enzymes In this example, the action of the GT#11 and GT#19 enzymes was tested separately.

[0069] The results of the various enzymatic reactions are shown in Table 2 below, which indicates the percentage of α-1,6, α-1,3 and α-1,4 linkages measured by proton NMR or the Hakomori method, as well as the percentage of hydrolysis (AOAC 2002.02) in the resulting reaction products.

[0070] [Table 2]

[0071] The inventors have observed that the enzyme GT#11 is able to reduce the proportion of linear α-1,4 bonds and increase the proportion of so-called "branched" α-1,3 and α-1,6 bonds.

[0072] On the other hand, the enzyme GT#19 was able to reduce the proportion of linear α-1,4 bonds and significantly increase the proportion of so-called "branched" α-1,6 bonds.

[0073] In both cases, an increased resistance to digestion (reflected by a decreased degree of hydrolysis) was observed, however the resulting products were not strong enough to be considered fibers.

[0074] Example 3: Simultaneous use of GT#11 and GT#19 enzymes In this example, we investigated the combined action of the two enzymes GT#11 and GT#19.

[0075] Therefore, the two enzymes were added simultaneously to the reaction mixture in various ratios as listed in the left column of Table 3.

[0076] The results of the various enzymatic reactions are shown in Table 3 below, which indicates the percentage of α-1,6, α-1,3 and α-1,4 linkages measured by proton NMR or the Hakomori method, as well as the percentage of hydrolysis (AOAC 2002.02) in the resulting reaction products.

[0077] [Table 3]

[0078] The inventors have observed that the simultaneous action of the enzymes GT#1 and GT#19 leads to a decrease in the proportion of linear α-1,4 bonds and an increase in the proportion of so-called "branched" α-1,3 and α-1,6 bonds. The results obtained are comparable or slightly inferior to the use of GT19 alone on 200 g / L of substrate.

[0079] This modification of the binding profile was observed to result in an increased resistance to digestion (reflected by a decreased degree of hydrolysis), however the resulting product is not sufficiently resistant (less than 40% hydrolysis according to the AGAC 2002.02 method) to be considered fiber.

[0080] Example 4: Sequential use of GT#11 and GT#19 enzymes In this example, we investigated the sequential action of two enzymes, GT#11 and GT#19.

[0081] Enzyme cascades represent a good strategy to increase the resistance of products to hydrolytic enzymes and achieve digestibility levels below 40%.

[0082] Within the framework of an enzyme cascade, the enzymes are used one after the other. Two different configurations of the two α-GTs were studied. -Glucidex19D is dissolved at 200 g.L-1 and the first α-GT is reacted at a concentration of 0.05 mg.mL-1 for 24 hours. The reaction is stopped by heating at 95°C for 5 minutes. The second enzyme is then reacted at the same concentration of 0.05 mg.mL-1. The reaction is stopped again by heating at 95°C for 5 minutes and incubating for 24 hours. -Glucidex19D 100g.L -1 First, 0.05 g / L of α-GT was added. -1 The reaction was stopped by heating at 95°C for 5 minutes. -1 Glucidex19D and then 0.05 gL of the second enzyme. -1 The reaction is stopped again by heating to 95° C. for 5 min and incubating for 24 h.

[0083] These various strategies make it possible to take advantage of the 4,3-α-glucanotransferase specificity of α-GT (No. 11) and to enhance its action compared to that of α-GT (No. 19). Indeed, in addition to the level of α-1,6 linkages, a non-negligible level of α-1,3 linkages can be achieved (Table 4).

[0084] We observed that a level of α-1,4 linkages of up to 50% was obtained under these conditions and that three types of glycosidic linkages (α-1,6, α-1,3 and α-1,4) were represented in the final product.

[0085] The results of the various enzymatic reactions are shown in Table 4 below, which shows the percentages of α-1,6, α-1,3 and α-1,4 linkages as determined by proton NMR or the Hakomori method, as well as the percentage of hydrolysis (AOAC 2002.02) in the resulting reaction products.

[0086] [Table 4]

[0087] The inventors have therefore demonstrated that the sequential use of the two enzymes makes it possible to obtain a product with less than 40% hydrolysis, regardless of the order of this sequence and with or without the addition of substrate between the two reactions. In other words, the sequential use of the α-glucanotransferases GT#11 and GT#19 made it possible to obtain a soluble fiber from a mixture of oligo- and polysaccharides with a DE of 19.

[0088] Example 5: Sequential use of GT#11 and GT#19 enzymes on a large scale In this example, we performed an upscaling to produce 1 g of fiber instead of the 50 mg produced in the previous example. 15 mL of 200 g.L-1 Glucidex 19D was subjected to a cascade reaction containing first α-GT (No. 11) for 24 h, followed by α-GT (No. 19) for 24 h. Each enzyme was used at 0.1 gL-1. Compared to the smaller volume equivalent test of Example 4, the same distribution in the types of bonds is obtained (Table 5).

[0089] [Table 5]

Claims

1. A method for preparing a mixture of α-glucans, comprising: - providing a substrate that is a mixture of oligosaccharides and polysaccharides having a polydispersity index of from -5 to 10, preferably from 6 to 9.5, more preferably from 7 to 9, from 8 to 8.5, and most preferably about 8.4; - a first incubation in the presence of a first enzyme; - a second incubation with a second enzyme; wherein - the first and second enzymes are α-glucanotransferases capable of cleaving α(1,4) glycosidic bonds to form α(1,3) glycosidic bonds and / or α-glucanotransferases capable of cleaving α(1,4) glycosidic bonds to form α(1,6) glycosidic bonds.

2. The substrate is - 40 to 50% oligosaccharides having a degree of polymerization (DP) of 1 to 9, - 15 to 20% polysaccharides having a DP of 10 to 20, - 35 to 40% polysaccharides having a DP greater than 20, where the percentages are expressed as relative percentages by mole and the total is 100%, according to the method of claim 1.

3. The substrate has a dextrose equivalent (DE) of 18 to 20, preferably 18 to 19, more preferably about 18.4, according to the method of claim 1.

4. The substrate is introduced at a concentration of 50 g / L to 500 g / L, preferably 100 g / L to 200 g / L, of the reaction medium, according to the method of claim 1.

5. The substrate is added during the first and second incubations, according to the method of claim 1.

6. The first enzyme is an α-glucanotransferase capable of cleaving the α(1,4) glycosidic bond to form an α(1,3) glycosidic bond, and the second enzyme is an α-glucanotransferase capable of cleaving the α(1,4) glycosidic bond to form an α(1,6) glycosidic bond, according to the method of claim 1.

7. The first enzyme is an α-glucanotransferase capable of cleaving the α(1,4) glycosidic bond to form an α(1,6) glycosidic bond, and the second enzyme is an α-glucanotransferase capable of cleaving the α(1,4) glycosidic bond to form an α(1,3) glycosidic bond, according to the method of claim 1.

8. ​ The method according to claim 1, wherein the α-glucanotransferase capable of cleaving the α(1,4) glycosidic bond to generate an α(1,6) glycosidic bond is a protein having the sequence of SEQ ID NO: 1, or a protein having at least 90% identity with the protein having SEQ ID NO:

1.

9. The method according to claim 1, wherein the α-glucanotransferase capable of cleaving the α(1,4) glycosidic bond to generate an α(1,3) glycosidic bond is a protein having the sequence of SEQ ID NO: 2, or a protein having at least 90% identity with the protein having the sequence of SEQ ID NO:

2.

10. The method according to claim 1, wherein each enzyme is at a concentration of 0.01 to 1 mg / mL, preferably 0.05 to 0.5 mg / mL, and even more preferably about 0.1 mg / mL of the reaction medium.

11. The method according to claim 1, wherein each incubation is carried out for a period of 12 to 48 hours, preferably about 24 hours, and / or at a temperature of 20 to 40 °C, preferably about 37 °C, and / or at a pH of 5 to 6.5, preferably about 5.

75.

12. A mixture of α-glucans obtainable by the method according to any one of claims 1 to 11.

13. A mixture of α-glucans, - having a content of hydrolyzable fiber of less than 45% by weight based on the total weight of the dry matter, - and / or at least 20% of α(1,6) bonds, - and / or at least 3% of α(1,3) bonds, characterized in that the fiber content corresponds to the hydrolyzable (i.e., non-resistant) fraction by the AOAC 2002.02 method, and the proportions of α(1,6) and α(1,3) bonds represent the respective molar percentages of α(1,6) and α(1,3) bonds with respect to the total number of glycosidic bonds measured by the Hakomori method, a mixture of α-glucans.

14. Use of the mixture of α-glucans according to claim 13 for the preparation of a food for human or animal nutrition.

15. Sequential use of a glucanotransferase capable of cleaving an α(1,4) glycosidic bond to produce an α(1,6) glycosidic bond and a glucanotransferase capable of cleaving an α(1,4) glycosidic bond to produce an α(1,3) glycosidic bond for reducing the digestibility of a mixture of α-glucans, wherein each of the glucanotransferases has the sequence of SEQ ID NO: 1 or a protein having at least 90% identity with the protein having SEQ ID NO: 1, and a protein having at least 90% identity with the protein having SEQ ID NO: 2 or SEQ ID NO: 2.