A method for producing alternan-oligosaccharides

Abstract

A method (S1) for producing alternan-oligosaccharides (8), comprising: contacting sucrose (9) with a catalytically effective amount of alternan sucrase (13) and an acceptor molecule (12) in a reactor (11), wherein the alternan sucrase (13) and the acceptor molecule (12) are present in the reactor (11) in an aqueous liquid (4), the sucrose (9) is continuously or semi-continuously fed into the reactor (11), and the sucrose (9) and the acceptor molecule (12) are converted into alternan-oligosaccharides (8), and fructose (6) is formed as a by-product; and continuously or semi-continuously removing at least a portion of the fructose (6) from the reactor (11) by membrane filtration (17).

Description

Technical Field

[0001] The present invention relates to a method for producing alternan-oligosaccharides. Background Art

[0002] Alternan-oligosaccharides are known as prebiotic ingredients. U.S. Patent 7,182,954 discloses that oligosaccharides produced by the reaction of sucrose with various acceptor sugars catalyzed by the enzyme alternansucrase can be effective as prebiotics to control enteric bacterial pathogens. By treating animals with a composition comprising one or more of these oligosaccharides in an amount effective to promote the growth of beneficial bacteria (e.g., lactobacilli, bifidobacteria), the number of enteric pathogenic bacteria can be significantly reduced or inhibited.

[0003] WO0047727A2 provides a nucleic acid molecule encoding alternansucrase. In addition, vectors, host cells and plant cells transformed by the nucleic acid molecule and plants containing them are provided. In addition, because the nucleic acid molecule encoding alternansucrase is inserted, a method for preparing a transgenic plant synthesizing the carbohydrate alternan is described. In addition, a method for preparing alternan and a product produced by the method are provided.

[0004] WO2009095278A2 relates to the use of alternan polysaccharides and alternan oligosaccharides as acidic food ingredients, and acidic foods comprising alternan as an ingredient. The disclosure also relates to the use of alternan as a heat-stable ingredient in food preparations, and foods comprising alternan as an ingredient, wherein the food is subjected to a heating step during its manufacture.

[0005] WO0047727A2 and WO2009095278A2 suggest producing alternan-oligosaccharides by a process wherein

[0006] a) contacting a solution containing sucrose with a catalytically effective amount of alternansucrase and an acceptor molecule under conditions that allow conversion of the sucrose to alternan-oligosaccharides and fructose; and

[0007] b) Separation of alternan-oligosaccharides and fructose from the solution.

[0008] The reaction is carried out in a batch reactor containing all educts and can be carried out between room temperature and 37°C and at a pH between about 4.7 and 7, and the reaction can be carried out until the sucrose is substantially consumed. The product is usually obtained in the form of a slurry, which can be further purified (i.e., by membrane filtration) and / or dried.

[0009] A problem with this process is the accumulation of by-products. Furthermore, the current methods cannot be used when preparing long-chain maltoalternan oligosaccharides, since the desired average chain length cannot or can hardly be achieved using the current methods.

[0010] Purpose of the Invention

[0011] It is an object of the present invention to provide an alternative process for producing alternan-oligosaccharides. Preferably, the process minimizes by-products and / or allows for better removal of by-products. Summary of the invention

[0012] The present invention provides the method according to claim 1.

[0013] The present invention provides a method for producing alternan-oligosaccharides, comprising

[0014] Sucrose is contacted with a catalytically effective amount of alternansucrase and an acceptor molecule in a reactor, wherein

[0015] The alternansucrase and the receptor molecule are present in an aqueous liquid in the reactor, the sucrose is fed continuously or semi-continuously into the reactor, and

[0016] The sucrose and the acceptor molecule are converted into alternan-oligosaccharides, with fructose being formed as a by-product;

[0017] At least a portion of the fructose is continuously or semi-continuously removed from the reactor by membrane filtration.

[0018] By the present invention, in its general form or in specific embodiments, one or more of the following benefits may be achieved.

[0019] Low fructose concentrations can always be achieved.

[0020] The formation of other unwanted by-products, such as alternan polymers or leucrose, can be reduced or minimized.

[0021] Fructose is not necessarily the only by-product. Other possible by-products are alternan polymers or leucrose, which can also be removed as described further below.

[0022] Compared to previous methods, longer chain alternan-oligosaccharides can be produced. The chain length can be adjusted via the amount of sucrose added.

[0023] The invention provides an alternan-oligosaccharide obtained by the method.

[0024] Further benefits are described in the specific examples included in the specification.

[0025] Detailed description

[0026] Alternan-oligosaccharides:

[0027] The degree of polymerization (DP) or the average degree of polymerization (ie the weight-average DPw) is determined by GPC-RI (gel permeation chromatography with refractive index detection) or by the alternative method HPAEC-PAD (high performance anion exchange chromatography with pulsed amperometric detection).

[0028] Alternan is a saccharide consisting of glucose units, or a saccharide consisting essentially of glucose units (in the presence of an acceptor molecule containing a structure other than glucose or glucose units). Glucose units are interconnected by α-1-3- and α-1-6-glycosidic bonds, and these two types of bonds mainly appear alternately. If there is an acceptor molecule containing glucose (one or more units), such as maltose, these can be connected in other ways. Alternan can contain branches (Seymour et al., Carbohydrate Research 74, (1979), 41-62). Alternan-polysaccharides and alternan-oligosaccharides are types of alternans, which differ in their degree of polymerization. Alternan-polysaccharide molecules may or may not contain acceptor molecules, preferably contain acceptor molecules.

[0029] Alternan in the present invention, in particular alternan-oligosaccharide, can be defined as a glucose-based sugar, wherein such sugar has a reducing end and D-glucose monomers linked by alternating α-1-6 and α-1-3 glycosidic bonds, wherein an acceptor molecule (also called "acceptor molecule unit", in particular a maltose unit) is present at the reducing end or in other words linked to the reducing end.

[0030] The alternan-oligosaccharide molecule preferably comprises one acceptor molecule.

[0031] The weight average degree of polymerization DPw of the alternan-oligosaccharides of the invention produced by the method of the invention is preferably 5-30, preferably 5-25, more preferably 10-30, or 10-20, still more preferably 10-18 or 12-18. A more preferred DPw range is 18-30 or 20-30. These values ​​are measured by GPC-RI. A detailed method description is given in the Examples section.

[0032] The average degree of polymerization DPw of the alternan-oligosaccharides of the present invention produced by the method of the present invention is preferably 7-32, 12-30, 12-20 or 12-18 as measured by HPAEC-PAD. A detailed description of the method is given in the Examples section. The average degree of polymerization may be greater than 13, 14, 15 or 16 (HPAEC-PAD). In some embodiments, the average degree of polymerization is greater than 17 (HPAEC-PAD). The average degree of polymerization may be less than 20, 19 or 18 (HPAEC-PAD).

[0033] The GPC-RI values ​​and HPAEC-PAD values ​​mentioned above are independent of each other. This means that the values ​​and ranges given above are not assigned to each other when comparing different methods. Products with different DPw can be produced, the DPw can be controlled, and the DPw of different products can be measured with different methods.

[0034] In the present invention, the singular term "alternan-oligosaccharide" denotes monodisperse alternan-oligosaccharides having molecules of only one degree of polymerization (DP) as well as polydisperse alternan-oligosaccharides having molecules of different degrees of polymerization.

[0035] The alternan-oligosaccharide of the present invention preferably consists of, or the alternan-oligosaccharide essentially comprises or essentially consists of, alternan molecules having a degree of polymerization (DP) in the range of 3 to 30. The DP endpoints of these ranges are not average values ​​but individual values.

[0036] The term "essentially comprises or essentially consists of..." means that the weight percentage of alternan molecules with a degree of polymerization (DP) in the range of 3-30 is greater than 90.0%, preferably greater than 95.0% or 97.0%, more preferably greater than 98.0%, still more preferably greater than 99.0%, most preferably greater than 99.5%, based on the total weight of alternan-oligosaccharides, in particular based on dry weight. A small amount of alternan molecules with a DP higher than 30 may be present in the alternan-oligosaccharides. The term "small amount" means that the weight percentage is less than 10.0%, or less than 5.0%, preferably less than 3.0%, more preferably less than 2.0%, more preferably less than 1.0%, most preferably less than 0.5%, based on the total weight of alternan-oligosaccharides.

[0037] The degree of polymerization (DP) of the alternan-oligosaccharide is defined as the number of D-glucosyl units directly or indirectly linked to the acceptor molecule plus the number of monosaccharide units of the acceptor molecule still present in the alternan-oligosaccharide.

[0038] According to the definition of the present invention, the DP of alternan-polysaccharide is higher than 30. This DP is not an average value but a single value. In the present invention, the singular term "alternan-polysaccharide" refers to monodisperse alternan-polysaccharides with molecules of only one degree of polymerization (DP) as well as polydisperse alternan-polysaccharides with molecules of different degrees of polymerization.

[0039] According to the definition of the present invention, the weight average molecular weight Mw of the alternan polysaccharide (determined by GPC RI or GPC MALLS) may be greater than 5000 g / mol. In another embodiment, the weight average molecular weight Mw of the alternan polysaccharide is in the range of 10000000 g / mol to 60000000 g / mol (determined by GPC MALLS), more preferably in the range of 12000000 g / mol to 50000000 g / mol.

[0040] Alternanose Sucrase:

[0041] The alternansucrases employed herein can be obtained from a variety of microorganisms, preferably strains of Leuconostoc, in particular strains of Leuconostoc mesenteroides, such as disclosed in WO 00 / 47727. In one embodiment, the enzyme is produced by strains that secrete a high ratio of alternansucrases to dextransucrases, such as those described in U.S. Pat. No. 5,702,942 to Leathers et al. In another embodiment, alternansucrases useful for producing alternan-oligosaccharides include Leuconostoc mesenteroides strains NRRL B 1355, 23185, 23186, 23188, 23311, 21297, 30821, 30894. These enzymes can additionally be cloned and recombinantly expressed, such as described in the document Gilles Joucla, Doctoral Dissertation, Ingenier INSA, Toulouse, France, 2003. The alternansucrase may be produced in an organism other than Leuconostoc mesenteroides, the organism comprising a recombinant nucleic acid encoding the alternansucrase. The preferred organism is Escherichia coli.

[0042] Any of the above microorganisms may be cultured using conventional techniques and produced under aerobic conditions effective to promote growth and production of the enzyme, such as described by Leathers et al. After cultivation, the enzyme may be isolated from the microorganism using conventional techniques, such as by centrifugation or filtration.

[0043] In one embodiment, the term "alternansucrase is present in the reactor in an aqueous liquid" means that the alternansucrase is dissolved, emulsified or suspended in the aqueous liquid, preferably dissolved. In one embodiment, the alternansucrase is not immobilized on a carrier material.

[0044] Receptor molecules:

[0045] An acceptor molecule is a molecule which the alternansucrase is able to catalyze in a chain elongation reaction. An alternan-oligosaccharide molecule comprises an acceptor molecule, preferably one acceptor molecule. An alternan-polysaccharide molecule preferably comprises an acceptor molecule, more preferably one acceptor molecule. Even if the singular term "molecule" is used herein, a plurality of acceptor molecules are used in the present invention, wherein these molecules may be chemically identical or different.

[0046] Alternan, in particular alternan-oligosaccharides, can be produced by reacting an acceptor molecule and sucrose with one or more alternan sucrases, which transfer glucose units from sucrose to the acceptor molecule and release fructose and alternan, in particular alternan-oligosaccharides. The acceptor molecule can accept glucose units from sucrose.

[0047] The acceptor molecule that can be added to the reaction mixture is preferably a carbohydrate or a carbohydrate derivative. The use of an external acceptor results in the production of low molecular alternating sugar-oligosaccharides. The acceptor molecule can be selected from a sugar or sugar alcohol, which has a free hydroxyl group at one or more of the carbons at positions 2, 3 and 6 that can accept the glucose unit from sucrose. The carbohydrate acceptor is preferably a saccharide selected from maltose, isomaltose, maltitol, (iso) maltotriose and methyl-α-D-glucan. Other preferred acceptor molecules are glucose, gentiobiose, raffinose, melibiose, isomaltitol, isomaltooligosaccharides, theanderose, kojibiose, glucosyltrehalose, cellobiose, maltotetraose, brown sugar, lactose, panose or a mixture thereof. The acceptor molecule is preferably maltose.

[0048] Depending on the specific acceptor chosen, the glucosyl units are typically added via an α(1,6) bond, or if an α(1,6) bond is already present then via an α(1,3) bond. The reaction typically produces a mixture of oligosaccharides with varying degrees of polymerization (DP).

[0049] In one embodiment, the term "the receptor molecule is present in the reactor in the aqueous liquid" means that the receptor molecule is dissolved, emulsified or suspended in the aqueous liquid, preferably dissolved.

[0050] Aqueous liquids:

[0051] The term "aqueous liquid" in the present invention refers to a liquid containing at least 80% by volume of water, preferably at least 90% by volume, or at least 95% by volume. The upper limit is 100% by volume. Preferably, the aqueous liquid is water.

[0052] Reactor:

[0053] The reactor may be a tank reactor.

[0054] The reaction may be carried out under agitation, e.g. stirring. In one embodiment, the reactor is a stirred tank reactor. In another embodiment, which may be combined with the foregoing, the agitation is accomplished by moving the contents of the reactor, preferably by pumping, preferably by circulating the contents of the reactor by a pump as described elsewhere in this specification.

[0055] When mixing (especially stirring):

[0056] - contacting sucrose with a catalytically effective amount of alternansucrase and an acceptor molecule in a reactor, wherein the alternansucrase and the acceptor molecule are present in an aqueous liquid in the reactor, the sucrose being fed continuously or semi-continuously into the reactor,

[0057] Carry out under stirring (especially agitation).

[0058] In another embodiment, removal of fructose and / or addition of sucrose induces turbulence in the reactor, thereby causing mixing of the components.

[0059] Sucrose (substrate) was added continuously or semi-continuously.

[0060] The term "semi-continuous" with respect to sucrose feeding means that the sucrose feeding is interrupted one or more times, preferably more than one time. In other words, "semi-continuous" means that there are at least two feeding time periods, preferably at least three feeding time periods, or at least four feeding time periods, wherein each feeding time period is separated from the subsequent feeding time period by a feeding interruption time period. The feeding time period of sucrose can be a constant time period or a non-constant time period (i.e., the length of the time period may be different from each other). The feeding interruption time period can be a constant time period or a non-constant time period. The feeding time period of sucrose and the feeding interruption time period can be time periods of the same length or time periods of different lengths.

[0061] The term "semi-continuous" with respect to fructose removal means that the removal of fructose is interrupted one or more times, preferably multiple times (more than once). In other words, "semi-continuous" means that there are at least two removal (or removal) time periods, preferably at least three removal time periods, or at least four removal time periods, wherein each removal time period is separated from the subsequent removal time period by a removal interruption time period. The fructose removal time period may be a constant time period or a non-constant time period. The fructose removal interruption time period may be a constant time period or a non-constant time period. The fructose removal time period and the fructose removal interruption time period may be time periods of the same length or time periods of different lengths.

[0062] In semi-continuous operation, the time period of feeding sucrose and the time period of removing fructose can be the same, overlapping or non-overlapping. In one embodiment of semi-continuous operation, sucrose feeding is performed when fructose removal is interrupted, and sucrose feeding is interrupted when fructose removal is performed. Specifically, this means that sucrose feeding and fructose removal are performed alternately.

[0063] Sucrose can be fed directly or indirectly to the reactor. For example, when direct feeding is performed, sucrose or a sucrose solution is added by a feed pipe that directly terminates in the reactor or in the reactor. For example, when indirect feeding is performed, sucrose or a sucrose solution is added by a feed pipe that terminates in another device that is fluidically connected to the reactor, such as another pipe, or another device, such as a device including a membrane mentioned below, or a reactor system including a reactor and a device including a membrane mentioned below can be fed. The feeding (or supply) of sucrose is not performed through a membrane. That is, when feeding sucrose, sucrose will not penetrate the cell membrane. If sucrose is fed to the above-mentioned device including a membrane, the feeding of sucrose does not pass through the membrane of the device. That is, sucrose will not penetrate the membrane of the device. The membrane is used for membrane filtration to remove fructose, allowing fructose and part of the aqueous liquid to penetrate, but not allowing sucrose to penetrate.

[0064] Before the addition of sucrose, a catalytically effective amount of alternansucrase and an acceptor molecule are present in the reactor. The sucrose can be added as a solid or preferably as a solution or suspension, preferably in an aqueous liquid. The provided sucrose can be directly converted by the alternansucrase.

[0065] In this process, the bioconversion, i.e. the enzymatic formation of alternan-oligosaccharides, takes place with continuous or semi-continuous removal of fructose. The removal takes place after the formation of alternan-oligosaccharides has started and when fructose is produced as a by-product.

[0066] In one embodiment, the fructose removal is performed while sucrose is fed to the reactor. Simultaneously means that one or more sucrose addition time periods and one or more fructose removal time periods overlap at least in part. Preferably, fructose is removed at least during the entire process of adding sucrose. After the addition of sucrose is stopped or when the sucrose addition ends, the fructose removal can continue.

[0067] Removing at least a portion of the fructose is related to the total amount of fructose formed in the process (as a by-product). Removing at least a portion of the fructose preferably means equal to or greater than 70 mol%, preferably equal to or greater than 80 mol%, more preferably equal to or greater than 90 mol% of the total fructose produced. The upper limit of removal is 100 mol%. Preferably, the entire amount of fructose produced (100 mol%) or substantially the entire amount is removed. Substantially all means equal to or greater than 95 mol%.

[0068] The removal of fructose is also called depletion.

[0069] In one embodiment, the removal of fructose is performed by diafiltration. That is, the membrane filtration is or is performed as diafiltration.

[0070] In one embodiment of the invention, removing at least a portion of the fructose comprises: continuously or semi-continuously circulating the contents of the reactor through a device comprising a membrane, and contacting the contents of the reactor with the membrane, wherein at least a portion of the fructose and a portion of the aqueous liquid pass through the membrane (as permeate), and returning the remainder (retentate) to the reactor.

[0071] The term "semi-continuous" with respect to a cycle means that the cycle is interrupted one or more times, preferably multiple times (more than once). In other words, "semi-continuous" means that there are at least two cycle time periods, preferably at least three cycle time periods, or at least four cycle time periods, wherein each cycle time period is separated from the subsequent cycle time period by a cycle interruption time period. The cycle time period may be a constant time period or a non-constant time period. The cycle interruption time period may be a constant time period or a non-constant time period. The cycle time period and the cycle interruption time period may be time periods of the same length or time periods of different lengths.

[0072] In a specific embodiment, the temperature in the reactor is in the range of 30-45°C or 30-40°C.

[0073] The device comprising the membrane may be a membrane tank, or a membrane module or a filtration tank comprising the membrane.

[0074] The device comprising the membrane is in fluid communication with the reactor. The contents of the reactor are directed to the device and returned from the device to the reactor. However, a portion of the fructose and a portion of the aqueous liquid pass through the membrane (as permeate) and are not directed back to the reactor.

[0075] The device including the membrane can be connected to the reactor by a connection suitable for conveying liquids (e.g., a conduit, a pipe, or a pipeline). A first connection may exist between the reactor outlet and the device inlet. A second connection may exist between the device outlet and the reactor inlet. A pump may be present for circulating the reactor contents. Circulating the contents of the reactor is accomplished through the reactor itself and the membrane device. Both are also referred to as a "reactor system" - the system includes the reactor and the device including the membrane. The circulating flow of the reactor contents may result in mixing of the components in the liquid phase (especially in turbulent flow).

[0076] In one embodiment, the continuous or semi-continuous removal of at least a portion of the fructose from the reactor is accomplished by nanofiltration. The membrane is preferably a nanofiltration membrane.

[0077] The fructose and aqueous liquid are continuously or semi-continuously removed by circulating continuously or semi-continuously through the device comprising the membrane.

[0078] In one embodiment, the pressure applied when performing membrane filtration to remove fructose is 5-30 bar. The temperature may be 30-40°C.

[0079] In another embodiment, the method includes feeding another aqueous liquid continuously or semi-continuously to a reactor or a reactor system including a reactor and a device including a membrane. By this measure, the aqueous liquid that leaves the reactor or reactor system as part of the permeate can be replaced. "Another aqueous liquid" refers to an aqueous liquid added to the reactor during the process. In other words, the other aqueous liquid refers to an aqueous liquid added in addition to the aqueous liquid mentioned in claim 1. The term "semi-continuously" with respect to feeding another aqueous liquid means that the feeding of the other aqueous liquid is interrupted once or more, preferably multiple times (more than once). In other words, "semi-continuously" means that there are at least two feeding time periods, preferably at least three feeding time periods, or at least four feeding time periods, wherein each feeding time period is separated from the subsequent feeding time period by a feeding interruption time period. The feeding time period of the additional aqueous liquid can be a constant time period or a non-constant time period. The feeding interruption time period of the additional aqueous liquid can be a constant time period or a non-constant time period. The feeding time period of the additional aqueous liquid and the feeding interruption time period of the additional aqueous liquid can be time periods of the same length or time periods of different lengths.

[0080] In one embodiment, the method further comprises removing at least a portion of the alternan-polysaccharide and at least a portion of the alternan sucrase formed as by-products, which is preferably accomplished by additional membrane filtration. In addition to fructose (also a by-product of the process), alternan-polysaccharide is formed as an additional by-product. This step is optionally carried out to separate the by-products. In the case of membrane filtration, alternan-oligosaccharides are obtained in the permeate. Alternan-polysaccharide and alternan sucrase are contained in the retentate. In one embodiment, this membrane filtration is ultrafiltration. The membrane is preferably an ultrafiltration membrane. In one embodiment, the pressure applied during this membrane filtration, in particular ultrafiltration, is 2-15 bar. The temperature during this additional membrane filtration can be 30-60°C.

[0081] The additional membrane filtration (for removing at least a portion of the alternan-polysaccharide and at least a portion of the alternansucrase) can be accomplished with an additional device comprising a membrane, which is an additional membrane.

[0082] In one embodiment, the method further comprises: concentrating the product obtained after removing at least a portion of the alternan-polysaccharide and after removing at least a portion of the alternan sucrase, wherein the product comprises alternan-oligosaccharides. In particular, the method may further comprise concentrating alternan-oligosaccharides, preferably in the permeate obtained by the further membrane filtration.

[0083] In the membrane filtration for removing fructose, or in the optional additional membrane filtration (for removing at least a portion of the alternan-polysaccharide and at least a portion of the alternansucrase), membranes of any known or commonly used materials can be used. Non-limiting examples are polyamide, polyethersulfone or polyvinylidene fluoride.

[0084] In one embodiment, the method further comprises drying the obtained product. As mentioned above, concentration means that the product is still in the liquid phase. Drying means obtaining a solid product.

[0085] Preferably, the product obtained after removing at least a portion of the alternan-polysaccharide and at least a portion of the alternansucrase formed as a by-product is dried. The above-mentioned concentration and drying can be carried out continuously.

[0086] The dried product may comprise at least 65% (w / w), or at least 70% (w / w), or at least 75% (w / w) or at least 80% (w / w) alternan-oligosaccharide (by dry weight), more specifically at least 85% (w / w) or at least 95% (w / w). A preferred upper limit that may be combined with each lower limit is 99.0% or 99.9% (w / w).

[0087] The dried product may contain 0.1 to 15% (w / w), or 0.1 to 10% (w / w), or 0.1 to 9% (w / w), or 0.1 to 8% (w / w), or 0.1 to 7% (w / w), or 0.1 to 6% (w / w), or 0.1 to 5% (w / w), or 0.1 to 3% (w / w), or 0.1 to 2% (w / w), or 0.1 to 1% (w / w), or 0.1 to 0.5% (w / w) of fructose equivalents (by dry weight). In some embodiments, the dried product contains less than 10% (w / w) of fructose equivalents (by dry weight). Preferably, the composition comprises less than 9% (w / w), less than 8% (w / w), less than 7% (w / w), less than 6% (w / w), less than 5% (w / w), less than 3% (w / w), less than 2% (w / w), less than 1% (w / w) or less than 0.5% (w / w) fructose equivalents (by dry weight).

[0088] In some embodiments, the fructose equivalent is leucrose, fructose and / or sucrose. In some embodiments, the fructose equivalent is leucrose. In some embodiments, the fructose equivalent is fructose. In some embodiments, the fructose equivalent is sucrose. In some embodiments, the fructose equivalent is leucrose and fructose. In some embodiments, the fructose equivalent is leucrose and sucrose. In some embodiments, the fructose equivalent is fructose and sucrose. In some embodiments, the fructose equivalent is leucrose, fructose and sucrose. In some embodiments, the fructose equivalent is leucrose, fructose and sucrose.

[0089] Fructose equivalents which are by-products of the process can be removed in the process. This has already been described above for fructose. In one embodiment, the process comprises continuously or semi-continuously removing at least a portion of the leucrose formed as a by-product from the reactor by membrane filtration. The membrane filtration is preferably the same as that used to remove fructose. Thus, fructose and leucrose can be removed in the same membrane filtration step.

[0090] Fructose equivalents belonging to the educts of the method can be removed in the method. In one embodiment, the method comprises removing at least a portion of sucrose as unreacted educts from the reactor continuously or semi-continuously by membrane filtration. The membrane filtration can be the same as that used to remove fructose. Therefore, fructose and sucrose can be removed in the same membrane filtration step.

[0091] In one embodiment of the process, the average degree of polymerization DPw or DPn of the alternan-oligosaccharides is adjusted by the amount of sucrose fed to the reactor.

[0092] In another embodiment, the sucrose feed is stopped when the desired average degree of polymerization DPw or DPn of the alternan-oligosaccharide is reached.

[0093] After cessation of sucrose addition, fructose removal may continue until the desired fructose concentration is or has been achieved.

[0094] The chain length produced can be adjusted by the amount of sucrose added. The degree of polymerization can vary with the concentration and relative proportion of sucrose and acceptor molecules. The reaction product usually consists of a mixture of alternating sugar oligosaccharides with different degrees of polymerization. In the case of relatively high sucrose: acceptor ratios, more glucosyl units are transferred to the dextran and a product with a higher degree of polymerization is obtained (i.e. the relative amount of high DP oligosaccharides in the product will increase). In contrast, in the case of low sucrose: acceptor ratios, the main reaction product is the product produced by the transfer of a single glucosyl unit to the acceptor.

[0095] Therefore, the yield of oligosaccharides having a desired degree of polymerization can be optimized by varying the sucrose:acceptor ratio (also known as the sucrose to acceptor ratio).

[0096] The sucrose:acceptor molecule ratio refers to the total amount of sucrose added to the method relative to the total amount of acceptor molecules used in the method. The ratio can be expressed as a mass ratio or a molar ratio.

[0097] The actual ratio in the reactor is lower than the above ratio because sucrose is fed continuously or semi-continuously instead of being fed all at once, and the fed sucrose is consumed.

[0098] The sucrose:acceptor molecule ratio expressed as a mass ratio (e.g., kg sucrose: kg acceptor molecule) is preferably 10: 1-30: 1, more preferably 15: 1-23: 1. Preferably, the acceptor molecule is maltose, but other acceptors may also be used within this range.

[0099] The sucrose:acceptor molecule ratio expressed as a molar ratio (e.g. mol sucrose molecule:mol acceptor molecule) is preferably 10:1-30:1, more preferably 15:1-23:1, wherein the acceptor molecule is preferably maltose, but other acceptors may also be used within this range.

[0100] Preferably, the feed rate or feeding pattern (eg continuously or semi-continuously) of sucrose is selected such that the fructose concentration in the reactor does not increase during the process or such that fructose does not accumulate.

[0101] Preferably, the feed rate of sucrose is chosen so that the sucrose concentration in the reactor does not increase during the process or so that sucrose does not accumulate. Preferably, the feed rate is chosen so that the fed sucrose is directly consumed in the reaction.

[0102] The preferred feed rate of sucrose is 50-1500 g / h, or 50-1000 g / h, or 50-800 g / h, or 50-500 g / h, or 50-250 g / hh, or 50 g / h-200 g / h, or the corresponding rate expressed in mol / h (molar mass of sucrose 342.30 g / mol). When sucrose is added to a solution or suspension, this ratio refers only to the weight of sucrose, not the weight of the entire solution or suspension.

[0103] In one embodiment of the method, additional alternansucrase is fed to the reactor, preferably continuously or semi-continuously. Thus, additional alternansucrase is added to the alternansucrase previously present in the reactor (the alternansucrase mentioned in claim 1). By this measure, higher total enzyme activity units of alternansucrase can be achieved and the reaction time can be reduced. Preferably, the amount of sucrose is adapted to a higher total enzyme unit by also increasing the amount of sucrose.

[0104] The term "semi-continuous" about the feeding of alternansucrase refers to that the enzyme feeding is interrupted once or multiple times, preferably more than once. In other words, "semi-continuous" refers to at least two feeding time periods, preferably at least three feeding time periods, or at least four feeding time periods, wherein each feeding time period is separated from the subsequent feeding time period by the feeding interruption time period. The feeding time period of the enzyme can be a constant time period or a non-constant time period (i.e., the length of the time period can be different from each other). The feeding interruption time period can be a constant time period or a non-constant time period. The feeding time period of the enzyme and the feeding interruption time period can be time periods of the same length or time periods of different lengths.

[0105] The preferred ratio of alternansucrase: sucrose (total amount of feed sucrose) is 1000-10000 units (enzyme activity): 1000 g sucrose, or 1000-7000 units (enzyme activity): 1000 g sucrose, or 1000-5000 units (enzyme activity): 1000 g sucrose, or 1000-2500 units (enzyme activity): 1000 g sucrose, preferably 1200-2300 units (enzyme activity): 1000 g sucrose, more preferably 1500-2000 units (enzyme activity): 1000 g sucrose. These ratios relate to the total enzyme activity units of the alternansucrase. Units (enzyme activity) refer to the total units of the alternansucrase used in the process. For example, if additional alternansucrase is fed to the reactor, the sum of (i) the alternansucrase present at the beginning and (ii) all alternansucrases further fed to the reactor refers to the total units.

[0106] Using the parameters mentioned herein, it is in particular possible to achieve preferred DPw or DPn of the oligosaccharides, some of which are mentioned in the description.

[0107] The reaction time associated with the method of claim 1 may depend on the selected sucrose:acceptor ratio, the selected sucrose feed rate, the selected enzyme activity and / or the selected total amount of sucrose. Typical time ranges are 10-150 hours, or 10-30 hours, or 10-24 hours, or 10-20 hours. Additional process steps mentioned elsewhere herein, such as removal of alternan-polysaccharide and alternansucrase formed as by-products by additional membrane filtration, or concentration of the retentate obtained in the additional membrane filtration, are not included.

[0108] The reaction temperature associated with the process of claim 1 is preferably in the range of 30-60° C. Specific temperature ranges or different temperatures may be applied in different process stages or steps.

[0109] In one embodiment, sucrose is fed continuously and the feed rate of sucrose (calculated as moles of sucrose per time) is equal or substantially equal to the continuous removal rate of fructose (calculated as moles of fructose per time), or the ratio of the feed rate of sucrose to the removal rate of fructose is in the range of 1.2: 1 to 1: 1. For each mole of sucrose consumed in the formation of alternan-oligosaccharides, the same mole of fructose is produced, which is preferably removed continuously.

[0110] In the following, the present invention is illustrated by examples which should not be interpreted as limiting the general idea of ​​the invention as described above and defined in the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0111] Figure 1a A schematic diagram showing a method according to the prior art;

[0112] Figure 1b A schematic diagram showing the method of the present invention;

[0113] Figure 2a The process design of the prior art method is shown;

[0114] Figure 2b The process design of the method of the present invention is shown;

[0115] Figure 3 A graph showing the increase in molecular weight over processing time when sucrose is fed at a constant rate. Example

[0116] method

[0117] Determination of DP using HPAEC-PAD

[0118] DP values ​​were measured by HPAEC-PAD after reduction and hydrolysis of glucose-based sugars. 2 mL of a solution containing 6 g / mL of a digestible carbohydrate composition was treated with 0.2 mL of a NaBH4 solution (40 mg / mL) in 0.5 M ammonia water at 40°C for 30 minutes. The reduced sample was then hydrolyzed with 0.5 mL of 2 M trifluoroacetic acid and heated at 121°C for 1 hour to release the monomers. The released monomers were quantified and the sample solution was injected into a CarboPac TM Thermo Scientific MA1 TM Dionex TM ICS-6000 ion chromatography system, and the eluent (water and NaOH 1000mM) was added at 0.4mL / min. The DP value was calculated using the following formula:

[0119]

[0120] Determination of DP by GPC-RI

[0121] DP value is also measured by GPC-RI.Sample is diluted with water and gel permeation chromatography (0.5mL / min) is performed in water using 2 x Tosoh TSK GELG2500PWXL posts connected in series.Oligosaccharides are separated by their relative molecular weight during penetration and quantified using refractive detection.Molecular weight quantification is based on external standard method using standard substances (PSS-polymer standards) in the range of 342 to 5000Da.Chromeleon Extension Pack V2.0 is used for calibration and calculation according to Dionex instructions.

[0122] Working Example

[0123] Example 1

[0124] Prior art methods are known, for example, from WO 0047727 A2 and WO 2009095278 A2 and include Figure 1a The following four steps P1-P4:

[0125] In step P1, the bioconversion 2 of sucrose and maltose is carried out in a batch reactor 1 at T = 37 ° C for about 20 hours. The sucrose: maltose ratio is selected to be 7:1 (w / w or mol / mol). Step P1 is completed in a batch reactor 1 for bioconversion 2, such as Figure 2a shown.

[0126] P2 is an ultrafiltration step 3 for the removal of alternan polymers (alternan polysaccharide) and alternansucrase (AlSu). Figure 2a The ultrafiltration device 3' is shown.

[0127] In P3, fructose is removed by nanofiltration 5. This step Figure 2a This is done in the nanofiltration device 5' shown. Here, water 4' is added and a mixture of water 4" and fructose 6 is removed.

[0128] In the final step P4, the product from P3 is concentrated by evaporation 7. Figure 2a The evaporation is completed in an evaporation device 7' and maltose-alternan oligosaccharides (MAOS) 8 are obtained.

[0129] In a specific embodiment, the method of the present invention comprises three steps S1-S3, such as Figure 1b As shown:

[0130] and Figure 1a Compared to the prior art method, the bioconversion 2 in step S1 includes continuous feeding 10 of sucrose 9 and continuous removal of fructose 6. Semi-continuous feeding and removal are possible. The mass ratio of sucrose 9 (total amount added) to maltose 12 is 19:1 (19 kg sucrose per 1 kg maltose) and the duration is about 72 hours. Step P3 of the prior art can be omitted because fructose 6 has been removed in step S1. Removal of fructose 6 makes it possible to use a higher ratio of sucrose 9 to maltose 12 and to achieve a higher degree of polymerization of maltose-alternan-oligosaccharides 8.

[0131] Step S1, biotransformation Figure 2bThe bioconversion is carried out in a reactor 11 in which maltose 12 and alternansucrase (AlSu) 13 are present in water 4. As described above, the bioconversion is carried out by continuous feeding 10 of sucrose 9, continuous removal of fructose 6, for about 72 hours (variable), T = 37 ° C, and a sucrose: maltose ratio of 19: 1 (w / w or mol / mol). Sucrose 9 is added as feed 10 (dissolved in water) and the contents of the reactor 11 are stirred. In the bioconversion in the reactor 11, alternan-oligosaccharides (also called maltoalternan oligosaccharides 8 (MAOS)) containing the acceptor molecule maltose are formed as the main product, and alternan polymers 14, fructose 6 and leucrose 15 are formed as by-products. The contents from the reactor 11 are continuously circulated through a membrane tank 16 (diafiltration tank), wherein water 4", fructose 6 and leucrose 15 are removed in a membrane filtration 17, which in this example is a nanofiltration, performed as a constant volume diafiltration. Compared to the prior art, the content of leucrose 15 is reduced from about 30% to less than 10%. The removed water 4" is replaced by a water 4' feed stream.

[0132] The reactor 11 and the membrane tank 16 form a combined bioconversion and nanofiltration device (also referred to as a reactor system).

[0133] The processing step S2 of this embodiment corresponds to the step P2 of the prior art. Here, alternan polysaccharide 14 (alternan polymer) and alternan sucrase 13 (AlSu) as by-products are removed by ultrafiltration 3 in the device 3'. This step is beneficial in the case where more alternan polymer 14 is formed as desired, or is beneficial for controlling the desired DPw of the remaining alternan substance.

[0134] The process step S3 of this embodiment corresponds to the step P4 of the prior art. Here, the product is concentrated by evaporation 7 in the device 7'.

[0135] The composition of the reaction solution used in the production process is shown in Table 1. 2.1 L of solution and the water in the system (dead volume) produced a total of about 5.6 L. The process was run without losses in the first hour to minimize the potential loss of maltose across the membrane. Subsequently, fructose was continuously lost through the nanofiltration membrane Filmtec NF270-2540 (DOW). After the chain extension was completed, the nanofiltration module was replaced with a TRISEP 2540-UE50-QXF ultrafiltration module (Microdyn Nadir). This separated the maltose-alternan oligosaccharide (MAOS) fraction from the longer aged chains and enzymes. The membranes used in the process and the process parameters used are summarized in Table 2. The filtrate was finally concentrated to a dry matter content of >72%.

[0136] Table 1: Composition of reaction solution

[0137] Element quantity Feed rate maltose 451g In batches sucrose 8500g ~100g / h Sodium acetate 57g In batches Alternanose Sucrase 1900U In batches water ad 2,1L In batches

[0138] Table 2: Membranes and parameters

[0139]

[0140] Figure 3 The increase in chain length over the entire process is shown. These values ​​were recorded by GPC-RI measurements. Figure 3 The relationship for calculating DPw from Mw value is as follows: DPw = Mw / (162 Da). It can be seen that at the end of the process, the DPw achieved is approximately 15.4 (2500 / 162).

[0141] To achieve an average chain length DPw of about 15, 19 kg sucrose (in total) were used per 1 kg maltose.

[0142] Comparative Example:

[0143] according to Figure 1a The alternan-oligosaccharides were prepared by the method shown. A DPw of 9.9 was obtained using a ratio of 21:1 (kg sucrose: kg maltose). For the DPw measurement, GPC-RI was used, but not exactly following the protocol mentioned in the methods section above. However, Figure 3 The results of 247 and 251 were compared, indicating that alternan with higher DPw can be obtained by the present invention.

[0144] The DPw values ​​of the two samples analyzed by the HPAEC-PAD method obtained by the method of the present invention are summarized in the following Table 3:

[0145] Table 3:

[0146]

[0147] Samples DCC-2 and DCC-3 were produced by the method of the present invention.

[0148] Glycosidic bonds of glucose-based oligosaccharides are measured by GC-MS with partially methylated sugar alcohol acetates. In brief, the sample is dissolved in anhydrous DMSO, deprotonated by adding n-butyl lithium (Sigma 230707) and methylated with methyl iodide (Sigma 289566). The methylated sample is subsequently hydrolyzed with 2N TFA (60 minutes at 121°C). The hydrolyzed sample is evaporated under a stream of nitrogen, redissolved in 1M ammonium hydroxide, and the aldehyde group is reduced with a DMSO solution containing sodium borodeuteride (20mg / ml). Glacial acetic acid is added dropwise to terminate the reaction, and acetylated by adding 1-methylimidazole and acetic anhydride. Partially methylated alditol acetates in acetone were quantitatively analyzed by GC-MS (7890A-5975C MSD, Agilent Technologies, Inc., Santa Clara, CA, USA) using a Supelco 24111-U SP-2380 capillary column (injector volume 0.5 μl; injector temperature 250°C; detector temperature 250°C; carrier gas, helium: 30 mL / min; split ratio, 40:1; temperature program, 100°C for 3 minutes, 4°C / min to 270°C for 20 minutes). Electron impact spectra were acquired at 69.9 eV in the mass range of 50-550 Da.

[0149]

[0150] The higher values ​​of 1,6-glycosidic bonds can be explained by the leucrose content in the digestible carbohydrate composition, which affects the amount of 1,5,6-tri-O-acetyl-1-deuterio-2,3,4-tri-O-methyl-D-glucitol observed. In addition, leucrose as well as monomeric glucose affect the amount of terminal -Glc in the digestible carbohydrate composition.

[0151] Embodiment 2:

[0152] In this example, the parameters were changed as shown in the table below and alternansucrase was added to the reactor in 4 equal portions, the first portion being present before sucrose was fed to the reactor.

[0153] parameter 1. Experimental - 72h process 2. Experiment - 24h process Sucrose Amount 15.5kg 16.5kg Feed rate 250g / h 775g / h Enzyme activity 56kU 79.2kU temperature 37℃ 43℃ time 72h 27h

[0154] The reaction time of the process according to the invention can be reduced by increasing the amount of sucrose, increasing the sucrose feed rate, increasing the enzyme activity and increasing the temperature (additional process steps such as removal of alternan-polysaccharide and alternansucrase by additional membrane filtration or concentration of the retentate obtained in the additional membrane filtration are not included here).

[0155] Reference Mark List:

[0156] P1 Batch bioconversion

[0157] P2 Ultrafiltration

[0158] P3 Nanofiltration

[0159] P4 Concentration

[0160] S1 Biotransformation

[0161] S2 Ultrafiltration

[0162] S3 Concentrate

[0163] 1 Batch Reactor

[0164] 2 Biotransformation

[0165] 3 Membrane filtration, in the example: ultrafiltration

[0166] 3' Membrane filtration device, in the embodiment: ultrafiltration device

[0167] 4 Aqueous liquid, in the embodiment: water

[0168] 4' Aqueous liquid, in the embodiment: water

[0169] 4” aqueous liquid, in the example: water

[0170] 5 nanometer filtration

[0171] 5' Nanofiltration Unit

[0172] 6. Fructose

[0173] 7 Evaporation

[0174] 7' Evaporation unit

[0175] 8 Alternan-oligosaccharides, in the examples: maltose alternan-oligosaccharides (MAOS)

[0176] 9. Sucrose

[0177] 10 Feed

[0178] 11 Reactor

[0179] 12 Receptor molecule, in the example: maltose

[0180] 13 Alternanose sucrase (AlSu)

[0181] 14 Alternan polymer (alternan polysaccharide)

[0182] 15 Leuconostoc

[0183] 16. A device comprising a membrane, in an embodiment: a membrane tank

[0184] 17 Membrane filtration; in the examples: bioconversion and nanofiltration (constant volume diafiltration)

Claims

1. A method for producing alternan-oligosaccharides, comprising: Sucrose is contacted with a catalytically effective amount of alternansucrase and an acceptor molecule in a reactor, wherein The alternansucrase and the receptor molecule are present in the reactor in an aqueous liquid, and the sucrose is fed continuously or semi-continuously into the reactor, and The sucrose and the acceptor molecule are converted into the alternan-oligosaccharide, with fructose being formed as a by-product; continuously or semi-continuously removing at least a portion of the fructose from the reactor by membrane filtration, wherein the receptor molecule is selected from maltose, isomaltose, maltitol, isomaltotriose and methyl-α-D-glucan, wherein the aqueous liquid comprises at least 90% by volume water.

2. The method of claim 1, wherein the membrane filtration is diafiltration.

3. The method of any one of claims 1 to 2, wherein removing at least a portion of the fructose comprises: circulating the contents of the reactor continuously or semi-continuously through a device comprising a membrane and contacting the contents of the reactor with the membrane, At least a portion of the fructose and a portion of the aqueous liquid pass through a membrane and the remainder is returned to the reactor.

4. The process of claim 1 further comprising continuously or semi-continuously feeding additional aqueous liquid into the reactor.

5. The method of claim 3, further comprising continuously or semi-continuously feeding additional aqueous liquid to the reactor system comprising the reactor and the device comprising a membrane.

6. The method according to claim 1, further comprising removing at least a portion of the alternan-polysaccharide formed as a further by-product, and at least a portion of the alternansucrase.

7. The process according to claim 1 , further comprising removing at least a portion of the alternan-polysaccharide formed as a further by-product, and at least a portion of the alternan sucrase, by further membrane filtration, wherein in the further membrane filtration the alternan-oligosaccharide is contained in the permeate.

8. The method of claim 1, further comprising concentrating the alternan-oligosaccharide.

9. The process according to claim 1, wherein the average degree of polymerization DPw or DPn of the alternan-oligosaccharides is adjusted by the amount of the sucrose fed to the reactor.

10. The process according to claim 9, wherein the feeding of the sucrose is stopped when the desired average degree of polymerization DPw or DPn of the alternan-oligosaccharide is reached.

11. The method according to claim 9, wherein the weight average degree of polymerization DPw of the alternan-oligosaccharide is 5 to 30 as determined by GPC-RI.

12. The method according to claim 9, wherein the weight average degree of polymerization DPw of the alternan-oligosaccharide is 10 to 20 as determined by GPC-RI.

13. The method according to claim 1, wherein the sucrose is fed continuously, and the feed rate of the sucrose based on the molar amount of sucrose each time is equal to the continuous removal rate of fructose based on the molar amount of fructose each time, or the ratio of the feed rate of the sucrose to the removal rate of the fructose is 1.2:1 to 1:

1.

14. The method of claim 1, wherein the molar ratio of sucrose:acceptor molecule is the total amount of feed sucrose relative to the total amount of acceptor molecules used in the method, ranging from 10:1 to 30:

1.

15. The method of claim 1, further comprising feeding additional alternansucrase to the reactor.

16. The method of claim 1, further comprising feeding additional alternansucrase continuously or semi-continuously into the reactor.

17. The method according to claim 1, wherein the ratio of alternansucrase:sucrose is 1000-10000 enzyme activity units: 1000 g sucrose, wherein the sucrose is the total amount of feed sucrose.

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