Improved methods for producing isomalto-oligosaccharides

Abstract

A method for producing improved isomalto-oligosaccharides (IMOs) from maltodextrins is described. The improved method involves the complete or partial replacement of the β-amylase used in conventional methods with a selected α-amylase. The resulting IMOs have longer chain lengths and reduced residual glucose content compared to IMOs produced using conventional methods.

Description

[Technical Field]

[0001] The present method is for producing improved isomalto-oligosaccharides (IMOs) from maltodextrins. The improved method involves the complete or partial replacement of the β-amylase used in conventional methods with a selected α-amylase. The resulting IMOs have longer chain lengths and reduced residual glucose content compared to IMOs produced using conventional methods. [Background technology]

[0002] Isomalto-oligosaccharides (IMOs) are partially digestible sugar-based food ingredients that offer health benefits to humans and other animals. IMOs are metabolized to a lesser extent than more widely used sugars, such as glucose, fructose, and sucrose, thereby providing texture and sweetness benefits with fewer calories compared to metabolizable sugars. IMOs may also provide the gut microbiota with a carbon source that influences the growth of desirable bacterial subpopulations. IMOs appear to lower intraluminal pH, inhibit the growth and activity of enteric pathogens, and stimulate the production of short-chain fatty acids in the gut. Because IMOs have a low glycemic index, they are desirable for diabetics, and because they are not metabolized by most oral bacteria, they are desirable for preventing dental caries.

[0003] Chemically, IMOs are a mixture of various oligosaccharides and glucose produced from maltodextrin. This mixture consists of linear oligosaccharides (malto-oligosaccharides) and branched oligosaccharides (isomalto-oligosaccharides). Traditionally, IMOs are produced from maltodextrin by the sequential or simultaneous action of β-amylase and transglucosidase. β-amylase produces maltose from maltodextrin, and maltose is the substrate for transglucosidase. Maltose is the donor molecule in the transglycosidation reaction, which hydrolyzes maltose, liberating one free glucose molecule and transferring the other glucose molecule to an acceptor.

[0004] The acceptor can be another maltose molecule, giving rise to a trisaccharide. The most abundant trisaccharide formed is panose. Glucose can also be transferred to higher sugars to give longer chain isomalto-oligosaccharides, to glucose to give rise to isomaltose formation, or to water to release it as another free glucose molecule. The rate at which the various oligosaccharides are formed depends on the concentrations of the various acceptors. Early in the reaction, high maltose concentrations are observed, resulting primarily in the formation of panose. As maltose concentrations decrease and panose concentrations increase later in the reaction, the more likely reaction product, the tetrasaccharide (Glc(α-1,6)Glc(α-1,6)Glc(α-1,4)Glc), will be formed. Summary of the Invention [Problem to be solved by the invention]

[0005] Unfortunately, each time a glucose molecule is transferred to an acceptor, a free glucose molecule is released from the donor maltose molecule. This results in an IMO syrup with a fairly high glucose content. While glucose can be removed from IMO syrup by chromatography, this process is expensive. A method is needed to reduce the amount of glucose present in IMO syrup made from starch substrates and improve the overall quality of the IMO. [Means for solving the problem]

[0006] Described herein are improved processes for producing isomalto-oligosaccharides (IMOs) that can yield longer chain IMOs and / or reduced amounts of glucose. Aspects and embodiments of the compositions and methods are described in the following independently numbered paragraphs. 1. In a first aspect, there is provided an improved process for producing isomalto-oligosaccharides (IMOs) from maltodextrin, comprising the steps of (i) contacting the maltodextrin with an α-amylase to produce malto-oligosaccharides, and (ii) contacting the malto-oligosaccharides with a transglucosidase to produce IMOs, wherein the process produces longer chain IMOs and / or reduced amounts of glucose compared to processes using β-amylase to produce IMOs from maltodextrin in step (i). 2. In some embodiments of the method of paragraph 1, step (i) is carried out in the presence of β-amylase at 660 saccharifying power (DP°) units per kg dry weight of maltodextrin or less. 3. In some embodiments of the method of paragraph 1, step (i) is carried out in the presence of β-amylase at 264 saccharifying power (DP°) units or less per kg dry weight of malto-oligosaccharides. 4. In some embodiments of the method of paragraph 1, step (i) is carried out in the presence of β-amylase having no more than 132 saccharifying power (DP°) units per kg dry weight of malto-oligosaccharides. 5. In some embodiments of the method of paragraph 1, step (i) is carried out in the presence of β-amylase at 66 saccharifying power (DP°) units or less per kg dry weight of malto-oligosaccharides. 6. In some embodiments of the method of paragraph 1, step (i) is carried out in the absence of β-amylase. 7. In some embodiments of the method of any of paragraphs 1-6, step (i) is carried out using an α-amylase that produces malto-oligosaccharides containing at least 15% DP3. 8. In some embodiments of the method of any of paragraphs 1-7, step (i) is carried out using an α-amylase that produces malto-oligosaccharides containing at least 10% DP4. 9. In some embodiments of the method of any of paragraphs 1-8, step (i) is carried out using an α-amylase that produces malto-oligosaccharides containing at least 5% DP5. 10. In some embodiments of the method of any of paragraphs 1-9, step (i) is carried out using an α-amylase that produces malto-oligosaccharides containing at least 40% DP2. 11. In some embodiments of the method of paragraphs 1-10, step (i) is carried out in the presence of pullulanase. 12. In some embodiments of the method of any of paragraphs 1-11, steps (i) and (ii) are performed sequentially. 13. In some embodiments of the method of paragraphs 1-11, steps (i) and (ii) are carried out simultaneously. 14. In some embodiments of the method of any of paragraphs 1-13, the maltodextrin is prepared from a starch-containing substrate using a liquefying α-amylase. 15. In some embodiments of the method of paragraph 14, the liquefying α-amylase and the α-amylase used in step (i) are the same. 16. In another aspect, an improved method for producing isomalto-oligosaccharides (IMOs) is provided, comprising the steps of: (i) contacting a starch-containing substrate with a liquefying α-amylase to produce maltodextrins; (ii) contacting the maltodextrins with a DP3+ producing α-amylase to produce malto-oligosaccharides; and (iii) contacting the malto-oligosaccharides with a transglucosidase to produce IMOs having longer chains compared to the IMOs produced using the DP3+ producing α-amylase in step (ii). 17. In some embodiments of the method of paragraph 16, the DP3+ producing α-amylase produces malto-oligosaccharides that contain at least 15% DP3, at least 10% DP4, at least 5% DP5, and / or up to 40% DP2. 18. In some embodiments of the method of paragraphs 16-17, steps (i) and (ii) and / or steps (ii) and (iii) are sequential, overlapping, or simultaneous. 19. In some embodiments of the method of any one of paragraphs 16-18, step (ii) is carried out in the presence of β-amylase having no more than 660 saccharifying power (DP°) units per kg dry weight of maltodextrin, no more than 264 saccharifying power (DP°) units per kg dry weight of maltodextrin, no more than 132 saccharifying power (DP°) units per kg dry weight of maltodextrin, or no more than 66 saccharifying power (DP°) units per kg dry weight of maltodextrin. 20. In some embodiments of the method of any one of paragraphs 16-19, step (ii) is carried out in the absence of β-amylase. 21. In some embodiments of the method of any one of paragraphs 16-20, step (ii) is carried out in the presence of pullulanase. 22. In some embodiments of the method of any one of paragraphs 16-21, the liquefying α-amylase and the DP3+ producing α-amylase used in step (ii) are the same. 23. In another aspect, there is provided an IMO produced by the method of any one of paragraphs 1-22.

[0007] These and other aspects and embodiments of the present compositions and methods will become apparent from the description and accompanying drawings herein. [Brief explanation of the drawings]

[0008] [Figure 1] 1 is a flow chart showing the steps and enzymes involved in a conventional process for preparing IMOs.

[0009] [Figure 2] 1 is a flow chart showing the steps and enzymes involved in the improved process of the present invention for preparing IMOs.

[0010] [Figure 3] FIG. 1 shows the transglucosidase reaction that occurs in a conventional process for preparing IMOs.

[0011] [Figure 4]FIG. 1 shows the reaction between a transglucosidase-glucose complex and a malto-oligosaccharide acceptor molecule to produce isomalto-oligosaccharides.

[0012] [Figure 5] FIG. 1 shows the reaction between a malto-oligosaccharide donor molecule and transglucosidase to produce a transglucosidase-glucose complex that can react with a malto-oligosaccharide acceptor molecule to produce isomalto-oligosaccharides. DETAILED DESCRIPTION OF THE INVENTION

[0013] I. Definition Before describing the processes and compositions of the present invention in detail, the following terms are defined for clarity. Terms not defined shall be accorded their ordinary meaning as used in the art.

[0014] As used herein, the term "starch" refers to a starch having the formula (CH 10 O5) x "Finally, refined starch" refers to any substance composed of complex polysaccharide carbohydrates from plants, which are composed of amylose and / or amylopectin having the formula (X), where X can be any number. Specifically, the term refers to any plant-based substance, including, but not limited to, grains, grasses, tubers, and roots, more specifically wheat, barley, corn, rye, rice, sorghum, bran, cassava, millet, potato, sweet potato, and tapioca. After purification of the complex polysaccharide carbohydrate from other plant components, it is called "refined starch."

[0015] The term "granular starch" refers to uncooked (raw) starch that has not undergone gelatinization.

[0016] As used herein, "maltodextrin" generally refers to oligosaccharides produced from starch by partial chemical or enzymatic hydrolysis. The size of the polysaccharides generally ranges from DP3 to DP20, but can be longer chains.

[0017] As used herein, "malto-oligosaccharide" refers to an oligosaccharide of glucose linked via an α-D-1,4 bond. Exemplary malto-oligosaccharides and their abbreviated IUPAC names (referring to the IUPAC terminology recommended by the IUB-IUPAC Joint Committee on Biochemical Nomenclature (JCBN) (1982) J. Biol. Chem. 257:3347-51) include, but are not limited to, maltose (Glc(α-1,4)Glc), maltotriose (Glc(α-1,4)Glc(α-1,4)Glc), and maltotetraose (Glc(α-1,4)Glc(α-1,4)Glc(α-1,4)Glc).

[0018] As used herein, "isomalto-oligosaccharides (IMOs)" generally refer to oligosaccharides of glucose containing α-D-1,6 linkages. Exemplary isomalto-oligosaccharides and their abbreviated IUPAC names (Id.) include, but are not limited to, isomaltose (Glc(α-1,6)Glc), isomaltotriose (Glc(α-1,6)Glc(α-1,6)Glc), and isomaltotetraose (Glc(α-1,6)Glc(α-1,6)Glc(α-1,6)Glc). Branched-chain oligosaccharides containing both α-D-1,4 and α-D-1,6 linkages, such as panose (Glc(α-1,6)Glc(α-1,4)Glc), are often considered to be IMOs as well. As used herein, IMOs may contain some α-D-1,4 linkages.

[0019] As used herein, the phrase "degree of polymerization (DP)" refers to the number of anhydroglucopyranose units (n) in a given sugar. An example of a DP1 is the monosaccharide glucose. Examples of DP2 are the disaccharides maltose and isomaltose.

[0020] As used herein, "α-amylase" is an intracellularly acting enzyme with the systematic name α-D-(1→4)-glucan glucanohydrolase and Enzyme Commission designation EC 3.2.1.1.

[0021] As used herein, a "starch processing enzyme" is an enzyme that depolymerizes starch substrates (including maltodextrins). Exemplary starch processing enzymes are α-amylase, glucoamylase, β-amylase, pullulanase, and α-glucosidase.

[0022] As used herein, a "maltogenic enzyme" is an enzyme that produces primarily maltose as a product. Such enzymes include extracellularly acting enzymes in the classification EC 3.2.1.2. Some primarily intracellularly acting enzymes, such as maltogenic α-amylases (EC 3.2.1.133), also produce significant amounts of maltose and would be considered "maltogenic enzymes" for purposes of the present invention.

[0023] As used herein, the term "malto-oligosaccharide-producing enzyme" refers to an enzyme that primarily produces malto-oligosaccharides with a degree of polymerization greater than 2. Such enzymes include, but are not limited to, EC 3.2.1.1, EC 3.2.1.116, EC 3.2.1.60, and 3.2.1.98.

[0024] As used herein, "transglucosidase" is synonymous with the terms α-glucosidase and the systematic name α-D-glucoside glucohydrolase, which has the Enzyme Commission designation EC 3.2.1.20.

[0025] As used herein, "pullulanase" is synonymous with the systematic name α-dextrin endo-1,6-alpha-glucosidase, which has a CAZY enzyme database designation of EC 3.2.1.41. Other debranching enzymes, such as isoamylase (EC 3.2.1.68), that have activity on branched-chain maltodextrins are considered "pullulanases" for the purposes of the present invention.

[0026] As used herein, "contacting" an enzyme with a substrate refers to bringing the enzyme and substrate together in a general aqueous environment, typically accompanied by mixing to achieve uniform distribution. The term "contacted" is used interchangeably with "treated."

[0027] As used herein, "producing" refers to producing a reaction product as a result of an enzymatic process.

[0028] As used herein, the singular articles "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. All references cited herein are incorporated by reference in their entirety. The following abbreviations / acronyms have the following meanings unless otherwise specified: ℃ Celsius temperature BBA Barley β-amylase DE Dextrose Equivalent DP Degree of Polymerization DP° saccharification power (unit of β-amylase activity) DP3+ DP3 or higher DPn DP with unknown values DS dry solid g or gm grams HPAE High-Performance Liquid Anion Exchange Chromatography HPLC High Performance Liquid Chromatography hr time IM2 Isomaltose IM3 isomaltotriose IM4 Isomaltotetraose IM5 Isomaltopentaose IM6 Isomalthexaose IM7 Isomaltoheptaose IMO isomalto-oligosaccharides IUPAC International Union of Pure and Applied Chemistry kg kilogram M mole mg milligram min mL and ml milliliters mm millimeters mM millimolar MT Metric Ton NaAc sodium acetate NaOH Sodium hydroxide PAD Pulsed Amperometric Detection PU pullulanase RI refractive index RPM or rpm Revolutions per minute TG transglucosidase U or u units w / v weight / volume μg microgram μL and μl microliters μm micrometer μM micromolar

[0029] II. Enzymatic Processes for Producing Improved IMOs An improved enzymatic process for producing isomalto-oligosaccharides (IMOs) from maltodextrins using transglucosidase is described, which utilizes α-amylase instead of β-amylase. The improved process produces longer chain IMOs using less glucose than conventional methods, allowing for more economical production of high-IMO, low-glucose specialty syrups.

[0030] The improved process can be visualized using the accompanying drawings. As shown in the flowchart in Figure 1, IMOs are conventionally produced from maltodextrins by the sequential or simultaneous action of β-amylase and transglucosidase. In the two-step process of the conventional method (left side of the flowchart), a starch slurry is converted to maltodextrins in a liquefaction process, and the maltodextrins are treated with β-amylase and pullulanase to produce maltose syrup, which is then treated with transglucosidase to produce IMOs. In the one-step process of the conventional method (right side of the flowchart), a starch slurry is converted to maltodextrins in a liquefaction process, and the maltodextrins are treated simultaneously with β-amylase, pullulanase, and transglucosidase to produce IMOs without isolating or separating the maltose syrup.

[0031] As shown in the flowchart in Figure 2, the improved process may continue the conversion of starch slurry to maltodextrins in the liquefaction process. However, the maltodextrins are currently treated with enzymes capable of producing malto-oligosaccharides longer than DP2 (i.e., maltose). Instead, DP3 (or longer-chain malto-oligosaccharide)-producing enzymes and pullulanase are used to produce a maltotriose (or longer-chain oligosaccharide)-rich syrup, which is then treated with transglucosidase to produce improved IMOs in a two-step process (left side of the flowchart). Alternatively, the maltodextrins are treated simultaneously with DP3 (or longer-chain malto-oligosaccharide)-producing enzymes, pullulanase, and transglucosidase to produce improved IMOs without isolating or separating the maltotriose (or longer-chain malto-oligosaccharide)-rich syrup in a one-step process (right side of the flowchart).

[0032] The advantages of the improved method will become apparent upon examination of the end products of the transglucosidase reaction. In Figures 3-5, glucose molecules are represented by circles, and glycosidic bonds are represented by lines connecting the circles. Glucose molecules with reducing ends are represented by solid circles, donor glucose molecules are represented by checkered circles, glucose acceptor molecules are represented by open circles, and unreactive glucose molecules are represented by gray circles. Free glucose molecules that have reducing ends but also function as acceptor glucose molecules are represented by half-open, half-black circles.

[0033] In conventional processes, especially early in the transglycosidation reaction, maltose produced from starch hydrolysate by β-amylase is abundant and serves as both the predominant donor and acceptor molecule for transglucosidase (Figure 3A). This, along with free glucose, results in the production of trisaccharides, the most abundant of which is panose. Later in the reaction, as maltose is depleted, longer-chain acceptor molecules become considerably more abundant, producing longer-chain IMOs along with the free glucose (Figure 3B). The free glucose itself can function as an acceptor for transglucosidase, with the release of isolated glucose, in which case short-chain IMOs are the product, but again simply a different free glucose (Figure 3C).

[0034] Figure 5 illustrates the benefits of the improved process. As noted above, when maltose is the donor malto-oligosaccharide, as in conventional IMO production processes, free glucose is formed during each transglucosidase reaction (Figure 5A). In contrast, when longer-chain donor malto-oligosaccharides, such as maltotriose (Figure 5B), maltotetraose (Figure 5C), or longer, are used, the transglucosidase reaction does not produce free glucose during the first part of the reaction to form a transglucosidase-glucose complex. The transglucosidase-glucose complex formed as in Figures 4B and 4C can interact with acceptor oligosaccharides of various chain lengths to produce IMOs. During this part of the reaction, free glucose is not produced (Figure 5).

[0035] III. Enzyme Composition A. DP3+-producing α-amylase DP3+ generating (also called DP3+ producing) α-amylases suitable for producing malto-oligosaccharides for use in the improved process are α-amylases that produce malto-oligosaccharides longer than DP2 (i.e., maltose) from maltodextrins. Such enzymes produce DP3, DP4, DP5, or longer malto-oligosaccharides. Enzymes that produce significant amounts of DP3 include, but are not limited to, α-amylases from Aspergillus species, such as A. kawachi, A. clavatus, and A. oryzae. Maltotriose-producing amylases have been identified in Streptomyces griseus, Bacillus subtilis, Microbacterium imperiae, and Chloroflexus aurantiacus. Enzymes that produce significant amounts of DP4 include, but are not limited to, amylases from Pseudomonas saccharophila. Enzymes that produce significant amounts of DP5 include, but are not limited to, α-amylases from several Bacillus species, including B. stearothermophilus and B. licheniformis, and Cytophaga species.

[0036] In general, a DP3+ producing α-amylase suitable for use in accordance with the present methods is any α-amylase that produces a sugar profile having a minimum of 15% DP3, a minimum of 10% DP4, or a minimum of 5% DP5, together with up to 40%, up to 30%, up to 20%, up to 10%, or even up to 5% DP2 (when the reaction is allowed to proceed for a sufficient time). Two or more DP3+ producing α-amylases can be used, in which case the combination of DP3+ producing α-amylases produces malto-oligosaccharides with the profile described above.

[0037] B. Transglucosidase The second enzyme crucial to the process for producing improved isomalto-oligosaccharides (IMOs) from malto-oligosaccharides is transglucosidase, also known as α-glucosidase and α-D-glucoside glucohydrolase. These molecules are classified as EC 3.2.1.20 enzymes in the CAZy family GH31 and have been identified in many organisms. Genbank contains over 400 entries for transglucosidases.

[0038] The enzyme exemplified herein is derived from Aspergillis niger and expressed in Trichoderma reesei. This enzyme is expressed at high levels but is not otherwise recognized to have unique properties compared to other transglucosidases studied. Therefore, numerous transglucosidases from numerous organisms are believed to be suitable for producing isomalto-oligosaccharides (IMOs) from maltodextrin.

[0039] The exemplified enzyme is commercially available as TRANSGLUCOSIDASE L2000® (DuPont Nutrition & Biosciences), with an activity of 1700 TGU (transglucosidase units) / g. 1 TGU is defined as the amount of enzyme required to produce 1 micromole of pamose per minute under the conditions of the assay. A minimum of 0.1 kg / MT of TRANSGLUCOSIDASE L2000® / MT(DS) is required. 1 kg / MT(DS) was used in all of the studies described herein.

[0040] C. Liquefying α-amylase Liquefying α-amylases for converting crude feedstocks, such as starch from grains and other plant materials, to maltodextrin are well known in the art and include enzymes derived from many microorganisms. Exemplary enzymes are commercially available, for example, under the tradename FUELZYME™ (BASF Enzymes LLC, San Diego, CA), LPHER A, AVANTEC, and LIQUOZYME products (Novozymes), and SPEZYME products (DuPont). More than one type of liquefying α-amylase can be used.

[0041] In some embodiments, the liquefying α-amylase may be further useful as a DP3+ producing enzyme for use in the improved process, depending on the profile of malto-oligomers produced, and thus the liquefying α-amylase may be or comprise a DP3+ producing enzyme.

[0042] The enzyme concentration required to produce such a glycoprofile depends on the type of reaction product it produces, the reaction conditions, and the reaction time. A trained person can determine the optimal amount. As an example, SPEZYME® ALPHA PF dosed at 0.2 kg / MT (DS) on a 12 DE liquefaction can produce a syrup with a DP5 of greater than 20% in about 7 hours.

[0043] The liquefaction of the starch can be carried out above, below or at the gelatinization temperature of the starch substrate. Other enzymes, such as proteases, can be present.

[0044] D. Enzyme Blends for Improved Process Implementation A preferred enzyme blend is one that produces a syrup with a high content of DP3 to DP5 malto-oligosaccharides from 12DE starch liquefaction in the absence of transglucosidase. High in this context means that at least 15%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% of the total malto-oligosaccharides are DP3, DP4, and / or DP5. Another way to define a high syrup is by the individual sugar components: at least 15% DP3, at least 10% DP4, at least 5% DP5, and / or up to 40%, 30%, 20%, 10%, or even 5% DP2.

[0045] Suitable enzyme blends are also or alternatively those that, in the presence of transglucosidase, produce a syrup from 12DE starch liquefaction with a content of greater than 4% isomaltopentaose, greater than 2% isomalthexaose and / or greater than 1% isomaltoheptaose as a percentage of total sugars, measured as described in the Examples.

[0046] E. No requirement for β-amylase activity An important feature of the improved processes and enzyme compositions is that they are carried out substantially in the absence of maltogenic activity, with the intention of minimizing the use of maltose as a donor for transglucosidase, thereby reducing the production of free glucose. By substantially in the absence of β-amylase activity, it is meant that enzymes classified as β-amylases or maltogenic amylases and / or enzyme compositions (e.g., blends) having β-amylase activity are not necessary or required to produce the improved IMOs described herein. Thus, β-amylase and / or β-amylase activity need not be applied to maltodextrins to produce the improved IMOs described herein.

[0047] β-Amylase activity is typically expressed in degrees of saccharification (DP°). One unit of diastase activity, expressed as DP° (degrees of DP), is defined as the amount of enzyme contained in 0.1 ml of a 5% solution of a sample enzyme preparation that will produce enough reducing sugars to reduce 5 ml of Fehling's solution when the sample is incubated with 100 ml of substrate at 20°C for 1 hour. The reducing sugars produced during the reaction are measured in an alkaline ferricyanide titration procedure. This enzyme assay measures both α-amylase and β-amylase activity present in a given sample.

[0048] Amounts of β-amylase activity that may be tolerated in the improved processes of the present invention, while still achieving the IMO quality advantages described herein, are up to about 660 DP° units of β-amylase per kg of starch hydrolysate (DS), up to about 264 DP° units of β-amylase per kg of starch hydrolysate (DS), up to about 132 DP° units of β-amylase per kg of starch hydrolysate (DS), and up to about 66 DP° units of β-amylase per kg of starch hydrolysate (DS). As noted above, measurable amounts of β-amylase need not be present.

[0049] F. Raw starch hydrolase Many amylolytic enzymes are active on raw starch, as described in U.S. Patent Nos. 7,037,704, 7,205,138, 7,303,899, and 7,378,256, and references therein. These enzymes are commonly referred to as raw amylolytic enzymes or granular starch hydrolyzing enzymes (GSHEs). GSHEs that liberate DP3 or longer chain sugars are suitable for use as described herein. GSHEs can be used in a two-step reaction in which raw starch is treated with GSHE, with or without pullulanase, to produce malto-oligosaccharides, which are then reacted with transglucosidase. GSHEs can also be used in a one-step reaction in which raw starch is treated with GSHE, with or without pullulanase, and simultaneously reacted with transglucosidase. Examples of enzymes capable of releasing oligosaccharides from raw starch include, but are not limited to, SPEZYME® ALPHA PF, SPEZYME® XTRA, Aspergillus karwachi α-amylase, and OPTIMALT® 4G.

[0050] IV. Improved IMO Features and Uses The improved process and enzyme composition allow for the production of isomalto-oligosaccharides (IMOs) from maltodextrin for several uses. The IMOs are longer chain than those produced using conventional processes, resulting in a lower glucose content in the syrup. The syrup can be physically separated into fractions having a desired DP range using methods similar to those used for conventional syrups. More specifically, the IMOs produced using the compositions and methods of the present invention are longer chain than those produced using conventional methods, resulting in an increased ratio of long-chain IMO molecule content (total sugars) to short-chain molecules. Longer-chain IMOs are likely to be less easily metabolized, providing greater health benefits to consumers and more food ingredient options for food producers.

[0051] These and other aspects and embodiments of the methods of the present invention will become apparent to those skilled in the art in light of this specification. The following non-limiting examples further illustrate the compositions and methods. [Example]

[0052] Example 1. Transglycosylation reaction using maltose and maltotriose Transglycosylation reactions were carried out with reagent-grade maltose and reagent-grade maltotriose (both purchased from Sigma-Aldrich) in water at 30% DS. A 30% DS solution of maltose or maltotriose was made and adjusted to pH 4.2. Approximately 2 g of maltose or maltotriose solution was weighed into an Eppendorf tube. Transglucosidase (TRANSGLUCOSIDASE® L-2000; DuPont) was added to each Eppendorf tube at a dose of 1 kg of product per 1 MT of substrate (DS). The tubes were incubated in a thermoblock at 60°C for 48 hours with a shaking speed of 750 rpm.

[0053] At the appropriate time points, samples were removed for HPLC analysis. A 100 μl aliquot was removed from the reaction medium, diluted 10 times with distilled water, and boiled. After filtration, 20 μl was injected into an HPLC system equipped with a Bio-Rad Aminex HPx-42A column (#1250096, 300 mm × 7.8 mm). The mobile phase was HPLC-grade distilled water and flowed at 0.6 ml / min for 22.5 minutes. The column temperature was 85°C, and detection was performed using an RI detector with a cell temperature of 40°C.

[0054] As summarized in Table 1, after 48 hours, most of the maltose and maltotriose had been consumed. The results further show that approximately half the glucose (DP1) was liberated after 48 hours when the reaction was performed on maltotriose compared to maltose. When maltose was treated with transglucosidase, branched maltotriose (DP3) was formed first, followed by the formation of branched oligosaccharides with higher degrees of polymerization (DPn), likely formed from DP3. When maltotriose was treated with transglucosidase, the more rapid formation of more highly branched oligosaccharides (DPn), likely DP4, occurred first. The results shown in Table 1 indicate that branched MOs with higher degrees of polymerization were formed by transglucosidase when the reaction started with malto-oligosaccharides with higher degrees of polymerization. This also resulted in a reduction in glucose formation. [Table 1]

[0055] Example 2. Transglycosylation reaction using DP2 and DP4 syrups Because conducting transglycosylation reactions using pure malto-oligosaccharides is commercially unattractive, the experiment in Example 1 was repeated using a starch hydrolysate enriched in DP4. This starch hydrolysate was prepared using a DP4-generating enzyme and compared with a maltose-rich starch hydrolysate prepared using β-amylase. Both starch hydrolysates were prepared from corn liquefaction with a DE of 11.28 at 32.5% DS. To produce the DP4-enriched hydrolysate, the liquefaction was incubated for 48 hours at pH 5.0 and 60°C with 0.9 kg / MT (DS) of DP4-generating α-amylase (OPTIMALT® 4G; DuPont) and 0.4 kg / MT (DS) of pullulanase (OPTIMAX® L-1000; DuPont). The same liquefact was treated with 0.9 kg / MT (DS) β-amylase (OPTIMALT® BBA; DuPont) plus 0.4 kg / MT (DS) pullulanase to produce a maltose-rich hydrolysate.

[0056] At appropriate time points, samples were removed for HPLC analysis. A 100 μl portion was removed from the reaction medium, diluted 10 times with distilled water, and boiled. After filtration, 20 μl was injected into an HPLC system equipped with a Bio-Rad Aminex HPx-42A column (#1250096, 300 mm × 7.8 mm). The mobile phase was HPLC-grade distilled water and flowed at 0.6 ml / min for 22.5 min. The column temperature was 85°C, and detection was performed in an RI detector with a cell temperature of 40°C. The sugar composition of the resulting syrup after 48 h of reaction time is summarized in Table 2. [Table 2]

[0057] These syrups were further reacted with transglucosidase to produce IMOs. The above DP2- or DP4-rich syrups were adjusted to pH 4.2 and 30% DS. A 2-g sample of either the DP4-rich syrup or the DP2-rich syrup was treated with 1 kg / MT (DS) of transglucosidase for 24 hours at 60°C. After 24 hours, the sample was removed for HPLC analysis. A 100 μl aliquot was removed from the reaction medium, diluted 10 times with distilled water, boiled, centrifuged, and filtered as described above, and then 20 μl was injected into an HPLC system equipped with the same Bio-Rad Aminex HPx-42A column using the same mobile phase, flow rate, and temperature. The sugar profiles of the starting DP2 and DP4 syrups and those obtained from the transglucosidase reaction after 24 hours are shown in Table 3. [Table 3]

[0058] The IMO syrup prepared with DP4-rich hydrolysate has a distinctly different composition compared to the syrup prepared with DP2-rich hydrolysate. DP1 levels are much lower after the TG reaction with DP4-rich syrup (17%) compared to DP2-rich syrup (28%). DP2 is also lower after the TG reaction with DP4-rich syrup, which is more enriched in longer-chain oligosaccharides, including DP5, DP6, DP7, DP8, and DP9.

[0059] Example 3. Selection of enzymes for producing DP2 to DP5 syrups The experiment described in Example 2 was repeated by applying a wider range of enzymes to maltodextrins in order to produce syrups enriched in DP2, DP3, DP4 and DP5 from maltodextrins.

[0060] The enzymes used to produce DP2-rich syrup were either 0.9 kg / MT (DS) β-amylase (OPTIMALT® BBA, as above) plus 0.4 kg / MT (DS) pullulanase (OPTIMAX® L1000, as above) or 0.5 kg / MT (DS) maltogenic amylase (OPTIMALT® 2G) with or without 0.4 kg / MT (DS) pullulanase. The enzyme used to produce DP3-rich syrup was 0.5 kg / MT (DS) DP3-producing α-amylase from Aspergillus kawachi (GC626; DuPont) with or without 0.4 kg / MT (DS) pullulanase. The enzyme used to produce DP4-rich syrup was 0.9 kg / MT (DS) of DP4-producing α-amylase (OPTIMALT® 4G, as described above) with or without 0.4 kg / MT (DS) of pullulanase. The enzyme used to produce DP5-rich syrup was either 6 μg purified protein / g (DS) of Cytophaga sp.-based α-amylase or 0.2 kg / MT (DS) of Bacillus stearothermophilus-based α-amylase (SPEZYME® ALPHA PF), with or without 0.4 kg / MT (DS) of pullulanase.

[0061] The amylase enzymes mentioned above are referred to as BBA, 2G, 626, 4G, CspAmy, and PF, respectively, in this and all remaining examples. The pullulanase used in all remaining examples was OPTIMAX® L1000, and the transglucosidase was TRANSGLUCOSIDASE L-2000, unless otherwise specified.

[0062] For all reactions, 10 g of corn liquefact was incubated with the enzymes identified above at the indicated dosages and pH for 48 hours at 60° C. A 100 μl aliquot was removed from the reaction medium and used to perform HPLC analysis, as described above.

[0063] In all cases, the addition of pullulanase to the reaction was clearly desirable to increase the level of desired sugars and reduce the amount of higher sugars (DPn). Consequently, only results using pullulanase will be discussed. Using a DP3-producing α-amylase (GC626), a syrup with approximately 32.5% DP3 and 39% DP2 was obtained. Using a DP4-producing α-amylase (OPTIMALT® 4G), a syrup containing approximately 44% DP4 was obtained. Using a DP5-producing α-amylase (CspAmy or SPEZYME® ALPHA PF), a syrup containing approximately 29% and 21% DP5, respectively, was obtained. The sugar composition (% of total sugars) of all these reactions is shown in Table 4. The enzyme abbreviations used in the table are readily apparent from the above descriptions. [Table 4]

[0064] In the second step, the above syrup was adjusted to pH 4.2 and 30% DS, and 2 g of each was further reacted with 1 kg / MT (DS) of transglucosidase at 60°C for 24 hours. As described above, a 100 μl aliquot was removed from the reaction medium and used for HPLC analysis. A second sample was prepared for analysis by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD). While other methods separate sugars based on size (monomer, dimer, etc.), HPAE-PAD can separate isomers such as maltotriose, panose, and isomaltotriose. Specifically, a 100 μl sample was removed, diluted 1,000-fold, and boiled for 10 minutes. After filtration, 10 μl of the sample was injected onto a Carbopac PA200 column (3 mm × 250 mm) equipped with a guard column at a flow rate of 0.5 ml / min and a temperature of 30°C. PAD was performed at a cell temperature of 25°C. The following conditions were used during the 60-minute chromatography run: (i) Prior to sample injection, the column was equilibrated with 10% 1M NaOH, 10% 500mM NaOAc, and 80% MilliQ water for 10 minutes. Sugar separation was performed by elution with a constant 10% 1M sodium hydroxide and 90% MilliQ water for 5 minutes. During the next 5 minutes, a gradient was initiated with 500mM NaOAc, increasing the % NaOAc in the mobile phase from 0% to 8% and decreasing the % MilliQ water from 90% to 82%. Over the next 50 minutes, the gradient changed, decreasing the % MilliQ water in the mobile phase from 82% to 0% and increasing the % NaOAc from 8% to 90%. The gradient is shown in Table 5. [Table 5]

[0065] Two analyses were required to calculate the IMO content. Using conventional HPLC methods (described above), the percentage content of various sugars was calculated by measuring the area of the DP1, DP2, DP3, DP4, DP5, DP6, DP7, DP8, DP9, DP10, and DPn peaks from the chromatogram. For clarity, 10% DP2 means that 10% by weight of the final sugar composition is DP2, etc. For the purposes of Table 5, DPn refers to ≥ DP11. As noted above, this analysis does not provide information about the isomers present. However, isomers present, for example, within the DP2 peak can be identified by HPAE-PAD analysis. The chromatogram from this HPAE-PAD analysis revealed peaks from various isomers that could be identified based on separate analysis of standard samples with known components. Because the concentration of each component in the standard mix is known, the content of specific components in the sample can be calculated. For example, if a sample contains 1.4% w / v maltose, 7.4% w / v isomaltose, 2.4% w / v kojibiose, and 1.6% w / v nigerose based on HPAE-PAD analysis, this means that the total DP2 contains 11% maltose, 58% isomaltose, 19% kojibiose, and 12% nigerose. For example, if the DP2 content in a syrup is 10% (measured by conventional HPLC), this means that the content of isomers in the total syrup is 1.1% maltose, 5.8% isomaltose, 1.9% kojibiose, and 1.2% nigerose. In this method, DP1 isomers, DP2 isomers, and DP3 isomers can be distinguished.

[0066] For DP1, DP2, and DP3 sugars, the majority of isomers likely to be formed can be conclusively identified because pure compounds can be purchased from chemical supply companies and used as standards. For longer-chain oligosaccharides, e.g., DP4 and above, not all isomers can be identified using readily available standards. Thus, for isomers above DP4, only linear malto-oligosaccharides up to DP10, i.e., malto-tetraose, malt-pentaose, malto-hexaose, malto-heptaose, malto-octaose, malto-nanoose, and malto-decaose, are identified. Additionally, linear isomalto-oligosaccharides up to DP7, i.e., isomalto-tetraose, isomalt-pentaose, isomalto-hexaose, and isomalto-heptaose, are identified. Other, more complex, branched-chain oligosaccharides appear in the chromatogram as unidentified peaks. As oligomers become longer, the likelihood of overlapping peaks on a chromatogram also increases, making quantitation uncertain. The lack of commercially available standards and methods for separating the longer chain isomers is understandable, as practical production of these IMOs is only possible in light of the improved methods of the present invention.

[0067] To identify the percentage of DP4 that is isomaltotetraose, we hypothesized that DP4 contains only maltotetraose and isomaltotetraose. Other unidentified branched-chain tetramers were not considered in the IMO content calculation. Because branched-chain oligosaccharides are often considered to be IMOs, this calculation results in a small underestimation of the total IMO content. This is also true for longer-chain malto-oligosaccharides. For the isomers DP8 to DP11, it is assumed that all of them are linear malto-oligosaccharides. This again results in a small underestimation of the total IMO content.

[0068] The results summarized in Table 6 show the sugar composition of the transglucosidase-treated syrup as a % of total sugars as measured by HPLC and related to DP number. The results summarized in Table 7 show the IMO content in the transglucosidase-treated syrup as a % of total sugars as measured by HPAE-PAD. In this table, IM2 represents isomaltose, IM3 represents isomaltotriose, etc. [Table 6] [Table 7]

[0069] Comparing the final IMO syrups summarized in Tables 4, 6, and 7, it is clear that the longer the donor molecule in the transglucosidase reaction, the smaller the increase in DP1 (free glucose) after treatment. Furthermore, the total DP1 content becomes lower as the length of the donor molecule increases.

[0070] In the two-step reaction, where a malto-oligosaccharide-rich syrup is first produced followed by transglucosidase treatment, there is little or no decrease in DPn levels, which is likely caused by the fact that pullulanase is added in the first reaction but not during the TG reaction.

[0071] Starting with syrups rich in DP4 or DP5, less DP2 is present after transglucosidase treatment, and there is little difference in the amount of DP3 and DP4 present after transglucosidase treatment with syrups rich in either malto-oligosaccharide. The amount of DP6 to DP10 after transglucosidase treatment increases with increasing donor molecule length. Transglucosidase reactions using syrups rich in longer chain sugars resulted in a decrease in the amount of isomaltose and an increase in the amounts of isomaltohexaose and isomaltoheptaose.

[0072] The IMO content (i.e., the sum of isomaltose, isomaltotriose, panose, isomaltotetraose, isomaltopentaose, isomaltohexaose, and isomaltoheptaose) is higher when starting from a syrup rich in DP3 compared to a syrup rich in DP2. This is not the case for syrups rich in DP4 and DP5, which have a higher content of DPn, possibly due to the lack of debranching activity during the transglycosylation reaction. These unavailable, presumably branched, maltooligosaccharides can be made available by the addition of pullulanase during TG treatment, and the IMO content after transglucosidase treatment with longer donor molecules would likely be higher.

[0073] Example 4. Simultaneous malto-oligosaccharide production and transglucosidation In conventional IMO production, it is not uncommon to simultaneously contact maltodextrin with β-amylase and transglucosidase to produce IMO in a one-step reaction. In this example, the experiment described in Example 3 was repeated, but instead of first producing a malto-oligosaccharide-rich syrup, malto-oligosaccharide production and transglucosidation were carried out simultaneously. The same liquefact, enzymes, and enzyme volumes were used in this one-step reaction as in Example 3. The temperature and pH were the same as in the first step of Example 3.

[0074] Samples were removed for HPLC and HPAE-PAD analysis at appropriate times during the one-step reaction, as described above. The sugar composition of the final IMO syrup is shown in Table 9. Table 8 shows the composition of isomalto-oligosaccharides (% of total sugars) as measured by HPAE-PAD and using the same calculations described in Example 3. Panose is listed separately in Table 9 and subsequent tables. [Table 8] [Table 9]

[0075] Comparing the final IMO syrups (at the end of the one-stage reaction) summarized in Tables 8 and 9, it is clear that the pullulanase active during the TG reaction reduced the amount of DPn in the entire reaction to a much lower level than in the two-stage reaction. The degradation of higher sugars clearly continues during the one-stage reaction, resulting in better utilization of the available oligosaccharides.

[0076] As above, the content of DP1 decreases with increasing donor molecule length. Overall, more DP1 is formed in the one-step process compared to the two-step process. Even during transglycosylation with longer donor molecules, glucose is ultimately liberated. The content of DP2–DP5 is similar to that in reactions using DP3+-producing enzymes but slightly lower than that in reactions using enzymes that produce maltose. The content of DP6–DP10 also increases with increasing donor molecule length in the one-step reaction, in the order PF > CspAmy2 > 4G, 626 > BBA / 2G.

[0077] The amount of shorter chain IMOs generally appears to decrease with increasing donor molecule length, while the levels of isomaltotriose, isomaltotetraose, and isomaltopentaose appear to remain roughly the same, whereas the levels of isomaltohexaose and isomaltoheptaose increase significantly with increasing donor molecule length.

[0078] Comparing the one-step reaction with the two-step reaction, it is clear that (a) more IMO is produced in the one-step reaction, (b) less panose is produced in the one-step reaction, (c) more isomaltose is produced in the one-step reaction, and (d) more IM5, IM6, and IM7 are produced in the one-step reaction.

[0079] Example 5. Desirable Amount of β-Amylase Activity In the above examples, IMO production was carried out without the use of β-amylase and compared to conventional IMO production using β-amylase. This experiment investigated how much β-amylase could be present in the improved IMO production process without negating the benefits of the improved process. The one-step reaction using OPTMALT® 4G (without β-amylase) described in Example 4 was repeated with a range of β-amylase dosages present.

[0080] Five grams of corn liquefact (DE 12.1, 33.1% DS) was incubated with 0.9 kg / MT (DS) of DP4-producing α-amylase (OPTIMALT® 4G) at pH 5.0 for 48 hours at 60°C, along with 0.16 kg / MT (DS) of pullulanase (OPTIMAX® L2500) and 1.0 kg / MT of transglucosidase. The same reactions were performed with increasing amounts of β-amylase (OPTIMALT® BBA) as indicated in Table 10. As a control, conventional one-stage saccharification was performed at pH 5.0 and 60°C using 0.9 kg / MT of β-amylase (OPTIMALT® BBA), 0.16 kg / MT of pullulanase (OPTIMAX® L2500), and 1.0 kg / MT of transglucosidase.

[0081] Samples were removed at appropriate time points during saccharification for HPLC and HPAE-PAD analysis as described in the previous examples. The sugar composition of the final IMO syrup is shown in Table 11. Table 12 shows the isomalto-oligosaccharide composition (% of total sugars) as determined by HPAE-PAD and using the same calculations as described in Example 3. [Table 10] [Table 11] [Table 12]

[0082] From Table 11, it is clear that the reaction with OPTIMALT® 4G, as seen in the previous examples, produces a syrup with a much lower DP1 than the conventional reaction with OPTIMALT® BBA. Table 11 also shows that small amounts of β-amylase can be present during the reaction with OPTIMALT® 4G without significantly affecting the results. Specifically, when up to 0.05 kg / MT of OPTIMALT® BBA is present, DP1 is as low as when BBA is absent. Only when 0.1 kg / MT of OPTIMALT® BBA is present does DP1 begin to increase, and further increase in a dose-dependent manner with the amount of β-amylase. At a dose of 0.5 kg / MT, DP1 levels are very close to those of the conventional reaction carried out using only β-amylase.

[0083] With the exception of DP11+, the amounts of other sugars produced follow the same trend. Up to a dosage of 0.05 kg / MT of OPTIMALT® BBA, little change in the sugar profile occurs compared to the reaction without OPTIMALT® BBA. With further increases in dosage, the difference between the improved and conventional methods becomes smaller as the dosage increases. The amount of DP11+ is lower for the reaction using both OPTIMALT® 4G and OPTIMALT® BBA compared to the conventional reaction using only OPTIMALT® BBA. Clearly, the longer-chain starch fraction is better hydrolyzed when both enzymes are present.

[0084] As shown in Table 12, the amount of longer chain IMOs (see, e.g., IM6 and IM7) was higher in the reactions with OPTIMALT® 4G than in the reactions with OPTIMALT® BBA. Additionally, less IM2 and more panose were measured in this example. Overall, the total amount of IMOs produced in this experiment was higher than in previous experiments, likely due to the use of different liquefacts.

[0085] When a small amount of β-amylase (OPTIMALT® BBA) is present in the reaction along with OPTIMALT® 4G, IM2 is higher than when it is not present. This amount remains unchanged when β-amylase doses up to 0.05 kg / MT are used. At higher doses of OPTIMALT® BBA, the IM2 content increases to levels similar to those in conventional reactions using OPTIMALT® BBA alone. Similar effects are seen with other sugars.

[0086] One surprising observation is that the total IMO content increases with 0.01 kg / MT of OPTIMALT® BBA present in the OPTIMALT® 4G reaction. This may be a substantial benefit to the improved process, since small amounts of β-amylase do not increase DP1 formation. However, larger amounts of β-amylase are clearly incompatible with the improved process of the present invention.

[0087] Example 6. Conversion of β-amylase dosage to β-amylase activity units In the above examples, enzyme dosages were expressed in kg enzyme / MT substrate, which is convenient for commercial products and is customary in the industry. For the purposes of defining improved methods, the dosage of β-amylase should be expressed in terms of activity units present in the reaction.

[0088] OPTIMALT® BBA has an average β-amylase activity of 1320 DP° / g of product, where DP° represents saccharifying power. DP° determination is based on a 30-minute hydrolysis of starch substrate at pH 4.6 and 20°C. Reducing sugars produced after hydrolysis are measured in an alkaline ferricyanide titration procedure. One unit of diastase activity, expressed as DP° (DP°), is defined as the amount of enzyme contained in 0.1 ml of a 5% solution of a sample enzyme preparation that will produce enough reducing sugars to reduce 5 ml of Fehling's solution when the sample is incubated with 100 ml of substrate for 1 hour at 20°C. As an example, 0.05 kg / MT of OPTIMALT® BBA is equivalent to 50 g / MT, which is equivalent to 50 x 1320 DP° units / MT or 66,000 DP° units / MT. This amount is 66 DP° units / kg or 66 DP° units per kg of dry solids in the starch hydrolysate.

[0089] The results described in Example 5 demonstrate that the improved process of the present invention is not adversely affected by the presence of up to 66 DP° units of β-amylase activity per kg of dry solids in the starch hydrolysate. With up to 66 DP° units present, DP1 is as low as in the absence of β-amylase, with the same or higher IMO content. Presence of as little as 13.2 DP° / kg is estimated to be potentially more beneficial. However, when increased amounts of β-amylase activity are present, the benefits of the improved process are diminished in a dose-dependent manner until the IMO profile resembles that obtained by the conventional process.

Claims

1. 1. A method for producing isomalto-oligosaccharides (IMO) from maltodextrin, comprising: (i) contacting maltodextrin with a DP3+ producing α-amylase in the presence of a β-amylase having no more than 66 saccharifying power (DP°) units per kg dry weight of maltodextrin, or in the absence of a β-amylase, to produce malto-oligosaccharides; (ii) contacting the malto-oligosaccharides with a transglucosidase to produce IMOs. and producing longer chain IMOs and / or reduced amounts of glucose compared to a process for producing IMOs from maltodextrin under the same conditions except for using β-amylase instead of the DP3+ producing α-amylase in step (i), In step (ii), a transglucosidase-glucose complex is formed, and the complex reacts with the malto-oligosaccharides to produce IMOs while reducing the amount of glucose produced; The method, wherein the DP3+ producing α-amylase produces malto-oligosaccharides containing at least 10% DP4 and at least 5% DP5.

2. 2. The method of claim 1, wherein the DP3+ producing α-amylase produces malto-oligosaccharides containing at least 15% DP3.

3. The method according to claim 1 or 2, wherein the DP3+ producing α-amylase produces malto-oligosaccharides containing 40% or less DP2.

4. 4. The method according to claim 1, wherein step (i) is carried out in the presence of pullulanase.

5. The method of any one of claims 1 to 4, wherein steps (i) and (ii) are carried out sequentially.

6. The method of any one of claims 1 to 4, wherein steps (i) and (ii) are carried out simultaneously.

7. 7. The method of any one of claims 1 to 6, wherein the maltodextrin is prepared from a starch-containing substrate using a liquefying α-amylase.

8. 8. The method of claim 7, wherein the liquefying α-amylase and the DP3+ producing α-amylase used in step (i) are the same.

9. 1. A method for producing isomalto-oligosaccharides (IMO), comprising: (i) contacting a starch-containing substrate with a liquefying α-amylase to produce maltodextrins; (ii) contacting the maltodextrin with a DP3+ producing α-amylase in the presence of a β-amylase having no more than 66 saccharifying power (DP°) units per kg dry weight of maltodextrin, or in the absence of a β-amylase, to produce malto-oligosaccharides; and (iii) contacting the malto-oligosaccharides with a transglucosidase to produce IMOs. and producing longer chain IMOs and / or reduced amounts of glucose compared to a process for producing IMOs under the same conditions except that in step (ii) β-amylase is used instead of DP3+ producing α-amylase, In step (iii), a transglucosidase-glucose complex is formed, and the complex reacts with the malto-oligosaccharides to produce IMOs while reducing the amount of glucose produced; The method, wherein the DP3+ producing α-amylase produces malto-oligosaccharides containing at least 10% DP4 and at least 5% DP5.

10. 10. The method of claim 9, wherein the DP3+ producing α-amylase produces malto-oligosaccharides containing at least 15% DP3 and / or up to 40% DP2.

11. 11. The method of claim 9 or 10, wherein steps (i) and (ii) and / or steps (ii) and (iii) are sequential, overlapping or simultaneous.

12. 12. The method according to any one of claims 9 to 11, wherein step (ii) is carried out in the presence of pullulanase.

13. 13. The method according to any one of claims 9 to 12, wherein the liquefying α-amylase and the DP3+ producing α-amylase used in step (ii) are the same.

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