Method for enzymatic production of soluble fibers

Abstract

The present invention relates to a process for preparing a non-digestible alpha-glucan mixture from a substrate rich in oligosaccharides having a degree of polymerization (DP) of 4.

Description

Technical Field

[0001] The present invention relates to a method for preparing a non-digestible α-glucan mixture from a substrate rich in oligosaccharides with a degree of polymerization (DP) of 4. In the present application, the substrate designates a syrup containing oligosaccharides, wherein the content of oligosaccharides with a degree of polymerization (DP) of 4 is at least 40%, preferably at least 45%, even more preferably at least 50%.

[0002] The present invention also relates to mixtures of non-digestible alpha-glucans.

[0003] The present invention also relates to the use of an α-glucanotransferase capable of producing α(1,6) glycosidic bonds for reducing the digestibility of an α-glucan mixture. Prior art

[0004] Dietary fiber plays an important role in human nutrition. Among dietary fibers, there is a distinction between soluble fibers that are soluble in water and have gelling ability and insoluble fibers. Soluble fibers (including branched maltodextrins) are particularly advantageous because they are not easily digestible. Therefore, incorporating them into the diet can reduce the glycemic index of food and prolong satiety. They also have prebiotic properties for intestinal flora; in other words, they can selectively promote the growth of certain probiotic types of bacteria or the activity of microflora, providing health benefits.

[0005] So far, soluble fibers including branched maltodextrins have been obtained mainly by physicochemical methods.

[0006] In particular, the applicant company uses the trade name The case of maltodextrin sold by FM10 as a water-soluble fiber.

[0007] There are other soluble fibers obtained by physicochemical methods, such as those sold by Tate and Lyl. Marketed by Dupont Nutrition and Biosciences or

[0008] Many studies have demonstrated that digestibility properties are directly related to the percentage of various types of glycosidic bonds within soluble fiber.

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

[0010] By increasing the percentage of α1→6 bonds or α1→3 bonds, the degree of branching of maltodextrins increases, which makes them more resistant to digestion.

[0011] Enzymatic methods using enzymes capable of promoting "branched" type bond formation have many advantages in terms of safety and environmental protection and also offer better specificity.

[0012] Initially, most enzymatic processes for producing soluble fibers were performed using sucrose as a substrate for the enzymes in order to create new bonds. For example, WO2015183714 describes an enzymatic reaction performed with a mixture of sucrose and α-glucan-type substrates.

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

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

[0015] The Applicant Company has then discovered that the target fibers in human and animal nutrition can be obtained by an enzymatic process from a syrup enriched in oligosaccharides having a degree of polymerization (DP) of 4. The Applicant Company has therefore developed a process using specific enzymes which enables the generation of α(1,6) bonds from a syrup enriched in DP4 oligosaccharides.

[0016] In a first aspect, the present invention relates to a method for preparing an α-glucan mixture (preferably a branched maltodextrin mixture), the method comprising the steps of mixing together a substrate and an enzyme, wherein the substrate is a syrup rich in oligosaccharides having a degree of polymerization (DP) of 4, and the enzyme is an α-glucanotransferase capable of cleaving α(1,4) glycosidic bonds and generating α(1,6) glycosidic bonds.

[0017] According to the present invention, the terms "α-glucan", "soluble fiber", "food soluble fiber" are used interchangeably. They define an oligosaccharide consisting of at least 3 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, usually expressed as the concept of "dextrose equivalent" (DE). In this regard, the definition of maltodextrin given in the Monograph Specifications of the Food Chemical Codex stipulates that the DE value of maltodextrin shall not exceed 20. Above 20, these are glucose syrups.

[0019] However, such DE measurements are not sufficient to accurately represent the molecular distribution of α-glucans. In fact, acid hydrolysis of completely random starch or enzymatic hydrolysis of slightly more ordered starch provides a mixture of glucose and glucose polymers that cannot be precisely defined using DE measurements alone and that contains short-sized, low-DP molecules as well as very long-sized, high-DP molecules.

[0020] In fact, the measurement of DE only gives an approximate idea of ​​the average DP of the mixture of constituent glucose polymers of glucose and α-glucan and hence their number average molecular weight (Mn). In order to complete the characterization of the molecular weight distribution of α-glucans, it is important to determine another parameter, namely, the weight average molecular weight (Mw).

[0021] In practice, (Mn) and (Mw) are experimentally determined by means of different analytical techniques such as, for example, the measurement method adapted for glucose polymers based on gel permeation chromatography on a column calibrated with pullulan of known molecular weight.

[0022] The Mw / Mn ratio is called the polynomial index (PI) and can characterize the overall molecular weight distribution of a polymer blend. Typically, the molecular weight distribution of standard maltodextrins results in IP values ​​between 5 and 10.

[0023] These various parameters are also a reflection of the α-glycosidic bond distribution of the α-glucan. In fact, a mixture of standard α-glucans has a very high percentage of "straight chain" α(1,4) bonds (greater than 90%) and a low percentage of so-called "branched" (α(1,2) bonds, α(1,3) bonds and α(1,6) bonds).

[0024] The process according to the invention makes it possible to reduce the percentage of α(1,4) bonds relative to α(1,6) bonds, which has the advantage of reducing the digestibility of the α-glucan mixture obtained by this process.

[0025] The α-glucan mixture prepared according to the method of the present invention is preferably a branched maltodextrin mixture.

[0026] For the purposes of the present invention, branched maltodextrin is understood to be a maltodextrin having a higher content of α(1,6) glycosidic bonds than standard maltodextrin.

[0027] Standard maltodextrins are defined as a purified and concentrated mixture of glucose and glucose polymers linked essentially at α(1,4) with only 4% to 5% α(1,6) glycosidic bonds, of widely varying molecular weight, completely soluble in water and with low reducing power.

[0028] According to one embodiment of the invention, the syrup enriched in DP4 oligosaccharides comprises at least 40%, preferably at least 45%, even more preferably at least 50% of DP4 oligosaccharides.

[0029] According to one embodiment of the present invention, the syrup enriched with oligosaccharides having a DP of 4 has a dextrose equivalent (DE) greater than 20.

[0030] According to a preferred embodiment of the present invention, the DP4-enriched syrup is a syrup having the characteristics as described in Table 1 below.

[0031] In a preferred embodiment of the invention, the substrate is present in the reaction medium in a concentration between 50 g / L and 500 g / L, preferably between 100 g / L and 200 g / L.

[0032] In a preferred embodiment of the present invention, the α-glucanotransferase capable of cleaving α(1,4) glycosidic bonds and producing α(1,6) glycosidic bonds is a protein 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 (hereinafter referred to as GT#19). 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 with the protein having the sequence SEQ ID No: 1. The sequence SEQ ID No: 1 corresponds to Genbank Accession No. WP_053069107.1.

[0033] As shown in the examples, the inventors have demonstrated that the enzyme GT#19 is able to modify DP4-rich syrup to render it non-digestible, with a hydrolysis percentage less than or equal to 45% according to method AOAC 2002.02).

[0034] According to one embodiment of the invention, the enzyme is added in a concentration between 0.01 mg / mL and 1 mg / mL reaction medium, preferably between 0.05 mg / mL and 0.5 mg / mL reaction medium, even more preferably about 0.1 mg / mL reaction medium.

[0035] According to one embodiment of the invention, the substrate and the enzyme are contacted for a period of between 12 hours and 48 hours, preferably about 24 hours.

[0036] According to one embodiment of the invention, the substrate and the enzyme are mixed together at a temperature between 20°C and 40°C, preferably about 37°C.

[0037] According to one embodiment of the invention, substrate and enzyme are mixed together at a pH between 5 and 6.5, preferably between 5.5 and 6, even more preferably about 5.75.

[0038] In one embodiment of the present invention, the method further comprises the step of enzymatic treatment by an α-glucanotransferase capable of cleaving α(1,4) glycosidic bonds and producing α(1,3) glycosidic bonds. For example, it can be a protein having the sequence SEQ ID No:2 or a protein having at least 90% identity with the protein having the sequence SEQ ID No:2 (hereinafter referred to as GT#11). 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 with the protein having the sequence SEQ ID No:2. The sequence SEQ ID No:2 corresponds to Genbank accession number AOR73699.1.

[0039] According to one aspect, the present invention also relates to an α-glucan mixture, preferably a branched maltodextrin mixture, obtainable by the process described above.

[0040] This alpha-glucan mixture is characterized by its low digestibility according to the AOAC 2002.02 method. Advantageously, the process according to the invention makes it possible to reduce the hydrolyzable fraction measured according to the AOAC 2002.02 method by at least 2 times, preferably at least 2.5 times, even more preferably at least 3 times relative to the starting substrate.

[0041] AOAC 2002.02 method can be used specifically by the company The assay was performed in the "HPAEC-PAD assay" section of the "Starch resistance, K-RSTAR06 / 18" kit sold by ELISA, as described in part 5 of Example 1 below.

[0042] The process according to the invention makes it possible to increase the percentage of α(1,6) bonds by at least 3 times, preferably at least 3.5 times, even more preferably at least 4 times, relative to the starting substrate.

[0043] The percentage of α(1,6) bonds can be measured by the Hakomori method (1964 HAKOMORI Rapid and complete methylation of glycolipids and polysaccharides 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.

[0044] According to one aspect, the present invention relates to an α-glucan mixture, preferably a branched maltodextrin mixture, characterized in that the α-glucan mixture has:

[0045] - a content of less than 55%, preferably less than 50%, even more preferably less than 45% of hydrolyzable fibers,

[0046] - and / or at least 20% α(1,6) bonds,

[0047] wherein the fiber content corresponds to the hydrolyzable (ie, non-resistant) fraction according to the AOAC 2002.02 method, and the percentage of α(1,6) bonds represents the molar percentage of α(1,6) bonds relative to the total number of glycosidic bonds measured by the Hakomori method.

[0048] Preferably, the content of hydrolyzable fibers is less than 44%, preferably less than 43%, even more preferably less than 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%.

[0049] Preferably, the content of hydrolyzable fibers is greater than 5%, preferably greater than 10%, 11%, 12%, 13%, 14%, 15%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%.

[0050] Preferably, the content of hydrolyzable fibers is between 5% and 45%, preferably between 10% and 45%, preferably between 20% and 44%, even more preferably between 30% and 45%.

[0051] Preferably, the percentage of α(1,6) bonds 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%.

[0052] Preferably, the percentage of α(1,6) bonds is at most 40%, preferably at most 36%, at most 35%, at most 34%, at most 33%, at most 32%, at most 31%.

[0053] Preferably, the percentage of α(1,6) bonds is between 20% and 40%, preferably between 20% and 35%, more preferably between 25% and 35%.

[0054] Preferably, the percentage of α(1,3) bonds is at least 2%, preferably at least 3%.

[0055] Preferably, the percentage of α(1,3) bonds is at most 6%, preferably at most 5%.

[0056] Preferably, the percentage of α(1,3) bonds is between 2% and 8%, preferably between 3% and 5%.

[0057] Preferably, the percentage of α(1,2) bonds is at least 1%, preferably at least 2%.

[0058] Preferably, the percentage of α(1,2) bonds is at most 8%, preferably at most 6%, at most 5%, at most 4% or at most 3%.

[0059] Preferably, the percentage of α(1,4) bonds is at most 80%, preferably at most 70%, at most 65%.

[0060] Preferably, the percentage of α(1,4) bonds is at least 50%, preferably at least 55%, at least 60%.

[0061] Preferably, the percentage of α(1,4) bonds is between 50% and 80%, preferably between 55% and 70%, more preferably between 55% and 65%.

[0062] Preferably, the α-glucan mixture, preferably a branched maltodextrin mixture, is characterized in that the α-glucan mixture has:

[0063] - a content of less than 55%, preferably less than 50%, even more preferably less than 45% of hydrolyzable fibers,

[0064] - and has one or more of the following characteristics:

[0065] - between 20% and 40% α(1,6) bonds,

[0066] - between 20% and 40% α(1,3) bonds,

[0067] - between 2% and 8% α(1,2) bonds,

[0068] - between 50% and 80% α(1,4) bonds,

[0069] wherein the fiber content corresponds to the hydrolyzable (i.e., non-resistant) fraction according to the AOAC 2002.02 method, and the percentage of α(1,6) bonds, α(1,3) bonds, α(1,2) bonds, or α(1,4) bonds represents the molar percentage of said type of bonds relative to the total number of glycosidic bonds as measured by the Hakomori method.

[0070] Preferably, the content of α(1,6) bonds, α(1,3) bonds, α(1,2) bonds or α(1,4) bonds is such that the sum of their molar percentages is equal to 100%.

[0071] The present invention also relates to the use of the α-glucan mixture obtained according to the process described above and the α-glucan mixture having the properties described above in the preparation of food for human or animal nutrition.

[0072] In general, the alpha-glucan mixture according to the present invention can be used to promote intestinal health, blood sugar management, satiety and weight management, and sustained energy release.

[0073] Finally, in another aspect, the present invention relates to the use of a glucanotransferase capable of cleaving α(1,4) glycosidic bonds and generating α(1,6) glycosidic bonds in order to reduce the digestibility of an α-glucan mixture, the 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.

[0074] In this aspect of the invention, the α-glucan mixture is preferably a syrup rich in oligosaccharides, in particular a syrup rich in oligosaccharides having a DP of 4 as described in the first aspect of the invention.

[0075] Preferably, the reduction in digestibility is at least 2-fold, preferably at least 2.5-fold, even more preferably at least 3-fold, reduction in the hydrolyzable fraction measured according to the AOAC 2002.02 method relative to the starting substrate.

[0076] The invention will be better understood with the aid of the following examples, which are intended to be illustrative and non-limiting.

[0077] Example 1: Preparation of branched maltodextrins from DP4-enriched syrup: Materials and methods

[0078] 1. Preparation of DP4 substrate solution

[0079] The starting substrate used was a syrup rich in DP4, having the characteristics described in Table 1:

[0080] [Table 1]

[0081] standard Sub-criteria Measurement method unit value Molecular weight Mn(pulp starch equivalent) MCL1439 Da 615 Molecular weight Mw (pulp starch equivalent) MCL1439 Da 1045 Carbohydrate distribution DP1 MCL190A relatively% 2.5 Carbohydrate distribution DP2 MCL190A relatively% 6.1 Carbohydrate distribution DP3 MCL190A relatively% 7.4 Carbohydrate distribution DP4 MCL190E relatively% 55.7 Carbohydrate distribution DP5 MCL190E relatively% 2.3 Carbohydrate distribution DP6 MCL190E relatively% 2.1 Carbohydrate distribution DP7 MCL190E relatively% 2.3 Carbohydrate distribution DP8 MCL190E relatively% 5.5 Carbohydrate distribution DP9 MCL190E relatively% 1.4 Carbohydrate distribution DP10 MCL190E relatively% 1.3 Carbohydrate distribution >DP10 MCL190E relatively% 13.4 Reducing sugar Dextrose Equivalent (DE) MCL050B 33.4 Dry Ingredients Mass loss after drying MCL209A ％ 30.2

[0082] Various substrate solutions (DP4-enriched syrup) were prepared at concentrations of 100 g / L, 200 g / L, or 400 g / L in 50 mM sodium acetate buffer (pH 5.75).

[0083] 2. Production of Recombinant Enzyme .

[0084] The following enzymes were recombinantly produced;

[0085] - Enzyme GT#11: α-4,3 glucanotransferase from Lactobacillus fermentum NC2970, the amino acid sequence of which has the sequence listed in Genbank under the reference number AOR73699.1 (SEQ ID No: 2).

[0086] - Enzyme GT#19: Glycoside hydrolase GH70 from Lactobacillus mucosae, the amino acid sequence of which has the sequence listed in Genbank under the reference number WP_053069107.1. (SEQ ID No: 1)

[0087] E. coli BL21 star cells containing plasmid pET-21a-enzyme (No.: X) were cultured in ZYM-5052 medium containing 1% glycerol and 1% lactose (for the production of various enzymes, including GT#11 and GT#19). At the end of the culture, the cells were centrifuged at 6500 g for 10 minutes, the cell pellet was resuspended in 20 mM phosphate buffer (pH 7.4) containing 300 mM NaCl and 20 mM imidazole at DO600nm of 80, and the cells were lysed by cold sonication using 30% amplitude, 4 cycles of 20 seconds, followed by standing for 4 minutes. Cell debris was separated from the dissolved protein by centrifugation at 10,000 g for 30 minutes.

[0088] 3. Enzyme Purification

[0089] Purification of the target protein was performed on a cobalt resin (Invitrogen) loaded with divalent cobalt ions (CO2+), to which the polyhistidine tag has an affinity. Elution was performed by creating competition between the polyhistidine tag and increasing concentrations of imidazole. In brief, 10 mL to 35 mL of Escherichia coli (E.coli) cell extract was contacted with 1 mL of cobalt resin for 1 hour, which was pre-balanced with 25 mL of 20 mM phosphate buffer (pH 7.4) containing 300 mM NaCl and 20 mM imidazole. Filtering the resin on a porous glass can remove all unbound proteins. 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, 3 mL of 20 mM phosphate buffer (pH 7.4) containing 300 mM NaCl and 250 mM imidazole was used to elute for 5 minutes to separate the target enzyme. The enzyme solution was then dialyzed (10 kDa molecular weight cut-off membrane) with 5 L 50 mM sodium acetate buffer (pH 5.75) (overnight, stirred at 4 ° C) to remove NaCl and imidazole. 2000 ultra-micro spectrophotometers (Thermofisher) were used to measure their absorbance at 280 nm, thereby measuring various protein solutions. The ProtParam tool application of the ExPASy bioinformatics resource portal website was used to measure the molecular extinction coefficient ε.

[0090] 4. Enzymatic reaction

[0091] The 0.1 mg / mL purified enzyme was reacted and dialyzed in 50 mM sodium acetate buffer (pH 5.75) in the presence of 10%, 20% or 40% substrate. The reactant was stirred and incubated at 37 ° C for 24 hours. The reaction was terminated by heating (95 ° C for 5 minutes). Samples were taken at the initial time and the final time to analyze the specificity of the enzyme using different analytical techniques (HPAEC-PAD, NMR).

[0092] 5. Digestibility Test

[0093] The transfer reaction was frozen at -80°C for 24 hours and then freeze dried. 25 mg of the freeze dried product was placed in 1 mL of 100 mM sodium maleate buffer containing 30 U pancreatic alpha-amylase and 3 U amyloglucosidase (starch resistance kit, Megazyme K-STAR 06 / 18, which implements AOAC 2002.02 method). The reaction was incubated at 37°C for 16 hours. The product was diluted in water before HPAEC PAD analysis.

[0094] 6. Chromatographic analysis

[0095] 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). TM CarboPac with Pa100 guard pre-column (2mm×50mm) TM The analysis was performed on a Thermo ICS6000 system with a PA100 analytical column (2 mm × 250 mm). A sodium acetate gradient in 150 mM sodium was applied at a flow rate of 0.250 ml.min-1 according to the following curve: 0 min-5 min, 0 mM; 5 min-35 min, 0 mM-300 mM; 35 min-40 min, 300 mM-450 mM; 40 min-42 min, 450 mM. Detection was performed using a gold working electrode and a pH Ag / AgCl reference cell. The samples were diluted to a total dry mass of 1 g.L-1 before injection.

[0096] 7.NMR .

[0097] spectrum 1 H. 13 C and HSQC were recorded on a Bruker Avance 500 MHz instrument with a 5 mm Z-gradient H-BB-DBBI probe at 298 K. Data were acquired and processed using TopSpin 3 software.

[0098] 8.Hakomori method

[0099] The Hakomori method (1964HAKOMORI Rapid and complete methylation of glycolipids and polysaccharides catalyzed by methylsulfinyl carbanion in dimethyl sulfoxide) enables the chemical characterization of glycosidic bonds by distinguishing between free OH groups and bonded groups. This is a destructive method that includes the steps of methylation, hydrolysis, reduction with NaBD4, acetylation and mass spectrometry analysis.

[0100] Example 2: Preparation of branched maltodextrin from DP4-enriched syrup: Results

[0101] The results of various enzymatic reactions are shown in Table 2 below, which shows the percentages of α-1,6 bonds; α-1,3 bonds and α-1,4 bonds measured by proton NMR or by the Hakomori method, and the hydrolysis percentage in the obtained reaction products (AOAC 2002.02).

[0102] [Table 2]

[0103]

[0104] RM: Raw material, = DP4 syrup

[0105] The inventors have demonstrated that the enzyme GT#19 is able to modify DP4-rich syrup to render it non-digestible (percent hydrolysis according to method AOAC 2002.02 less than or equal to 45%).

[0106] The product obtained by this enzymatic treatment with enzyme GT#19 contains significantly fewer α-1,4 bonds and more α-1,6 bonds than the starting point. The number of α-1,2 bonds and α-1,3 bonds remains unchanged. Therefore, enzyme GT#19 is a 4,6-α-glucanotransferase.

[0107] In contrast, treatment with another GT (enzyme GT#11) resulted in a decrease in the percentage of α-1,4 bonds and the appearance of α-1,3 bonds. The digestibility of the product obtained by treatment with enzyme GT#11 was also reduced (53% and 55% at initial concentrations of 100 mg / mL and 200 mg / mL, respectively, compared to 84% and 88% of the untreated substrate).

[0108] Advantageously, the alpha-glucan mixture according to the invention also has a target in vitro digestibility profile according to the Englyst method.

Claims

1. A method for preparing an α-glucan mixture, the method comprising the steps of mixing a substrate and an enzyme together, the substrate being a syrup rich in oligosaccharides having a degree of polymerization (DP) of 4, and the enzyme being an α-glucanotransferase capable of cleaving α(1,4) glycosidic bonds and generating α(1,6) glycosidic bonds.

2. The method according to claim 1, wherein the syrup enriched in oligosaccharides with a DP of 4 comprises at least 40%, preferably at least 45%, even more preferably at least 50% of oligosaccharides with a DP of 4.

3. The method according to claim 1 or 2, wherein the dextrose equivalent (DE) of the syrup enriched in oligosaccharides having a DP of 4 is greater than 20.

4. The method according to any one of the preceding claims, wherein the concentration of the substrate is between 50 g / L reaction medium and 500 g / L reaction medium, preferably between 100 g / L reaction medium and 200 g / L reaction medium.

5. The method according to any one of the preceding claims, wherein the α-glucanotransferase capable of cleaving α(1,4) glycosidic bonds and capable of generating α(1,6) glycosidic bonds is a protein 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.

6. The method according to any of the preceding claims, wherein the concentration of the enzyme is between 0.01 mg / mL and 1 mg / mL reaction medium, preferably between 0.05 mg / mL and 0.5 mg / mL reaction medium, even more preferably about 0.1 mg / mL reaction medium.

7. A method according to any one of the preceding claims, characterised in that The substrate and the enzyme are mixed together for a period of between 12 hours and 48 hours, preferably about 24 hours, and / or at a temperature comprised between 20°C and 40°C, preferably about 37°C, and / or at a pH between 5 and 6.5, preferably about 5.

75.

8. A method according to any one of the preceding claims, characterised in that The method further comprises the step of enzymatic treatment by an α-glucanotransferase capable of cleaving α(1,4) glycosidic bonds and generating α(1,3) glycosidic bonds.

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

2.

10. An alpha-glucan mixture obtainable by a process according to any one of the preceding claims.

11. An α-glucan mixture, characterized in that The α-glucan mixture has: - a content of less than 55%, preferably less than 50%, even more preferably less than 45% of hydrolyzable fibers, - and / or at least 20% α(1,6) bonds, The fiber content corresponds to the hydrolyzable portion according to the AOAC 2002.02 method, and the percentage of α(1,6) bonds indicates the molar percentage of α(1,6) bonds relative to the total number of glycosidic bonds measured by the Hakomori method.

12. Use of the alpha-glucan mixture according to any one of claims 10 or 11 for the preparation of a food for human or animal nutrition.

13. Use of a glucanotransferase capable of cleaving α(1,4) glycosidic bonds and generating α(1,6) glycosidic bonds in order to reduce the digestibility of an α-glucan mixture, the glucanotransferase having the sequence SEQ ID No: 1 or a sequence having at least 90% identity with the protein having the sequence SEQ ID No: 1.

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