Process for producing alternan oligosaccharides

Abstract

In order to solve the problems of conventional production methods, a method for producing alternan-oligosaccharides (8) is provided, comprising a specific step (S1), in which sucrose (9) is contacted in a reactor (11) with a catalytically effective amount of an alternan sucrase enzyme (13) and an acceptor molecule (12), which are present in the reactor (11) in the form of an aqueous liquid (4). Sucrose (9) is supplied to the reactor (11), and the sucrose (9) and the acceptor molecule (12) are converted into alternan-oligosaccharides (8), with fructose (6) being produced as a by-product. The method further comprises a step of removing fructose (6) from the reactor (11) by membrane filtration (17).

Description

[Technical field]

[0001] The present invention relates to alternan oligosaccharides. of manufacturing The process for Regarding. [Background technology]

[0002] Alternan-oligosaccharides are described as prebiotic ingredients. teeth disclose that oligosaccharides generated by the alternansucrase enzyme-catalyzed reaction of sucrose with various acceptor sugars are effective as prebiotics for controlling enteric bacterial pathogens. (For example, lactic acid bacteria, bifidobacteria, etc.) one of these oligosaccharides in an amount effective to promote the growth of Or multiple The method of treating an animal with a composition comprising can be .

[0003] Patent Document 2 provides a nucleic acid molecule encoding an alternan sucrase. Also Also provided are vectors, host cells, plant cells transformed with the described nucleic acid molecules, and plants containing them.Furthermore, methods are described for preparing transgenic plants that synthesize carbohydrate alternan by inserting a nucleic acid molecule encoding an alternan sucrase.Furthermore, methods for preparing alternan and products obtained therefrom are provided.

[0004] Patent Document 3 teeth The present invention is directed to the use of alternan polysaccharides and alternan oligosaccharides as ingredients in acidic foods, and to acidic foods containing alternan as an ingredient. Also, Disclosure ContentIt also covers the use of alternan as a heat-stable ingredient in food formulations and food products containing alternan as an ingredient, where the food product has been subjected to a heating step during its manufacture.

[0005] Patent Documents 2 and 3 propose methods for producing alternan oligosaccharides, which include the steps of: a) contacting a sucrose-containing solution with a catalytically effective amount of an alternan sucrase enzyme and an acceptor molecule under conditions that allow the conversion of sucrose to alternan oligosaccharides and fructose; b) The Alternan-oligosaccharides and fructose are separated from the solution.

[0006] The reaction is carried out in a batch reactor containing all the extract and can be carried out at room temperature to 37°C and at a pH of about 4.7 to 7. Can , sucrose Almost It can be progressed until it is consumed. can The resulting product is usually obtained as a syrup, which can be further refined. (i.e. by membrane filtration) , Dry Will be thing There is . [Prior art documents] [Patent documents]

[0007] [Patent Document 1] U.S. Patent No. 7,182,954 (Claims, etc.) [Patent Document 2] International Publication No. 2000 / 47727 (Claims, etc.) [Patent Document 3] International Publication No. 2009 / 095278 (Claims, etc.) Summary of the Invention [Problem to be solved by the invention]

[0008] Accumulation of by-products has been an issue in this process. preparation In this case, the processes used heretofore cannot be used because the desired average chain length is not obtained at all or is hardly obtained at all.

[0009] Therefore, the object of the present invention is to For manufacturing alternative process The purpose of this project is to provide Preferably , This process According to the company, it minimizes the production of by-products. Can , and / or By-products of More efficient To Removal will be able to . [Means for solving the problem]

[0010] The present invention teeth , as claimed in claim 1 process The present invention provides alternan oligosaccharides. of manufacturing A process for In a reactor, sucrose is contacted with a catalytically effective amount of an alternan sucrase enzyme and an acceptor molecule. Including, The alternansucrase enzyme and the acceptor molecule are placed in a reactor. In aqueous liquid and sucrose but , In the reactor Continuous or semi-continuous supply And, sucrose and The acceptor molecule is converted to an alternan oligosaccharide, fructose but and continuously or semi-continuously removing at least a portion of the fructose from the reactor by membrane filtration. That Contains nothing Characterized by Provide a process .

[0011] The present invention provides the general form Or in certain embodiments, one of the following advantages: Or multiple can be achieved. Consistent low fructose concentration can be achieved . The production of further unwanted by-products, such as alternan polymer or leucrose, can be reduced or minimized. The by-products are not necessarily only fructose, but also possible products include alternan polymers or leucrose. The following are some of the reasons: , As explained below, You can also remove them It is possible . Conventional Process Longer chain than of Alternan oligosaccharides can be produced. The chain length can be adjusted by the amount of sucrose added. The present invention teeth , This process The alternan oligosaccharide obtained in of provide. Further advantages include: Detailed Description This is described in a specific embodiment.

[0012] [Alternan oligosaccharides] The degree of polymerization, DP, or the weight-average degree of polymerization, DPw, can be measured by GPC-RI (refractive index detection) as an alternative method. by gel permeation chromatography), or HPAEC-PAD (pulsed amperometric detection) by Measured using high-performance anion exchange chromatography Will be .

[0013] Alternan is a sugar composed of glucose units, or essentially of glucose units when glucose or other structures are present. The glucose units are linked together by α-1-3- and α-1-6-glycosidic bonds ( linkage When it is called too ), and these two types of bonds mainly alternate. When glucose-containing acceptor molecules such as maltose are present, they are bonded in a different way. There are cases Alternan contains branches. There are cases(Seymour et al., Carbohydrate Research 74, (1979), 41-62). Alternan polysaccharides and alternan oligosaccharides are types of alternan that are differentiated by their degree of polymerization. Alternan polysaccharide molecules can be synthesized by the addition of acceptor molecules. and , but preferably includes an acceptor molecule.

[0014] The alternan in the present invention, particularly the alternan oligosaccharide, is a glucose-based saccharide. definition To be Can, The sugars are composed of reducing ends and α-1-6 and α-1-3 glycosides. linkage The acceptor molecule is a D-glucose monomer with alternating unit Acceptor molecules, also called maltose, Units are , is present at the reducing end, or in other words is attached to the reducing end.

[0015] Preferably, the alternan-oligosaccharide molecule comprises at least one acceptor molecule.

[0016] Produced by the method of the present invention The present invention Alternan oligosaccharides is preferably Weight average degree of polymerization DPw but 5 to 30 range Located , Preferably 5~25 and more preferably in the range 10~30 or 10~20 range and more preferably 10-18 or 12-18 range To There is. preferable DPw Range The range is 18 to 30 or 20 to 30. value Measured by GPC-RI will be done. More detailed methods are described in the Examples section below.

[0017] Produced by the method of the present invention The present inventionThe alternan oligosaccharides had average degrees of polymerization (DPw) of 7-32, 12-30, 12-20, and 14-26, as measured by HPAEC-PAD. or The average degree of polymerization is preferably in the range of 12 to 18. The detailed method will be described in the Examples section below. The average degree of polymerization is preferably 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 is less than 20, less than 19, or less than 18 (HPAEC-PAD). It is okay .

[0018] The above GPC-RI and HPAEC-PAD values ​​should be considered independently. this is ,When comparing different methods, the above values ​​and ranges but Correspond to each other It means that It is possible to produce products with different DPw, to manipulate the DPw, and to measure the DPw of different products in different ways.

[0019] In the present invention, the singular term "alternan oligosaccharides" refers both to monodisperse alternan oligosaccharides, having molecules with only one degree of polymerization (DP), and to polydisperse alternan oligosaccharides, having molecules with different degrees of polymerization.

[0020] The alternan oligosaccharide of the present invention preferably consists of alternan molecules having a degree of polymerization (DP) in the range of 3 to 30, or preferably contains alternan molecules having a degree of polymerization (DP) substantially in the range of 3 to 30, or preferably consists of alternan molecules having a degree of polymerization (DP) substantially in the range of 3 to 30. range The endpoints are meant to represent a single value, not an average value.

[0021] "Essentially …… Contains nothing (comprise) mosquito , Or teeth 、 In effect ……fromThe term "consists" refers to an amount of at least 90.0% by weight, particularly on a dry basis, of alternan molecules having a degree of polymerization (DP) in the range of 3 to 30, based on the total weight of alternan oligosaccharides, preferably at least 95.0% by weight or at least 97.0% by weight, more preferably at least 98.0% by weight, even more preferably at least 99.0% by weight, most preferably at least 99.5% by weight. Amount of This means that alternan oligosaccharides have a higher DP Here, the term "small amount" means an amount of less than 10.0% by weight, or less than 5.0% by weight, preferably less than 3.0% by weight, more preferably less than 2.0% by weight, even more preferably less than 1.0% by weight, and most preferably less than 0.5% by weight, based on the total weight of alternan oligosaccharides.

[0022] The degree of polymerization (DP) of an alternan oligosaccharide is defined as the number of D-glucosyl units directly or indirectly bound to the acceptor molecule plus the number of monosaccharide units of the acceptor molecule remaining in the alternan oligosaccharide. definition will be done.

[0023] The present invention By definition Alternan polysaccharides have a DP higher than 30. this DP does not mean an average value but a single numerical value. In the present invention, the singular term "alternan polysaccharide" refers to both monodisperse alternan polysaccharides having molecules with only one degree of polymerization (DP) and polydisperse alternan polysaccharides having molecules with different degrees of polymerization.

[0024] The present invention By definition Alternan polysaccharides have a weight average molecular weight (Mw) of 5000 g / mol or more ( (Measured by GPC RI or GPC MALLS) Have Good According to another embodiment, the alternan polysaccharide comprises: Weight average molecular weight (Mw) 10000000g / mol~60000000g / mol(GPC MALLS Measured at ) range and , More preferablyWithin the range of 12,000,000 g / mol to 50,000,000 g / mol is .

[0025] [Alternan sucrase enzyme] Alternansucrases for use herein can be prepared from various microorganisms, preferably strains of the genus Leuconostoc, in particular from the strains of the genus Leuconostoc, as disclosed, for example, in WO 02 / 04311. L. It can be obtained from strains of L. mesenteroides. can According to one embodiment, The The enzymes are produced by strains secreting a high ratio of alternan sucrase to dextransucrase, as described in U.S. Patent No. 5,702,942 to Leathers et al. According to another embodiment, alternan sucrase enzymes that can be used to produce alternan oligosaccharides include strains of Leuconostoc mesenteroides NRRL B 1355, 23185, 23186, 23188, 23311, 21297, 30821, 30894. These enzymes can be further cloned and recombinantly expressed, as described in the doctoral thesis of Gilles Joucla (Ingenier INSA, Toulouse, France, 2003). Also , the alternan sucrase enzyme, L. It can be produced in organisms other than Mesenteroides. 、 The organism contains a recombinant nucleic acid encoding the alternan sucrase enzyme. nothing. A preferred organism is E. coli.

[0026] The production of alternansucrase can be carried out using conventional techniques. and, Leathers et al. literature The method may be carried out by culturing any of the above microorganisms under aerobic conditions effective to promote the growth and production of the enzyme as described in can After incubation, the enzyme For example, centrifugation or filtrationIt is possible to isolate or separate from a microorganism using conventional techniques. can .

[0027] In one embodiment, the alternan sucrase enzyme is In aqueous liquid The term "present in" means that the alternan sucrase enzyme is dissolved, emulsified or suspended in the aqueous liquid, preferably dissolved. In one embodiment the alternan sucrase enzyme is not immobilized on a carrier material.

[0028] [Acceptor molecule] The acceptor molecule is alternansucrase. but Chain-extending reaction Can be catalyzed Meaning Molecular It is understood that The alternan oligosaccharide molecule contains an acceptor molecule. fruit , Preferably Contains one acceptor molecule. Alternan polysaccharide molecule teeth , Preferably Contains an acceptor molecule fruit , More preferably Although the singular term molecule is used, the present invention contemplates a plurality of acceptor molecules, which may be chemically identical or different. good .

[0029] Alternan, especially alternan oligosaccharides, transfers glucose units from sucrose to the acceptor molecule, releasing fructose and alternan, especially alternan oligosaccharides. 、 1 One or more By reacting with the alternansucrase enzyme, vinegar To be able to can The acceptor molecule accepts a glucose unit from sucrose. can be .

[0030] The acceptor molecule that can be added to the reaction mixture is preferably a carbohydrate or carbohydrate derivative. The use of an external acceptor leads to the production of low molecular weight alternan oligosaccharides. The acceptor molecule can accept a glucose unit from sucrose. can , one of carbon position numbers 2, 3, and 6 Or multiple The sugar or sugar alcohol may be selected from those having a free hydroxyl group at the can Preferably, the carbohydrate acceptor is a sugar selected from the group consisting of 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, nigerose, lactose, panose or mixtures thereof. Preferably, the acceptor molecule is maltose.

[0031] Depending on the particular acceptor selected, glucosyl units are generally added via an α(1,6) linkage or, if an α(1,6) linkage is already present, via an α(1,3) linkage. This reaction typically produces a mixture of oligosaccharides with different degrees of polymerization (DP).

[0032] In one embodiment, "the acceptor molecule is in the reactor In aqueous liquid The term "present in" means that the acceptor molecule is dissolved, emulsified or suspended in the aqueous liquid, preferably dissolved.

[0033] [Aqueous liquid] The term "aqueous liquid" in the present invention means a liquid containing at least 80 vol.% water, preferably at least 90 vol.% water, or at least 95 vol.% water, with an upper limit of 100 vol.%. Preferably, the aqueous liquid is water.

[0034] [Reactor] The reactor is a tank reactor. That's good. . The reaction is stirred , e.g. by stirring carried out Good In one embodiment, the reactor is tank type In another embodiment, which can be combined with the previous embodiment, the stirring is performed by moving the contents of the reactor. This is preferably done using a pump, preferably This is accomplished by circulating the reactor contents with a pump, as described elsewhere in this specification. When stirring, especially agitating, "Contacting sucrose with a catalytically effective amount of an alternan sucrase enzyme and an acceptor molecule in a reactor, the alternan sucrase enzyme and the acceptor molecule being present in an aqueous liquid within the reactor, and the sucrose being continuously or semi-continuously fed to the reactor" is understood to mean that the process is carried out by stirring, in particular agitation. It will be held. In another embodiment, the removal of fructose and / or the addition of sucrose creates turbulence inside the reactor, which allows the components to be separated. but mixture Will be . The sucrose (substrate) is added continuously or semi-continuously. R .

[0035] The term "semi-continuously" in relation to sucrose supply means that the supply of sucrose is Or multiple times , preferably interrupted twice or more. That is, the term "semi-continuously" means that there are at least two feeding periods, preferably at least three feeding periods, or at least four feeding periods, each of which is interrupted by a feeding interruption period. Next supply period and It means that it is separated. of The supply period is a fixed period. But often , for a non-fixed period of time It is okay (i.e., period of The lengths are different Good ). supply of The interruption period is a fixed period. But often , for a non-fixed period of time It is okay The sucrose supply period and the supply interruption period were of equal length. But often , with periods of different lengths It is okay .

[0036] The term "semi-continuously" with respect to fructose removal means that the removal of fructose is Or multiple times , preferably Multiple times ( More than once ) The term "semi-continuously" means interrupted. At least two removal ( or removing ) Period, preferably at least three Removal period, or at least four Removal Each removal period is divided into two periods: Next removal period and It means separated. Fructose of The removal period is a fixed period. But often , for a non-fixed period of time It is okay Fructose of The removal suspension period is a fixed period. But often , for a non-fixed period of time It is okay Fructose of Elimination Periods and Fructose of The removal suspension period is a period of equal length. But often , with periods of different lengths It is okay .

[0037] In semi-continuous operation, the period during which sucrose is fed and the period during which fructose is removed are the same. But often , duplicate Even if No duplication It is not necessary In one embodiment of semi-continuous operation, sucrose is fed when fructose removal is interrupted and fructose removal is performed. R At times, the sucrose supply is interrupted, which in particular means that sucrose supply is alternated with fructose removal.

[0038] The supply of sucrose to the reactor can be direct or indirect. can Direct feeding is, for example, when sucrose or a sucrose solution is added to the reactor. At the or inside the reactor Ends soonIndirect feeding occurs when the sucrose or sucrose solution is added through a feed pipe, whereas indirect feeding occurs when, for example, the sucrose or sucrose solution is added through a feed pipe that terminates in another device in fluid communication with the reactor, such as a further pipe, or a further device, such as a device containing a membrane, as described later in this specification. Added Alternatively, the feed is to a reactor system that includes a reactor and a device that includes a membrane, as described later in this specification. Good The supply (or delivery) of sucrose is not via the membrane. That is, in the supply of sucrose, the sucrose does not penetrate the membrane. When sucrose is supplied to the device described above that includes a membrane, the supply of sucrose does not penetrate the membrane of the device. Not through That is, sucrose does not pass through the membrane of the device. The membranes described above used in membrane filtration to remove fructose are membranes that allow fructose and a portion of the aqueous liquid to pass through, but do not allow sucrose to pass through.

[0039] A catalytically effective amount of alternansucrase enzyme and acceptor molecule are present inside the reactor before sucrose is added. As may be added, Preferably solution or As a suspension , preferably in an aqueous liquid addition Will be The supplied sucrose is directly converted by alternansucrase. Be It is possible.

[0040] Books process In the present study, the bioconversion, i.e. the enzymatic production of alternan-oligosaccharides, is carried out under continuous or semi-continuous removal of fructose, which is carried out after the start of the production of alternan-oligosaccharides, at the point when fructose is produced as a by-product.

[0041] In one embodiment, the removal of fructose is carried out simultaneously with the addition of sucrose to the reactor, where simultaneously means one Period or multiple Period and fructose elimination One or more periods The period is at least PartiallyIt is preferred that fructose is removed at least during the entire period that sucrose is added. Fructose removal is initiated after sucrose addition is stopped. or Sucrose addition was terminated. Time ,continuation May be .

[0042] The removing of at least a portion of the fructose may include in a process: ( As a by-product ) The removal of at least a portion of the fructose is preferably at least 70 mol %, more preferably at least 80 mol %, of the total fructose produced. twist Preferably, it means 90 mol% or more. The upper limit of removal is 100 mol%, and it is preferable that the entire amount (100 mol%) or substantially the entire amount of the produced fructose is removed. Here, substantially the entire amount means 95 mol% or more.

[0043] The removal of fructose is called depletion. Also called .

[0044] The removal of fructose is, in one embodiment, performed by diafiltration. By To be carried out That is, such membrane filtration is or is carried out as diafiltration.

[0045] 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 reactor contents with a membrane. At least Part of the fructose and part of the aqueous liquid are ( As permeate ) Passes through the membrane rest( retentate ) is returned to the reactor.

[0046] The term "semi-continuously" in relation to a cycle means that the cycle is Or multiple times , preferably Multiple times ( More than once ) "Semi-continuously" means interrupted by at least two circulation Period, preferably at least three circulation period, or at least four circulation There is a period, each circulation The duration depends on the period of interruption of circulation. The next cycle period and It means that the cycle is divided into periods. But often , for a non-fixed period of time It is okay . circulation of The interruption period is a fixed period. But often , for a non-fixed period of time It is okay Circulation period and cycle of The interruptions are of equal length. But often , with periods of different lengths It is okay .

[0047] The temperature inside the reactor is in the range of 30 to 45°C, or 30 to 40°C, in certain embodiments.

[0048] The device containing the membrane is called a membrane cell, a membrane module, or a filtration cell containing a membrane. may be .

[0049] The membrane-containing device is in fluid communication with the reactor. The contents of the reactor are passed through the device. Sent However, a portion of the fructose and a portion of the aqueous liquid are returned from the apparatus to the reactor. ( As permeate ) It does not pass through the membrane and is not returned to the reactor.

[0050] The membrane-containing device is connected to the reactor by a connection suitable for transporting liquids, such as a tube, pipe, or pipeline. Good The first connection is between the outlet of the reactor and the inlet of the device. Good The second connection is between the outlet of the device and the inlet of the reactor. GoodThere is a pump to circulate the contents of the reactor. Good The circulation of the reactor contents is carried out through the reactor itself and through the membrane device. Both are called "reactor systems" and are systems that include a reactor and a device that includes a membrane. The circulation flow of the reactor contents teeth , which causes mixing of the components in the liquid phase, especially in turbulent flows There are cases .

[0051] In one embodiment, the continuous or semi-continuous removal of at least a portion of the fructose from the reactor is performed by nanofiltration. Preferably, the membrane is a nanofiltration membrane.

[0052] Fructose and aqueous liquid are removed continuously or semi-continuously by continuous or semi-continuous circulation through a device containing the membrane.

[0053] In one embodiment, the pressure applied to the membrane filtration to remove fructose is in the range of 5 to 30 bar. The temperature is 30 to 40° C. It may be .

[0054] In a further embodiment, the present invention process comprises continuously or semi-continuously feeding the further aqueous liquid to the reactor or to the reactor system comprising the reactor and the device comprising the membrane. This procedure allows replacing the aqueous liquid leaving the reactor or the reactor system as part of the permeate. By "further aqueous liquid" is meant an aqueous liquid which is added to the reactor during the process. That is, by further aqueous liquid is meant an aqueous liquid which is added in addition to the aqueous liquid according to claim 1. The term "semi-continuously" with respect to the feeding of further aqueous liquid means that the feeding of further aqueous liquid is performed once. Or multiple times , preferably Multiple times ( More than once ) That is, "semi-continuously" means that there are at least two feeding periods, preferably at least three feeding periods, or at least four feeding periods, each feeding period being a feeding period. of Next supply period due to interruption period Separated byThe period during which the additional aqueous liquid is supplied is a fixed period of time. But often , for a non-fixed period of time It is okay The period during which further supply of aqueous liquid is suspended is a fixed period of time. But often , for a non-fixed period of time It is okay The period during which the additional aqueous liquid is supplied and the period during which the supply of the additional aqueous liquid is discontinued are time periods of equal length. But often , with periods of different lengths It is okay .

[0055] In one embodiment, process at least a portion of the alternan polysaccharide produced as a by-product and at least a portion of the alternan sucrase enzyme, Preferably by further membrane filtration Remove thing Further including nothing . Books In addition to the fructose by-product of the process, alternan polysaccharides are produced as a further by-product. This step is optionally carried out in order to separate the by-products. In the case of membrane filtration, alternan oligosaccharides are obtained in the permeate, whereas alternan polysaccharides 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 in such membrane filtration, in particular in ultrafiltration, is in the range of 2-15 bar. The temperature in this further membrane filtration is in the range of 30-60° C. That's good. .

[0056] ( For removing at least a portion of the alternan polysaccharide and at least a portion of the alternan sucrase enzyme ) Further membrane filtration is Further film A membrane The method is carried out in a further apparatus including Good .

[0057] In one embodiment, processfurther comprises concentrating the product obtained after removing at least a portion of the alternan polysaccharides and after removing at least a portion of the alternan sucrase enzyme, said product comprising alternan oligosaccharides. process teeth, Preferably In the permeate obtained by the further membrane filtration described above, 、 and further concentrating the alternan oligosaccharides. Good .

[0058] Membrane filtration to remove fructose, or ( For removing at least a portion of the alternan polysaccharide and at least a portion of the alternan sucrase enzyme ) In the optional further membrane filtration, membranes of any known or conventional material may be used. can . non-limiting As an example , polyamide, polysulfone, or poly(vinylidene) fluoride.

[0059] In one embodiment, process Dry the resulting product. thing As mentioned above, concentrating means concentrating the product in a liquid phase. As it is Drying is intended to obtain a solid product. meaning is doing.

[0060] It is preferable to dry the product obtained after removing at least a part of the alternan polysaccharides produced as by-products and at least a part of the alternan sucrase enzyme. The above-mentioned concentration and drying are preferably carried out successively. Can .

[0061] The dry product contains at least 65% (w / w), or at least 70% (w / w), or at least 75% (w / w), or at least 80% (w / w) alternan-oligosaccharides on a dry basis. But well , More specifically, Contains at least 85% (w / w) or at least 95% (w / w) alternan oligosaccharides It's okayThe upper limit that can be combined with each lower limit is preferably 99.0% or 99.9% (w / w).

[0062] The dried product has a fructose equivalent content, on a dry basis, of 0.1-15% (w / w), or 0.1-10% (w / w), or 0.1-9% (w / w), or 0.1-8% (w / w), or 0.1-7% (w / w), or 0.1-6% (w / w), or 0.1-5% (w / w), or 0.1-3% (w / w), or 0.1-2% (w / w), or 0.1-1% (w / w), or 0.1-0.5% (w / w) May include In some embodiments, the dry product comprises less than 10% (w / w) fructose equivalents on a dry basis. Preferably, such compositions comprise 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 on a dry basis.

[0063] 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.

[0064] This process The by-product fructose equivalents are process This can be removed in That isThe fructose has already been described. In one embodiment, process comprises continuously or semi-continuously removing at least a portion of the leucrose produced as a by-product from the reactor by membrane filtration, preferably the same membrane filtration as that used to remove fructose, so that fructose and leucrose can be removed in the same membrane filtration step.

[0065] Books Process The fructose equivalents belonging to the extract are process In one embodiment, the present invention can be removed by process The method includes continuously or semi-continuously removing at least a portion of the sucrose from the reactor as unreacted extract by membrane filtration. Such membrane filtration is the same as that used for the removal of fructose. That's good. Therefore, fructose and sucrose can be removed in the same membrane filtration step.

[0066] Books process of one In an embodiment, the average degree of polymerization DPw or DPn of the alternan oligosaccharides is adjusted by the amount of sucrose fed to the reactor.

[0067] In a further embodiment, the alternan oligosaccharides are purified when or after the desired average degree of polymerization DPw or DPn is reached. attitude Ta Sometimes , Sucrose supply Stop vinegar do.

[0068] After sucrose addition is stopped, the fructose concentration is increased until or after the desired fructose concentration is reached. Until the time , Fructose removal continuation May .

[0069] The length of the chains produced varies with the amount of sucrose added. difference Adjust It is possible. The degree of polymerization can be varied depending on the concentration and relative ratio of sucrose and acceptor molecules. beThe reaction products generally consist of a mixture of alternan oligosaccharides with different degrees of polymerization. At relatively high sucrose:acceptor ratios, more glucosyl units are transferred to the glucan, resulting in a product with a higher degree of polymerization. ( That is, the relative amount of high DP oligosaccharides in the product increases. ) On the other hand, at low sucrose:acceptor ratios, the reaction product is the transfer of a single glucosyl unit to the acceptor. Major It becomes.

[0070] Therefore, by varying the sucrose:acceptor ratio (sometimes referred to as the sucrose to acceptor ratio), the desired degree of polymerization can be achieved. have Oligosaccharides yield It is possible to optimize can .

[0071] The ratio of sucrose:acceptor molecules is process The amount of the acceptor molecule used in the During the process This refers to the total amount of sucrose added. This ratio is expressed as a mass ratio or a molar ratio. Can be done .

[0072] The actual ratio in the reactor will be lower than the ratio stated above because the sucrose is not fed all at once, but is fed continuously or semi-continuously, and the sucrose fed is consumed.

[0073] The ratio of sucrose:acceptor molecule is preferably within the range of 10:1 to 30:1, more preferably within the range of 15:1 to 23:1, by mass ratio (e.g., kg sucrose:kg acceptor molecule). The acceptor molecule is preferably maltose, although other acceptors within this range are also acceptable. of It can also be applied can .

[0074] The ratio of sucrose:acceptor molecules is preferably in the range of 10:1 to 30:1, more preferably in the range of 15:1 to 23:1, in terms of molar ratio (e.g., Mol sucrose molecules: Mol acceptor molecules). The acceptor molecule is preferably maltose, although other acceptors within this range are also acceptable. of It can also be applied can .

[0075] Sucrose supply rate or supply method (e.g. continuous or semi-continuous) is preferably selected so that the concentration of fructose in the reactor does not increase during the process or so that fructose does not accumulate.

[0076] The sucrose feed rate is preferably selected so that the concentration of sucrose in the reactor does not increase during the process or so that sucrose does not accumulate, and is preferably selected so that the sucrose fed is directly consumed in the reaction.

[0077] The supply rate of sucrose is preferably 50 to 1500 g / hr, or 50 to 1000 g / hr, or 50 to 800 g / hr, or 50 to 500 g / hr, or 50 to 250 g / hr, or 50 to 200 g / hr, or each rate expressed in mol / hr (molar mass of sucrose: 342.30 g / mol). When sucrose is added to a solution or suspension, this rate refers to the weight of sucrose only, not the weight of the entire solution or suspension.

[0078] Books process In one embodiment, the further alternan sucrase enzyme is Preferably The feed is made to the reactor continuously or semi-continuously. In other words , inside the reactor already Further alternan sucrase enzyme is added to the existing alternan sucrase enzyme (the alternan sucrase enzyme according to claim 1). treatmentBy increasing the amount of sucrose, a higher total unit of alternansucrase enzyme activity can be achieved and the reaction time can be shortened. The amount of sucrose To accommodate higher total enzyme units but preferable.

[0079] With respect to the supply of the alternansucrase enzyme, the term "semi-continuously" means that the supply of the enzyme is Or multiple times , preferably interrupted twice or more. That is, the term "semi-continuously" means that there are at least two feeding periods, preferably at least three feeding periods, or at least four feeding periods, each of which is interrupted by an interruption period. and It means that it is separated. of The supply period is a fixed period. It is okay to have , for a non-fixed period of time It is okay (i.e., period of The lengths are different (May be) The interruption in supply will be for a certain period of time. It is okay to have , for a non-fixed period of time It is okay .enzyme of Supply period and supply of The interruptions are of equal length. But often , with periods of different lengths It is okay .

[0080] A preferred ratio of alternan sucrase enzyme:sucrose (total amount of sucrose provided) 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. the law of nature Preferably, the enzyme activity is 1,200 to 2,300 units per 1,000 g of sucrose, and more preferably, the enzyme activity is 1,500 to 2,000 units per 1,000 g of sucrose. twist These ratios are related to the total units of enzymatic activity of alternansucrase. Such units (enzymatic activity) are processFor example, when additional alternan sucrase enzyme is fed to the reactor, the total units of alternan sucrase enzyme used in the reactor are the total units of (i) the alternan sucrase enzyme initially present and (ii) all the additional alternan sucrase enzyme fed to the reactor. and The sum of these represents the total unit.

[0081] Using the above parameters it is particularly possible to arrive at a preferred DPw or DPn of the oligosaccharides, some of which are of It is mentioned in the specification.

[0082] Claim 1 process The reaction time associated with depends on the selected sucrose:acceptor ratio, the selected sucrose feed rate, the selected enzyme activity, and / or the selected total amount of sucrose. Can Typical reaction times are in the range of 10 to 150 hours, or 10 to 30 hours, or 10 to 24 hours, or 10 to 20 hours. Alternan polysaccharides and alternan sucrase enzymes produced as by-products are removed by further membrane filtration or the retentate obtained by the further membrane filtration may be concentrated, as described elsewhere herein. of No steps are included.

[0083] Claim 1 Related to the process of The reaction temperature is preferably within the range of 30 to 60° C. A specific temperature range or a different temperature but , different processes Stage or applied in steps Good .

[0084] In one embodiment, the sucrose is fed continuously; in moles of sucrose per hour The sucrose supply rate is equal to or substantially equal to the continuous removal rate of fructose in terms of moles of fructose per hour, or the ratio of the sucrose supply rate to the fructose removal rate is within the range of 1:2 to 1:1. It is preferred that the same molar amount of fructose is produced per mole of sucrose consumed in the production of alternan-oligosaccharides, and that this molar amount is continuously removed.

[0085] The present invention will now be illustrated by means of examples which should not be construed as limiting the general idea of ​​the invention as described above and in the claims. [Brief description of the drawings]

[0086] [Figure 1] Figure 1(a) shows a schematic of the prior art process, and Figure 1(b) shows a schematic of the process of the present invention. [Diagram 2] Figure 2(a) shows the prior art process design, and Figure 2(b) shows the inventive process design. [Diagram 3] FIG. 3 is a chart showing molecular weight increase versus process time at a constant sucrose feed rate. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0087] [method] (HPAEC-PAD by DP measurement) DP values ​​were measured using HPAEC-PAD after reduction and hydrolysis of glucose-based sugars. Two milliliters of a solution containing 6 g / mL of digestible carbohydrate composition was in 0.5M ammonia The reduced sample was treated with 0.2 mL of NaBH4 solution (40 mg / mL) at 40 °C for 30 min. The reduced sample was hydrolyzed with 0.5 mL of 2 M trifluoroacetic acid heated at 121 °C for 1 h to obtain the monomer. release He made him do so. release The separated monomers were quantified by injecting the sample solution into a Thermo Scientific™ Dionex™ ICS-6000 ion chromatograph system equipped with a CarboPac™ MA1 and feeding the eluent (water and 1000 mM NaOH) at 0.4 mL / min. The DP value was calculated using the following formula:

[0088]

number

[0089] (DP measurement by GPC-RI) DP value was measured by GPC-RI instead. Samples were diluted with water, Two Tosoh poles connected in series TSK GEL G2500 PWXL column of Use , underwater (0.5mL / min) The oligosaccharides were subjected to gel permeation chromatography. During the permeation, the oligosaccharides were separated by their relative molecular weight and quantified using refractometric detection. The quantification of the molecular weight was based on the external standard method using standards (PSS-Polymer Standards) ranging from 342 to 5000 Da. For calibration and calculation, Chromeleon Extension Pack V2.0 was used according to the instructions of Dionex. EXAMPLES

[0090] [Example 1] Prior art processes are described, for example, in US Pat. and 3 and includes the following four steps P1 to P4 as shown in FIG. 1(a).

[0091] 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 7:1 (w / w or mol / mol). but Step P1 is carried out in a batch reactor 1 for bioconversion 2 shown in FIG.

[0092] Step P2 is an ultrafiltration step 3 for removing alternan polymers (alternan polysaccharides) and alternan sucrase enzyme (AlSu). This step is carried out in an ultrafiltration device 3' shown in Figure 2(a).

[0093] In step P3, fructose is removed by nanofiltration 5. This step is carried out in a nanofiltration device 5' as shown in FIG. 2(a). 4´ is added and a mixture of water 4″ and fructose 6 is removed.

[0094] In the final step P4, the product from step P3 is concentrated by evaporation 7. This is carried out in evaporator 7' in Figure 2(a) to obtain maltose alternan oligosaccharides (MAOS) 8.

[0095] In a particular embodiment, the process of the present invention comprises three steps S1 to S3 shown in FIG. 1(b).

[0096] In comparison with the prior art process of FIG. 1(a), the bioconversion 2 in step S1 consists of a continuous feed 10 of sucrose 9 and a continuous removal of fructose 6. Semi-continuous feed and removal too Yes. The mass ratio of sucrose 9 (total amount added) to maltose 12 is 19:1 (19 kg of sucrose to 1 kg of maltose). and , the duration is about 72 hours. Since fructose 6 has already been removed in step S1, step P3 in the prior art can be omitted. By removing fructose 6, a higher ratio of sucrose 9 to maltose 12 can be used; and It is possible to increase the degree of polymerization of maltose alternan oligosaccharide 8. It becomes possible .

[0097] The bioconversion step S1 is carried out in a reactor 11 in FIG. 2(b), where maltose 12 and the enzyme alternansucrase (AlSu) 13 are converted into water 4. Medium As mentioned above, biotransformation teeth , a continuous supply 10 of sucrose 9 and a continuous removal of fructose 6, for a duration of about 72 h (variable), T = 37°C, with a sucrose to maltose ratio of 19:1 (w / w or mol / mol). (dissolved in water)The feed 10 is added and the contents of the reactor 11 are stirred. In the bioconversion in the reactor 11, alternan oligosaccharides containing the acceptor molecule maltose, also called maltose alternan oligosaccharides 8 (MAOS), are produced as the main product, and alternan polymers 14, fructose 6 and leucrose 15 are produced as by-products. The contents from the reactor 11 are continuously circulated through a membrane cell (diafiltration cell) 16, In this embodiment, With nanofiltration Yes, performed as constant volume diafiltration In the membrane filtration 17, water 4", fructose 6 and leucrose 15 are removed. The content of leucrose 15 is reduced from about 30% to less than 10% compared to the prior art. The removed water 4" is replaced by a feed stream of water 4'.

[0098] Reactor 11 and membrane cell 16 constitute a combined bioconversion and nanofiltration device, also referred to as a reactor system.

[0099] In this embodiment Process Step S2 corresponds to step P2 of the prior art, where the by-product alternan polysaccharide (alternan polymer) 14 and the enzyme alternan sucrase (AlSu) 13 are removed by ultrafiltration 3 in a device 3'. This step is used if more alternan polymer 14 than desired is produced. or , it is useful to manipulate the desired DPw of the remaining alternan species.

[0100] In this embodiment Process Step S3 corresponds to step P4 of the prior art, where the product is concentrated by evaporation 7 in device 7'.

[0101] The composition of the reaction solution used during production is shown in Table 1. The 2.1 L solution, together with the water (dead volume) in the system, will be approximately 5.6 L. The process is carried out at 40° C. for the first hour to minimize the potential loss of maltose through the membrane. Reduce The nanofiltration membrane is then Filmtec NF270-2540(DOW) Always reduce After chain extension is complete, the nanofiltration module is TRISEP 2540-UE50-QXF The ultrafiltration module was replaced with a new one (Microdyn Nadir). R The maltose alternan oligosaccharides (MAOS) fraction was separated from the longer retrograded chains and enzymes by the ELISA. The membranes and process parameters used in this process are summarized in Table 2. The filtrate was finally concentrated to a dry matter content of more than 72%.

[0102] [Table 1]

[0103] [Table 2]

[0104] Figure 3 shows the process Progression The increase in chain length with increasing The value is , recorded by GPC-RI measurement 。 Mw in Figure 3 value Relation for calculating DPw from formula teeth, below As stated above.

[0105]

number

[0106] Therefore, it is understood that at the end of the process, a DPw of approximately 15.4 is reached (2500 / 162).

[0107] 19 kg of sucrose (total) for every 1 kg of maltose to give an average chain length of about DPw 15. but use was done .

[0108] [Comparative Example] Alternan-oligosaccharides were produced according to the process shown in Figure 1(a) in a ratio of 21:1 (kg sucrose:kg maltose). of The DPw was measured using GPC-RI, and the protocol for the method described in the method section above was used. Did not fully comply with Nevertheless, as compared with the results in FIG. 3, it is shown that according to the present invention, alternan having a higher DPw can be obtained.

[0109] The DPw values ​​of the two samples obtained by the method of the present invention, analyzed by the HPAEC-PAD method, are summarized in Table 3 below.

[0110] [Table 3]

[0111] Samples DCC-2 and DCC-3 are process It was manufactured by

[0112] The glycosidic linkage profile of glucose-based oligosaccharides was measured by GC-MS using partially methylated alditol acetates. Briefly, samples were dissolved in anhydrous DMSO, deprotonated by the addition of n-butyllithium (Sigma 230707), and methylated with methyl iodide (Sigma 289566). The methylated samples were then hydrolyzed with 2N TFA (121 ℃ The hydrolyzed samples were evaporated under a nitrogen air draft, redissolved in 1 M ammonium hydroxide, and the aldehyde groups were reduced with a solution of sodium borodeuteride (20 mg / ml) in DMSO. The reaction was stopped by dropwise addition of glacial acetic acid and acetylated by adding 1-methylimidazole and acetic anhydride. The partially methylated alditol acetates in acetone were purified 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 In °C 3 minutes, 4 ℃ / min to 270 In °C 20 min) using GC-MS (7890A-5975C MSD, Agilent Technologies, Inc., Santa Clara, CA, USA). transformation Electron impact spectra were acquired at 69.9 eV over the mass range of 50–550 Da.

[0113] [Table 4]

[0114] The higher values ​​of 1,6-glycosidic bonds are explained by the leucrose content in the digestible carbohydrate composition, which contributes to the amount of 1,5,6-tri-O-acetyl-1-deuterio-2,3,4-tri-O-methyl-D-glucitol observed. Furthermore, leucrose, like monomeric glucose, contributes to the amount of terminal Glc in the digestible carbohydrate composition.

[0115] [Example 2] this In the examples, the parameters were changed as shown in the table below, and the alternansucrase was fed to the reactor in 4 equal portions: first was present before the sucrose was fed into the reactor.

[0116] [Table 5]

[0117] The process of the present invention In Reaction time is (Further steps such as removing alternan polysaccharides and alternan sucrase enzyme by further membrane filtration, or concentrating the retentate obtained by further membrane filtration are not included here.) , can be shortened by increasing the amount of sucrose, increasing the sucrose feed rate, increasing the enzyme activity and increasing the temperature. [Explanation of symbols]

[0118] P1: Batch biotransformation P2: Ultrafiltration P3: Nanofiltration P4: Concentration S1: Biotransformation S2: Ultrafiltration S3: Concentration 1: Batch reactor 2: Biotransformation 3: Membrane filtration (ultrafiltration in the example) 3': Membrane filtration device (ultrafiltration device in the embodiment) 4, 4', 4': Aqueous liquid (water in the embodiment) 5: Nanofiltration 5´: Nanofiltration device 6: Fructose 7: Evaporative dehydration 7´: Evaporation dehydration device 8: Alternan oligosaccharide (in the embodiment, maltose alternan oligosaccharide (MAOS)) 9: Sucrose 10: Supply 11: Reactor 12: Acceptor molecule (maltose in this example) 13: Alternansucrase (AlSu) 14: Alternan polymer (alternan polysaccharide) 15: Roy Close 16: Membrane-containing device (membrane cell in the embodiment) 17: Membrane filtration (examples include bioconversion and nanofiltration (constant volume diafiltration))

Claims

1. A method for producing a sucrose-based catalyzed catalysis comprising contacting sucrose (9) with a catalytically effective amount of an alternan sucrase enzyme (13) and an acceptor molecule (12) in a stirred tank reactor (11) with stirring, the alternan sucrase enzyme (13) and the acceptor molecule (12) are present in an aqueous liquid (4) in the reactor (11), the alternan sucrase enzyme being dissolved, emulsified or suspended in the aqueous liquid, and the sucrose (9) is continuously or semi-continuously fed to the reactor (11), the sucrose (9) and the acceptor molecule (12) are converted to alternan oligosaccharides (8) with fructose (6) being produced as a by-product; Continuously or semi-continuously removing at least a portion of the fructose (6) from the reactor (11) by membrane filtration (17), removing at least a portion of the fructose (6) comprises continuously or semi-continuously circulating the contents of the reactor (11) through a device (16) containing a membrane and contacting the contents of the reactor (11) with the membrane, wherein at least a portion of the fructose (6) and a portion of the aqueous liquid (4'') pass through the membrane and the remainder is returned to the reactor, a molar ratio of sucrose (9):acceptor molecules (12), i.e., the total amount of sucrose (9) provided relative to the total amount of acceptor molecules (12) used, of 10:1 to 30:1; A method for producing alternan oligosaccharides (8), the alternan oligosaccharides having an average degree of polymerization DPw of greater than 14.

2. 2. The method of claim 1, wherein the membrane filtration (17) is diafiltration.

3. 3. The process of claim 1 or 2, further comprising continuously or semi-continuously feeding additional aqueous liquid to the reactor.

4. 10. The method of claim 1, further comprising continuously or semi-continuously feeding additional aqueous liquid to a reactor system comprising the reactor (11) and the device (16) comprising a membrane.

5. The method according to any one of claims 1 to 4, further comprising removing (S2) at least a part of the alternan polysaccharides (14) produced as a further by-product and at least a part of the alternan sucrase enzyme (13), preferably by a further membrane filtration (3), in which the alternan oligosaccharides (8) are contained in the permeate.

6. The method according to any one of claims 1 to 5, further comprising concentrating (S3) the alternan-oligosaccharides (8).

7. The method according to any one of claims 1 to 6, wherein the average degree of polymerization DPw or DPn of the alternan-oligosaccharides (8) is adjusted by the amount of the sucrose (9) fed to the reactor (11).

8. The method according to any one of claims 1 to 7, wherein the supply of sucrose (9) is stopped when a desired average degree of polymerization DPw or DPn of the alternan-oligosaccharides (8) is reached.

9. The method according to any one of claims 1 to 8, wherein the average degree of polymerization DPw of the alternan oligosaccharides (8) is in the range of 5 to 30 as measured by GPC-RI.

10. The method according to any one of claims 1 to 8, wherein the average degree of polymerization DPw of the alternan oligosaccharides (8) is in the range of 10 to 20 as measured by GPC-RI.

11. The method according to any one of claims 1 to 10, wherein the acceptor molecule (12) is a maltose molecule.

12. 12. The method according to any one of claims 1 to 11, characterized in that the sucrose (9) is fed continuously and the feed rate of sucrose (9) in molar amounts of sucrose per hour is equal or substantially equal to the continuous removal rate of fructose (6) in molar amounts of fructose per hour, or the ratio between the feed rate of sucrose (9) and the removal rate of fructose (6) is in the range of 1.2:1 to 1:

1.

13. The process according to any one of the preceding claims, wherein further alternan sucrase enzyme is preferably continuously or semi-continuously fed to the reactor (11).

14. The method according to any one of claims 1 to 13, wherein the ratio of alternan sucrase enzyme to the total amount of sucrose (9) supplied is 1000 to 10000 units (enzyme activity):1000 g of sucrose (9).

15. A method according to any one of claims 1 to 14, wherein the average degree of polymerization DPw of the alternan oligosaccharides is within the range of 14 to 32.

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