Method for producing complex branched isomalto-oligosaccharides

Abstract

Described herein is a method for producing complex branched isomalto-oligosaccharides (IMO) from maltodextrins. The method involves an enzyme having α-1,6-branching activity in combination with enzymes having transglucosidase and pullulanase activity to produce more complexly branched long-chain length IMO compared to IMO produced using a conventional method.

Description

[0001] METHOD FOR PRODUCING COMPLEX BRANCHED ISOMALTOOLIGOSACCHARIDES

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003]

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 597,094 filed November 8, 2023, the disclosure of which is incorporated by reference herein in its entirety.

[0004] INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0005]

[0002] The Sequence Listing submitted in an XML file, in accordance with 37 C.F.R. §§2412 is incorporated herein by reference. The xml file name is “NB42098_WO_PCT.xml”, the date of creation of the xml file is October 17, 2024, and the size of the xml file in bytes is 6,807.

[0006] TECHNICAL FIELD

[0007]

[0003] Provided herein are methods for producing complex branched isomalto-oligosaccharides (IMO) from maltodextrins. The method involves an enzyme having a-l,6-branching activity and or an amylomaltase in combination with enzymes having transglucosidase and pullulanase activity to produce more complexly branched long-chain length IMO compared to IMO produced using a conventional method.

[0008] BACKGROUND

[0009]

[0004] Isomalto-oligosaccharides (IMO) are partially digestible sugar-based food ingredients that offer health benefits to humans and other animals. IMO are metabolized to a lower extent than more widely used sugars, such as glucose, fructose and sucrose, thereby providing texture and sweetness benefits at the cost of fewer calories compared to metabolizable sugars. IMO may also supply intestinal bacterial flora with a carbon source affecting the proliferation of desirable bacterial subpopulations. IMO appear to stimulate the production of short-chain fatty acids in the intestine, lowering the intra-luminal pH and inhibiting the growth and activity of enteropathogens. IMO have a low glycemic index, making them desirable for consumption by diabetics, and are not metabolized by most oral bacteria, making them desirable for avoiding cavities.

[0010]

[0005] Chemically, IMO is a mixture of different oligosaccharides, and glucose, that is produced from maltodextrins. The mixture consists of linear oligosaccharide (malto-oligosaccharides) and branched oligosaccharides (isomalto-oligosacharides). IMO is conventionally produced from maltodextrins by the sequential or simultaneous action of a P-amylase and a transglucosidase. The P-amylase produces maltose from the maltodextrins, which is a substrate for the transglucosidase. Maltose is the donor molecule in the transglycolysation reaction, which hydrolyzes maltose, releasing one free glucose molecule and transferring the other glucose molecule to an acceptor.

[0011]

[0006] The acceptor can be another maltose molecule, resulting in a trisaccharide. The most abundant trisaccharide formed is panose. The glucose can also be transferred to a higher sugar, resulting in longer chain isomalto-oligosaccharide, transferred to glucose, resulting in isomaltose formation, or transferred to water, releasing it as another free glucose molecule. The rate at which different oligosaccharides are formed depends on the concentration of the different acceptors. Initially in the reaction, there is a high maltose concentration, resulting primarily in the formation of panose. Later in the reaction, when the maltose concentration is reduced, and the panose concentration increased, the formation of a tetrasaccharide (Glc(a-l,6)Glc(a- l,6)Glc(a-l,4)Glc), will be the more likely reaction product.

[0012]

[0007] Koops recently described an improved enzymatic process for producing isomaltooligosaccharides (IMO) from maltodextrins using transglucosidase and a-amylase, providing advantages over the conventional use of transglucosidase and P-amylase

[0013] (W02021011793A1 / US2022259630A1). The improved process resulted in longer-chain IMO with reduced amounts of glucose, which are better suited for the production of high-IMO, low- glucose specialty syrups.

[0014] SUMMARY

[0015]

[0008] Described is a process for making complex branched long-chain isomalto-oligosaccharides (IMO), which are well-suited for the production of next generation IMO specialty syrups. Aspects and embodiments of the compositions and methods are described in the following, independently-numbered paragraphs.

[0009] Provided herein is a method for producing complex branched isomalto-oligosaccharides from maltodextrins, comprising: (i) contacting maltodextrins with an enzyme to produce maltooligosaccharides, and (ii) contacting the malto-oligosaccharides with one or more of an enzyme having transglucosidase activity, an enzyme having pullulanase activity and an enzyme having a- 1,6-branching activity to produce complex branched IMO, wherein the method produces more complex branched IMO compared to an otherwise identical method for producing IMO from maltodextrins in the substantial absence of an enzyme having a-l,6-branching activity.

[0016]

[0010] In some embodiments, the enzyme in (i) comprises an enzyme with a-amylase and / or [3- amylase activity. In some embodiments, the method further comprises contacting the maltodextrins in (i) or the malto-oligosaccharides in (ii) with an enzyme having amylomaltase activity. In some embodiments, the enzyme having a- 1,6-b ranching activity comprises a dose enzyme concentration between about 0.5 -20 mg branching enzyme / liter reaction medium. In some embodiments, the enzyme having a-l,6-branching activity is glycosyl hydrolase. In some embodiments, the glycosyl hydrolase is a GH13 or a GH57 enzyme. In some embodiments, the glycosyl hydrolase comprises a sequence that is at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 1 or SEQ ID NO:2. In some embodiments, step (i) is performed in the substantial absence of a P-amylase. In some embodiments, wherein steps (i) and (ii) are performed sequentially. In some embodiments, (i) and (ii) are performed simultaneously. In some embodiments, the maltodextrins are prepared from a starch-containing substrate using a liquefying a-amylase. In some embodiments, the liquefying a-amylase and the a-amylase used in step (i) are the same.

[0017] [OH] Provided herein is a method for producing complex branched isomalto-oligosaccharides from maltodextrins, comprising: (i) contacting maltodextrins with an enzyme to produce maltooligosaccharides, and (ii) contacting the malto-oligosaccharides with one or of an enzyme having transglucosidase activity, an enzyme having pullulanase activity and an enzyme having amylomaltase activity to produce complex branched IMO,

[0018]

[0012] wherein the method produces more complex branched IMO compared to an otherwise identical method for producing IMO from maltodextrins in the substantial absence of an enzyme having amylomaltase activity.

[0013] In some embodiments, the enzyme in (i) comprises an enzyme with a-amylase and / or P- amylase activity. In some embodiments, the method further comprises contacting the maltodextrins in (i) or the malto-oligosaccharides in (ii) with an enzyme having a-l,6-branching activity. In some embodiments, the enzyme having amylomaltase activity comprises a dose enzyme concentration between about 0.1-20 mg amylomaltase / L reaction medium. In some embodiments, the enzyme having amylomaltase activity is glycosyl hydrolase. In some embodiments, the glycosyl hydrolase is a GH77 enzyme. In some embodiments, the glycosyl hydrolase comprises a sequence that is at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, step (i) is performed in the substantial absence of a -amylase. In some embodiments, steps (i) and (ii) are performed sequentially. In some embodiments, steps (i) and (ii) are performed simultaneously. In some embodiments, the maltodextrins are prepared from a starch-containing substrate using a liquefying a-amylase. In some embodiments, the liquefying a-amylase and the a-amylase used in step (i) are the same. In some embodiments, the method produces less glucose compared to an otherwise identical method for producing IMO from maltodextrins in the substantial absence of an enzyme having amylomaltase and / or a-l,6-branching activity.

[0019]

[0014] Provided herein is an IMO produced by any of the methods disclosed herein. In some embodiments, the resistance to digestibility is increased relative to an IMO not produced by any of the methods described herein. In some embodiments, the IMO is more branched, longer, and the content of glucose in the syrup is lower, compared to an IMO produced from maltodextrins by an otherwise identical method but in the substantial absence of an enzyme having amylomaltase and / or a-l,6-branching activity.

[0020]

[0015] These and other aspects and embodiments of present compositions and methods will be apparent from the description and accompanying drawings.

[0021] BRIEF DESCRIPTION OF THE DRAWINGS

[0022]

[0016] FIG. l is a flowchart illustrating the steps and enzymes involved in a conventional process for preparing IMO.

[0023]

[0017] FIG. 2 is a flowchart illustrating the steps and enzymes involved in the improved process for preparing IMO as described in (W02021011793A1 / US2022259630A1).

[0018] FIGs. 3 A-3C are diagrams illustrating transglucosidase reactions that occur in a conventional process for preparing IMO from maltose.

[0024] [0191 FIG. 4 is a diagram illustrating reactions between a transglucosidase-glucose complex and malto-oligosaccharide acceptor molecules to produce isomalto-oligosaccharides.

[0025]

[0020] FIGs. 5A-5C are diagrams illustrating reactions between malto-oligosaccharide donor molecules and transglucosidase to generate transglucosidase-glucose complexes that can react with malto-oligosaccharide acceptor molecules to produce isomalto-oligosaccharides.

[0026]

[0021] FIG. 6 is a diagram illustrating reactions catalysed by an amylomaltase and a branching enzyme.

[0027] DETAILED DESCRIPTION

[0028] II. Definitions

[0029]

[0022] Prior to describing the present process and compositions in detail, the following terms are defined for clarity. Terms not defined should be accorded their ordinary meanings as used in the relevant art.

[0030]

[0023] As used herein the term “starch” refers to any material comprised of the complex polysaccharide carbohydrates of plants, comprised of amylose and / or amylopectin with the formula (CeHioC jx, wherein X can be any number. In particular, the term refers to any plantbased material including but not limited to grains, grasses, tubers and roots and more specifically wheat, barley, com, rye, rice, sorghum, legumes, cassava, millet, potato, sweet potato, and tapioca. After purification of the complex polysaccharide carbohydrates from the other plant components, it is called “refined starch.”

[0031]

[0024] The term “granular starch” refers to uncooked (raw) starch, which has not been subject to gelatinization.

[0032]

[0025] As used herein, “maltodextrins” refer to oligosaccharides that are generally produced from starch by partial chemical or enzymatic hydrolysis. The size of the polysaccharides generally ranges from DP3 to DP20, but can be longer.

[0033]

[0026] As used herein, “malto-oligosaccharides” refers to oligosaccharides of glucose linked via a-D-1,4 bonds. Exemplary malto-oligosaccharides, and their condensed IUPAC name (refer to 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(a-l,4)Glc), maltotriose (Glc(a-l,4)Glc(a-l,4)Glc), and maltotetraose (Glc(a-

[0034] 1.4)Glc(a- 1 ,4)Glc(a- 1 ,4)Glc).

[0035]

[0027] As used herein, “isomalto-oligosaccharides (IMO)” generally refer to oligosaccharides of glucose that include a-D-1,6 bonds. Exemplary isomalto-oligosaccharides, and their condensed IUPAC name (Id), include but are not limited to, isomaltose (Glc(a-l,6)Glc), isomaltotriose (Glc(a-l,6)Glc(a-l,6)Glc), and isomaltotetraose (Glc(a-l,6)Glc(a-l,6)Glc(a-l,6)Glc). Branched oligosaccharides having both a-D-1,4 and a-D-1,6 bonds, for example panose (Glc(a-l,6)Glc(a-

[0036] 1.4)Glc) are often considered IMO as well. As used herein, IMO may include some a-D-1,4 bonds.

[0037]

[0028] As used herein, the phrase “degree of polymerization” (DP) refers to the number (n) of anhydroglucopyranose units in a given saccharide. An example of DPI is the monosaccharide glucose. Examples of DP2 are the disaccharides maltose and isomaltose.

[0038]

[0029] As used herein, an “a-amylase” is an c r>-acting enzyme having the systematic name a- D-( l ^4)-glucan glucanohydrolase) and the Enzyme Commission designation EC 3.2.1.1.

[0039]

[0030] As used herein, a “starch processing enzyme” is an enzyme that depolymerizes a starch substrate (including maltodextrin). Exemplary starch processing enzymes are a-amylase, glucoamylase, P-amylase, amylomaltase, branching enzymes, pullulanase, and a-glucosidase.

[0031] As used herein, a “maltogenetic enzyme” is an enzyme that produces mainly maltose as products. Such enzymes include exo-acting enzymes of the classifications EC 3.2.1.2, Some predominantly end -acting enzymes such as maltogenic a-amylases (EC 3.2.1.133) also produce significant amounts of maltose and will be considered to be “maltogenetic enzymes” for the present purposes.

[0040]

[0032] As used herein, a “maltooligosaccharide producing enzyme” is an enzyme that produces mainly malto-oligsaccharides with a degree of polymerization of longer than two. 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.

[0041]

[0033] As used herein, a “transglucosidase” is synonymous with the term a-glucosidase and the systematic name a-D-glucoside glucohydrolase, having the Enzyme Commission designation EC 3.2.1.20.

[0042]

[0034] As used herein, a “pullulanase” is synonymous with the systematic name a-dextrin endo- 1,6-alpha-glucosidase., having the CAZY enzyme database designation EC 3.2.1.41. Other debranching enzymes such as isoamylases (EC 3.2.1.68), having activity on branched maltodextrins, are considered “pullalanases” for the present purposes.

[0043] [0351 As used herein, an “amylomaltase” is a 4-a-glucanotransferase catalyzing glucan chain transfer from one a-1,4 glucan to another a-1,4 glucan or 4-hydroxyl of glucose. Synonym(s): D enzyme (plants), dextrin glycosyltransferase, dextrin transglycosylase, disproportionating enzyme. Amylomaltases (E.C. 2.4.1.25) belong to the glucosyl hydrolase family 77 (GH77). Enzymes with similar activities are also present in the GH13 and GH57 families.

[0044]

[0036] As used herein, a “branching enzyme” is a 1,4-a-glucan branching enzyme catalyzing transfer of a segment of a a-1,4 glucan chain to the C-6 hydroxyl in a similar glucan chain. Branching enzymes are found in both higher plants (Q-enzyme) and in microorganisms. Most branching enzymes belong to glycoside hydrolase family 13 (GHB) according to the Carbohydrate-Active enZYmes (CAZy) classification. In addition, certain microorganisms also possess GH57 branching enzymes.

[0045]

[0037] As used herein, “contacting” an enzyme with a substrate refers to bringing the enzyme and substrate together in a common aqueous environment, typically accompanied by mixing to achieve uniform distribution. The term “contacted” is used interchangeably with “treated.”

[0038] As used herein, “generating” refers to producing a reaction product as the result of an enzymatic process.

[0046]

[0039] As used herein, “substantial” or “substantially” encompasses an amount that is large in size and / or value. As used herein, the term “substantial absence” can mean that the amount of an enzyme found would be only de minimis or in irrelevant amounts.

[0047]

[0040] As used herein, the singular articles “a,” “an” and “the” encompass the plural referents unless the context clearly dictates otherwise. All references cited herein are hereby incorporated by reference in their entirety. The following abbreviations / acronyms have the following meanings unless otherwise specified:

[0048] °C degrees Centigrade

[0049] BBA barley P-amylase

[0050] DE dextrose equivalents

[0051] DP degree of polymerization

[0052] DP° degrees diastatic power (units of P-amylase activity)

[0053] DP3+ DP3 or longer DPn DP with unknown value

[0054] DS dry solids g or gm gram

[0055] HPAE high-performance anion-exchange chromatography

[0056] HPLC high performance liquid chromatography hr hour

[0057] IM2 isomaltose

[0058] IM3 isomaltotriose

[0059] IM4 isomaltotetraose

[0060] IM5 isomaltopentaose

[0061] IM6 isomaltohexaose

[0062] IM 7 isomaltoheptaose

[0063] IMO isomalto-oligosaccharides

[0064] IUPAC International Union of Pure and Applied Chemistry kg kilogram

[0065] M molar mg milligram min minute mL and ml milliliter mm millimeter mM millimolar

[0066] MT metric ton

[0067] NaAc sodium acetate

[0068] NaOH sodium hydroxide

[0069] PAD pulsed amperometric detection

[0070] PU pullulanase

[0071] RI refractive index

[0072] RPM or rpm revolutions -per minute

[0073] TG transglucosidase

[0074] U or u unit w / v weight / volume pig microgram pL and pl microliter m micrometer pM micromolar

[0075] II. Enzymatic process for making complex branched IMO

[0076]

[0041] The present method allows for producing complex branched isomalto-oligosaccharides (IMO) from maltodextrins. The method involves an enzyme having branching activity and or amylomaltase activity in combination with enzymes having transglucosidase and pullulanase activity to produce more complexly branched long-chain length IMO compared to IMO produced using a conventional method.

[0077]

[0042] The present method expands and improves on the method recently described by Koops (W02021011793A1 / US2022259630A1, the contents of which is incorporated by reference herein in its totality) in that it unexpectedly combines enzymatic debranching and branching activity with transglucosidase activity to produce complex long IMO. The described IMO are further useful and well-suited for the production of specialty syrups.

[0078]

[0043] Without wishing to be bound by theory, it is proposed that the enzyme having branching activity (or “branching enzyme,” for brevity), transfers a glucosyl group or alpha 1-4 chain from a donor oligosaccharide to an acceptor oligosaccharide and introduces a new branchpoint in the process. It is hypothesized that the newly formed, more branched oligosaccharide, is less suitable as a donor molecule for transglycosylation but can still act as an acceptor molecule. In this way, it can be expected that adding a branching enzyme will result in a higher content of complex branched and longer oligosaccharides while continuing to produce less glucose than formed with conventional IMO production methods.

[0079]

[0044] The process provided herein provides advantages over described methods. As shown in the flowchart in FIG. 1, IMO is conventionally produced from maltodextrins by the sequential or simultaneous action of a P-amylase and a transglucosidase. In the conventional two-step process (left side of flowchart), a starch slurry is converted to maltodextrins in a liquefaction process after which the maltodextrins are treated with P-amylase and pullulanase to produce a maltose syrup, which is then treated with transglucosidase to produce IMO. In the conventional one-step process (right side of flowchart), a starch slurry is converted to maltodextrins in a liquefaction process and the maltodextrins are treated simultaneously with P-amylase, pullulanase and transglucosidase to produce IMO, without isolating or separating the maltose syrup.

[0080] [0451 As shown in the flowchart in FIG. 2, the process begins with the conversion of a starch slurry to maltodextrins in a liquefaction process. Matodextrins are then treated with a DP3 or longer malto-oligosaccharide-producing alpha-amylase enzyme and pullulanase to produce maltotriose or longer oligosaccharide-rich syrup, which is then treated with transglucosidase to produce improved IMO in a two-step process (left side of flowchart) or in a one-step process (right side of flowchart). The advantages of the method outlined in FIG. 2 are apparent upon examining end-products of the transglucosidase reactions.

[0081]

[0046] In the conventional process, particularly early in the stages of the transglycosilation reaction, maltose produced from the starch hydrolysate by P-amylase is abundant and serves as both the predominant donor molecule and acceptor molecule for the transglucosidase (FIG. 3A, glucose molecules are represented by circles and glycosidic bonds are represented by lines connecting the circles. Glucose molecules having reducing ends are filled solid black, donor glucose molecules are filled with a checked pattern, glucose acceptor molecules are filled solid white, and non-reacting glucose molecules, are shown in grey. Free glucose molecules having a reducing end, but which also serve as acceptor glucose molecules, are filled half-white and halfblack). This results in the production of a tri saccharide, the most abundant being panose, along with a free glucose. Later in the reaction, when maltose becomes depleted, longer acceptor molecules are relatively more abundant, producing longer IMO, along with a free glucose (FIG. 3B) Free glucose itself can also serve as an acceptor for transglucosidase (FIG. 3C), in which case a short IMO is product, but again with the release of what is merely a different free glucose.

[0047] When maltose is the donor malto-oligosaccharide as in a conventional IMO production process, a free glucose is formed with every transglucosidase reaction (FIG. 5A). In contrast, when a longer donor malto-oligosaccharide is used, such as maltotriose (FIG. 5B), maltotetraose (FIG. 5C), or higher, the transglucosidase reaction produces no free glucose during the first part of the reaction which forms the transglucosidase-glucose complex. In addition, a transglucosidase-glucose complex formed as in FIG. 4 can interact with acceptor oligosaccharides of various lengths to produce IMO. ITT. Enzymatic compositions

[0082] A. Branching enzyme

[0083]

[0048] Branching enzymes are found in both plants and microorganisms. In higher plants the enzymes belong to the glycosyl hydrolase family GH13. In microorganisms, the branching enzymes or glycogen branching enzymes (GBEs) are classified in two glycoside hydrolase (GH) families, 13 and 57 (Zhang et al. Carbohydrate polymers 216 (2019) 231-237). Branching enzymes can cleave a- 1,4-glycosidi clinkages in a linear starch, glycogen or maltooligosaccharide chains. The cleaved fragment is then attached onto the 6-hydroxyl group of an anhydroglucose moiety located in a chain segment of a-l,4-linked anhydroglucose residues. These enzymes are specifically generating a-l,6-linkages and no other linkages. These enzymes have a-l,6-banching activity. The cleaved fragment is attached to the same chain from which it was cleaved. In some embodiments, the branching enzyme acts on an amylose chain, making a branched amylose (FIG. 6). In some embodiments, the enzymes exemplified herein are GH57 UF1957 domain-containing proteins from Anaerolineaceae bacterium (BR5), and / or GH13 1,4- alpha-glucan branching enzymes GlgB from Pontibacter akesuensis (BRIO). In some embodiments, BR5 is expressed in Bacillus subtilis. In some embodiments, BRIO is expressed in Bacillus subtilis.

[0084]

[0049] In some embodiments, the branching enzyme is a glycosyl hydrolase family GH I 3 enzyme. In some embodiments, the branching enzyme contains a sequence that is at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 1. In some embodiments, the branching enzyme is glycosyl hydrolase family GH57 enzyme. In some embodiments, the branching enzyme contains a sequence that is at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO:2. In some embodiments, the branching enzyme contains a sequence that is at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in any of the SEQ ID Nos described in Table 1.

[0085] B. Amylomaltase

[0086]

[0050] Amylomaltases (E.C. 2.4.1.25) belong to the glucosyl hydrolase family 77 (GH77). Enzymes with similar activities, with different sequence characteristics, are also present in the GH13 and GH57 families. Amylomaltases transfer a segment of a-l,4-D-glucan to a new 4- position in an acceptor carbohydrate via a disproportionation reaction. Amylomaltase can transfer a- 1,4 glucosidic linkages from amylose to amylopectin or from linear maltooligosaccharides to other maltooligosaccharides. (FIG. 7). In some embodiments, any of the enzymes exemplified herein are Aquifex aelocius. In some embodiments, any of the enzymes exemplified herein are Aquifex aelocius Genbank Accession No AMI - AAC06897.1. In some embodiments, any of the enzymes exemplified herein are Thermits ihermophilus amylomaltase. In some embodiments, any of the enzymes exemplified herein are Thermits thermophilus amylomaltase Genbank Accession No AM3 - AB016244.1. In some of any embodiments, AMI is expressed in Bacillus subtilis. In some of any embodiments, AM3 is expressed in Bacillus subtilis. Genbank Accession No’s are: AMI : AAC06897.1 and AM3 : AB 016244.1

[0087]

[0051] In some embodiments, the amylomaltase is a glycosyl hydrolase family GH77 enzyme. In some embodiments, the amylomaltase contains a sequence that is at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO:3. In some embodiments, the amylomaltase enzyme contains a sequence that is at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO:4. In some embodiments, the branching enzyme contains a sequence that is at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in any of the SEQ ID Nos described in Table 2.

[0088] C. DP3+ generating a-amylases

[0089]

[0052] In some embodiments, any of the processes provided herein can use DP3+ generating (also called DP3+ producing) a-amylases for producing malto-oligosaccharide. In some embodiments, any of the DP3+ generating (also called DP3+ producing) a-amylases include those that produce malto-oligosaccharides longer than DP2 (i.e., maltose) from maltodextrins. In some embodiments, the enzymes are enzymes that produce DP3, DP4, DP5, or longer, maltooligosaccharides. Enzymes that produce significant amounts of DP3 include, but are not limited to, a-amylases from Aspergilus e.g.. A. kawachi, A clavatus and A. oryzae. In some embodiments, the enzymes are maltotriose-producing amylases derived from Streptomyces griseus. Bacillus subtilis, Microbacterium imperiaie and Chloroflexus aurantiacus. In some embodiments, the enzymes are enzymes that produce significant amounts of DP4 which include, but are not limited to, an amylase from Pseudomonas saccharophila. In some embodiments, the enzymes are enzymes that produce significant amounts of DP5, and include but are not limited to, a-amylases from several Bacillus spp., including B. stearothermophilus and B. licheniformis, as well as enzymes from Cytophaga spp.

[0090]

[0053] In some embodiments, a DP3+ generating a-amylase suitable for use according to the present methods is any a-amylase that produces a sugar profile that (when the reaction is left to process for sufficient time) has a minimum of 15% DP3, a minimum of 10% DP4 or a minimum of 5% DP5, along with a maximum of 40%, a maximum of 30%, a maximum of 20%, a maximum of 10% or even a maximum of 5% DP2. More than one DP3+ generating a-amylase can be used, in which case the combination of DP3+ generating a-amylases produces the described profile of malto-oligosaccharides.

[0091] D. Transglucosidases

[0092]

[0054] Provided herein are methods that use transglucosidase in the production of isomaltooligosaccharides (IMO) from malto-oligosaccharides or maltose. Transglucosidase is also known as a-glucosidase and a-D-glucoside glucohydrolase. The molecules are classified as EC 3.2.1.20 enzymes in CAZy Family GH31 and have been identified in numerous organisms. Genbank includes over 400 entries for transglucosidases.

[0093]

[0055] In some embodiments, any of the exemplary enzymes described herein are from Aspergillis niger and are expressed in Trichoderma reesei. The enzyme expresses at high levels but is otherwise not recognized as having unique properties compared to other transglucosidases studied. Accordingly, a large number of transglucosidases, derived from many organisms, are suitable for producing isomalto-oligosaccharides (IMO) from maltodextrins.

[0094]

[0056] In some embodiments, the exemplified enzyme is commercially available as TRANSGLUCOSIDASE L2000® (IFF Health & Biosciences) with an activity of 1700 transglucosidase units (TGU) / g. One TGU is defined as the amount of enzyme required to produce one micromole of panose per minute under the conditions of the assay. A minimum of 0.1 kg / MT of TRANSGLUCOSIDASE L2000®7MT of DS is needed. In all the work described herein, 1 kg / MT DS was used.

[0095] E. Liquefying a-amylase

[0096]

[0057] In some embodiments, a liquefying a-amylase for converting crude feedstocks, such as a starch from grains and other plant materials, to maltodextrins can be any liquefying a-amylase known to one of skill in the art. In some embodiments, the liquefying a-amylase includes enzymes derived from numerous microorganisms. Exemplary enzymes are commercially available as, e.g., FUELZYME™ (BASF Enzymes LLC, San Diego, CA), LPHERA®, AVANTEC® and LIQUOZYME® products, LpHera® products (Novozymes) and;

[0097] SPEZYME® products (IFF). In some embodiments, more than one liquefying a-amylase can be used.

[0098]

[0058] In some embodiments, the liquefying a-amylase may additionally be useful as the DP3+ generating enzyme for use in the improved process, depending on the profde of malto-oligomers generated. Accordingly, the liquefying a-amylase(s) may be, or may include, the DP3+ generating enzyme(s).

[0099]

[0059] The enzyme concentration needed to produce such a sugar profde depends on the type of reaction products it produces, the reaction conditions and the reaction time. A trained person can determine the optimal amount. As an example, SPEZYME® ALPHA PF dosed of 0.2 kg / MT DS on a 12 DE liquefact can produce a syrup with over 20% DP5 in about 7 hours.

[0100]

[0060] Starch liquefaction can be performed above, at or below the gelatinization temperature of the starch substrate. Other enzymes may be present, e.g., proteases.

[0101] G. Raw starch hydrolyzing enzymes

[0102]

[0061] Many starch degrading enzymes are active on raw starch, as described in, e.g., US Pat. Nos. 7037704, 7205138, 7303899, 7378256, which are incorporated by reference herein in their totality, and references contained within. These enzymes are generally referred to as raw starch hydrolyzing enzymes or granular starch hydrolyzing enzymes (GSHE). GSHE that liberate DP3 or longer sugars are suitable for use as described. GSHE can be used in a two-step reaction where raw starch is treated with a GSHE, with or without pullulanase, to produce a maltooligosaccharide which is then reacted with transglucosidase. GSHE can also be used in a one- step reaction where the raw starch is treated with a GSHE, with or without pulluanase, and simultaneously reacted with transglucosidase. Examples of enzymes that can liberate oligosaccharides from raw starch include, but are not limited to, SPEZYME® ALPHA PF, SPEZYME® XTRA, Aspergillus kctrwachi alpha-amylase, and OPTIMALT® 4G.

[0103] IV. Features and uses of complex branched IMO

[0104]

[0062] Provided herein is a process and enzymatic compositions that allow the production of complex branched isomalto-oligosaccharides (IMO) from maltodextrins for any number of uses.

[0063] In some embodiments, the method includes (i) contacting maltodextrins with an enzyme to produce malto-oligosaccharides, and (ii) contacting the malto-oligosaccharides with one or more of an enzyme that has transglucosidase activity, an enzyme that has pullulanase activity and an enzyme that has branching activity to produce complex branched IMO. In some embodiments, the method includes (i) contacting maltodextrins with an enzyme to produce malto-oligosaccharides, and (ii) contacting the malto-oligosaccharides with one or more enzymes having transglucosidase, pullulanase and branching activity to produce complex branched IMO.

[0064] In some embodiments, the method produces more complex branched IMO compared to an otherwise identical method for producing IMO from maltodextrins in the substantial absence of an enzyme having branching activity. In some embodiments, the enzyme having branching activity has an dose enzyme concentration between about 0.5 -20 mg branching enzyme / liter reaction medium. In some embodiments, the enzyme having branching activity has a dose enzyme concentration of between about 0.5, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.1, 15.2, 15.3, 15.4, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5 and 20 mg branching enzyme / liter reaction medium. In some embodiments, the enzyme having branching activity has a dose enzyme concentration of between about 1.3 and 15.1 mg branching enzyme / liter reaction medium.

[0105]

[0065] In some embodiments, the method includes (i) contacting maltodextrins with an enzyme to produce malto-oligosaccharides, and (ii) contacting the malto-oligosaccharides with one or of an enzyme having transglucosidase activity, an enzyme having pullulanase activity and an enzyme having amylomaltase activity to produce complex branched IMO. In some embodiments, the method produces more complex branched IMO compared to an otherwise identical method for producing IMO from maltodextrins in the substantial absence of an enzyme having amylomaltase activity.

[0106] [0661 Insome embodiments, the enzyme in (i) is an enzyme with a-amylase and / or P-amylase activity. In some embodiments, the method further includes contacting the maltodextrins in (i) or the malto-oligosaccharides in (ii) with a branching enzyme. In some embodiments, the enzyme having amylomaltase activity has an dose enzyme concentration between about 0.1 -20 mg amylomaltase / liter reaction medium. In some embodiments, the enzyme having amylomaltase activity has a dose enzyme concentration of between about 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 1.1, 1.2,

[0107] I.3, 1.4, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5,

[0108] I I.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.1, 15.2, 15.3, 15.4, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5 and 20 mg amylomaltase / liter reaction medium. In some embodiments, the enzyme having amylomaltase activity has a dose enzyme concentration of between about 0.5 and 11.5 mg branching enzyme / liter reaction medium.

[0109]

[0067] The IMO are more branched, longer, and the content of glucose in the syrup is lower, than with a conventional process. The syrup may be physically separated into fractions having a desired DP range, using methods similar to those used for conventional syrup. More complex and longer IMO are likely to be more poorly metabolized, offering greater health benefits to consumers and more food ingredient options to food producers. In some embodiments, the method produces less glucose compared to an otherwise identical method for producing IMO from maltodextrins in the substantial absence of an amylomaltase and / or branching enzyme.

[0110]

[0068] In some embodiments, the method produces more complex branched IMO compared to an otherwise identical method for producing IMO from maltodextrins in the substantial absence of an enzyme having branching activity. In some embodiments, the method produces more complex branched IMO compared to an otherwise identical method for producing IMO from maltodextrins in the substantial absence of an enzyme having amylomaltase and / or branching activity.

[0111]

[0069] These and other aspects and embodiments of the present methods, and compositions resulting, therefrom, will be apparent to the skilled person in view of the present description. The following examples are intended to further illustrate, but not limit, the compositions and methods.

[0112] EXEMPLARY EMBODIMENTS

[0113] 1. A method for producing complex branched isomalto-oligosaccharides from maltodextrins, comprising:

[0114] (i) contacting maltodextrins with an enzyme to produce malto-oligosaccharides, and

[0115] (ii) contacting the malto-oligosaccharides with one or more of an enzyme having transglucosidase activity, an enzyme having pullulanase activity and an enzyme having a- 1,6- branching activity to produce complex branched IMO, wherein the method produces more complex branched IMO compared to an otherwise identical method for producing IMO from maltodextrins in the substantial absence of an enzyme having a-l,6-branching activity.

[0116] 2. The method of embodiment 1, wherein the enzyme in (i) comprises an enzyme with a-amylase and / or P-amylase activity.

[0117] 3. The method of embodiment 1 or embodiment 2, wherein the method further comprises contacting the maltodextrins in (i) or the malto-oligosaccharides in (ii) with an enzyme having amylomaltase activity.

[0118] 4. The method of any one of embodiments 1-3, wherein the enzyme having a-l,6-branching activity comprises a dose enzyme concentration between about 0.5 -20 mg branching enzyme / liter reaction medium..

[0119] 5. The method of any one of embodiments 1-4, wherein the enzyme having a-l,6-branching activity is glycosyl hydrolase.

[0120] 6. The method of embodiment 5, wherein the glycosyl hydrolase is a GH13 or a GH57 enzyme.

[0121] 7. The method of embodiment 5 or embodiment 6, wherein the glycosyl hydrolase comprises a sequence that is at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 1 or SEQ ID NO:2.

[0122] 8. The method of embodiment 1 or embodiment 2, wherein step (i) is performed in the substantial absence of a P-amylase.

[0123] 9. The method of any of embodiments 1-8, wherein steps (i) and (ii) are performed sequentially. 10. The method of any of embodiments 1 -8, wherein steps (i) and (ii) are performed simultaneously.

[0124] 11. The method of any of embodiments 1-10, wherein the maltodextrins are prepared from a starch-containing substrate using a liquefying a-amylase.

[0125] 12. The method of embodiment 11, wherein the liquefying a-amylase and the a-amylase used in step (i) are the same.

[0126] 13. A method for producing complex branched isomalto-oligosaccharides from maltodextrins, comprising:

[0127] (i) contacting maltodextrins with an enzyme to produce malto-oligosaccharides, and

[0128] (ii) contacting the malto-oligosaccharides with one or of an enzyme having transglucosidase activity, an enzyme having pullulanase activity and an enzyme having amylomaltase activity to produce complex branched IMO, wherein the method produces more complex branched IMO compared to an otherwise identical method for producing IMO from maltodextrins in the substantial absence of an enzyme having amylomaltase activity.

[0129] 14. The method of embodiment 13, wherein the enzyme in (i) comprises an enzyme with a- amylase and / or P-amylase activity.

[0130] 15. The method of embodiment 13 or embodiment 14, wherein the method further comprises contacting the maltodextrins in (i) or the malto-oligosaccharides in (ii) with an enzyme having a-l,6-branching activity.

[0131] 16. The method of any one of embodiments 13-15, wherein the enzyme having amylomaltase activity comprises a dose enzyme concentration between about 0.1-20 mg amylomaltase / L reaction medium.

[0132] 17. The method of any one of embodiments 13-16, wherein the enzyme having amylomaltase activity is glycosyl hydrolase.

[0133] 18. The method of embodiment 17, wherein the glycosyl hydrolase is a GH77 enzyme.

[0134] 19. The method of embodiment 17 or embodiment 18, wherein the glycosyl hydrolase comprises a sequence that is at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO:3 or SEQ ID NO:4. 20. The method of embodiment 13 or embodiment 14, wherein step (i) is performed in the substantial absence of a P-amylase.

[0135] 21. The method of any of embodiments 13-20, wherein steps (i) and (ii) are performed sequentially.

[0136] 22. The method of any of embodiments 13-20, wherein steps (i) and (ii) are performed simultaneously.

[0137] 23. The method of any of embodiments 1-22, wherein the maltodextrins are prepared from a starch-containing substrate using a liquefying a-amylase.

[0138] 24. The method of embodiment 23, wherein the liquefying a-amylase and the a-amylase used in step (i) are the same.

[0139] 25. The method of any one of embodiments 1-24, wherein the method produces less glucose compared to an otherwise identical method for producing IMO from maltodextrins in the substantial absence of an enzyme having amylomaltase and / or a-l,6-branching activity.

[0140] 26. An IMO produced by the method of any of embodiments 1-25.

[0141] 27. The IMO of embodiment 26, wherein the resistance to digestibility is increased relative to an IMO not produced by the method of any one of embodiments 1-26.

[0142] 28. The IMO of embodiment 26 or embodiment 27, wherein the IMO is more branched, longer, and the content of glucose in the syrup is lower, compared to an IMO produced from maltodextrins by an otherwise identical method but in the substantial absence of an enzyme having amylomaltase and / or a-l,6-branching activity.

[0143] EXAMPLES

[0144] Example 1. IMO production in the presence of amylomaltase

[0145]

[0070] To produce IMO, a corn liquefact having a DE of 12.8 at 32% DS was incubated with an a-amylase (SPEZYME® ALPHA PF; IFF) at 0.65 kg / MT DS, a pullulanase (OPTIMAX® L- 1000; IFF) at 0.5 kg / MT DS and a transglucosidase (TRANSGLUCOSIDASE® L2000, IFF) at 0.9 kg / MT DS at pH 5.0 and 60°C for 48 hr. These conditions were previously determined to be optimal for IMO production as described in W02021011793 / US2022259630.

[0146]

[0071] To test the hypothesis that amylomaltase can further improve IMO production, the aforementioned reactions was performed in the presence and absence of two different amylomaltases, the first from Aquifex Aelocius (i.e AMI) and the second from Thermus thermophihis amylomaltase (i.e., AM3). The amylomaltases were dosed at 50 pl in each of two each 2-g reactions. For AMI and AM3 the 50 pl volumes added are equal to 5.31 and 11.47 mg amylomaltase protein per liter reaction mixture respectively.

[0147]

[0072] Samples of the resulting syrups were taken periodically for HPLC analysis. A 100 pl portion was taken from the reaction medium, diluted 10 times with distilled water and boiled. Following filtration, 20 pl of each diluted sample was injected into an HPLC apparatus equipped with a Bio-Rad Aminex HPx-42A column (Part No. 1250096, 300 mm x 7.8 mm). The mobile phase was HPLC-grade distilled water, running at 0.6 ml / min for 22.5 minutes, the temperature of the column was 85°C and detection was performed in a RI detector at a cell temperature of 40°C. The sugar compositions of the syrups, following 48 hr of reaction time, are summarized in Table El.

[0148] Table El. Sugar compositions of syrups produced from com liquefacts in the absence and presence of amylomaltase

[0073] Addition of either of the two amylomaltases results in a decreased DPI content, where AMI had the largest effect. For clarity, low glucose is beneficial in IMO production. In the presence of AMI DP2 is also lower than with the reference. Surprisingly, all the higher sugars (DP3 and above) are increased with AMI compared to the reference, suggesting that IMO synthesis in the presence of amylomaltase results in increased branched maltooligosaccharides that cannot act as donor for subsequent transglycosilation reactions. This effect is less apparent with AM3.

[0149]

[0074] A second sample was prepared for analysis by high-performance anion-exchange chromatography using pulsed amperometric detection (HPAE-PAD). Where other analytical methods separate saccharides based on size (i.e., monomers, dimers etc.), HPAE-PAD is capable of separating isomers such as maltotriose, panose and isomaltotriose. Specifically, 100 pl sample was collected, diluted 1,000 times and boiled for 10 min. Following filtration, a 10 pl sample was injected on a Carbopac PA200 column (3 mm x 250 mm) installed with a guard column at a flow rate of 0.5 ml / min and a temperature 30°C. PAD was performed with a cell temperature of 25°C. During the 60 min chromatographic run the following conditions were used: (i) prior to sample injection, the column was equilibrated for 10 min with 10% 1 M NaOH and 90% MilliQ water. Separation of the sugars was accomplished by elution with constant 10% 1 M sodium hydroxide and 90% MilliQ water for 5 min. A gradient with 500 mM NaOAc was started, where the % NaOAc in the mobile phase increased from 0 to 8%, and % of MilliQ water decreased from 90 to 82%. In the following 50 minutes the gradient changed and the amount of MilliQ in the mobile phase decreased from 82% to 0% and % NaOAc increased from 8 to 90%. Features of the gradient are summarized in Table E2.

[0150] Table E2. Chromatographic gradient

[0151]

[0075] To calculate IMO content, two analyses were required. With the conventional HPLC method (described, above), the content (%) of the different saccharides was calculate by measuring the area of the DPI, DP2, DP3, DP4, DP5, DP6, DP7, DP8, DP9, DP10 and DPn peaks from the chromatogram. 10% DP2 means that 10% by weight of the sugars present in the final sugar composition is DP2 (and so forth). For the purposes of Table 1, DPn refers to >DP11. As mentioned, this analysis does not provide information regarding the isomers present.

[0076] However, the isomers present in, for example, the DP2 peak, can be distinguished by HPAE-PAD analysis. Chromatograms from this HPAE-PAD analysis revealed peaks from different isomers that could be identified based on the separate analysis of a standard sample with known components. Since the concentration of each component in the standard mix is known, the content of that particular component 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 (w / v) based on HPAE-PAD analysis, this means that the total DP2 contains 11%, maltose, 58% isomaltose, 19% kojibiose and 12% nigerose. If for example the DP2 content in the syrup is 10% (measured by conventional HPLC), this means that the content of the isomers in the total syrup are: 1.1 % maltose, 5.8% isomaltose, 1.9% kojibiose and 1.2% nigerose. In this manner, DPI, DP2 and DP3 isomers can be distinguished.

[0152]

[0077] In the case of DPI, DP2 and DP3 sugars, the presence of most isomers predicted to be formed was confirmed based on comparisons with commercially available pure components as standards. For longer oligosaccharides, e.g., DP4 and higher, not all isomers can be identified using readily available pure components as standards. Accordingly, in the case of DP4 and higher isomers, only linear malto-oligosaccharides up to DP10 were distinguished, z.e., malto- tetraose, malto-pentaose, malto-hexaose, malto-heptaose, malto-octaose, malto-nonaose and malto-decaose. In addition, linear isomalto-oligosaccharides up to DP7 are distinguished, z.e., isomalto-tetraose, isomalto-pentaose, isomalto-hexaose and isomalto-heptaose are identified. Other, more complex, branched oligosaccharides show up in the chromatogram as unidentified peaks. As the oligomers become longer, there is also a greater likelihood that the peaks overlap on the chromatogram, making quantitation problematic.

[0153]

[0078] Identifying the fraction of DP4 that is isomalto-tetraose illustrates the problem with quantitation. The assumption is made that DP4 only contains malto-tetraose and isomalto- tetraose. Other unidentified branched tetramers are not taken into account for the IMO content calculation. Since branched oligosaccharides are often considered IMO, this calculation likely results in an underestimation of the total IMO content. The same applies for DP5 - DP7. For isomers of DP8 - DPI 1 , it is assumed that all isomers are linear malto-oligosaccharides. This again leads to underestimation of the total IMO content.

[0154] [0791 Results for IMO analysis are summarized in Table E3.

[0155] Table E3. IMO content as percent of total sugars, of above syrups as measured by HPAE-PAD analysis.

[0156]

[0080] Addition of AM3 in IMO production increased total IMO content by 1.5%. This increase was mainly from IM2 and panose at the expense of lower IM3 and IM5. Addition of AMI also resulted in higher panose content than the reference but a lower total IMO content. It is hypothesized that the complex reaction of IMO synthesis in the presence of AMI creates more branched structures (e.g., higher DP3 - DPI 1+ content at end of reaction) than in the reference reaction (and with AM3). In the IMO analysis, such branched oligosaccharides are not counted as IMO in the total EMO content. It is therefore not surprising that measured EMO content is lower with AMI.

[0157] Example 2. IMO production in the presence of amylomaltase at different dosages

[0158]

[0081] The experiment described in Example 1 was modified to measure dose response to amylomaltose. AMI was added at 5, 15, 30 and 50 pl per 2-gram reaction. For AMI the 5, 15, 30 and 50 pl volumes added are equal to 0.53, 1.59, 3.19 and 5.31 mg amylomaltase protein per liter reaction mixture respectively. At appropriate time points, samples were taken for HPLC and HPAE-PAD analysis as described above. The sugar compositions of the resulting syrups, following 48 hr reaction time, are summarized in Table E4. Results for IMO analysis are summarized in Table E5.

[0159] Table E4. Sugar composition of syrups produced from corn liquefact with the improved IMO method in the absence and presence amylomaltase.

[0160]

[0082] Increasing the amount of AMI in the transglycosylation reaction reduced resulting DPI and DP2, as described in Example 1. At the highest amount (5.31 mg / 1), the production of all longer sugars (DP3 - DPI 1+) was increased compared to without AMI. Dose-response behavior was readily apparent with increasing amounts of AMI. Lower DPI content may have resulted from diminishing transglucosidase products as available substrate is lower in the presence of AMI , and because part of the substrate is converted in the elongation reaction catalyzed by amylomaltase. Table E5. IMO content of above syrups as measured by HPAE-PAD analysis.

[0161]

[0083] Adding AMI to the transglycosilation reaction increased total IMO content, so long as no more than 1.59 mg / 1 was added in the reaction. At a higher dose the total IMO content was equal (3.19 mg / 1) or significantly lower (5.31 mg / 1). At the lowest dose, IM2 - IM5 production increased, while at the highest dose production of these sugars decreased.

[0162]

[0084] Amylomaltase hydrolyses alpha 1-4 chains and transfer a small linear alpha 1-4 chain to an acceptor. When the acceptor is another alpha 1-4 chain, the chain becomes elongated. The acceptor can also be an alpha 1 -6 linked glucosyl group, in which case the 1-6 linkage-containing acceptor is elongated with an alpha 1-4 chain. It is hypothesized that at low dose, the amylomaltase increases the available substrate for transglycosilation by elongating alpha 1-4 chains. At higher dose, the addition of alpha 1-4 chains to alpha 1-6 branches (generated by transglucosidase) may reduce the available substrate and thus reduce the IMO content.

[0163]

[0085] Non-linear branched structures produced by elongation of a branched chain, are not counted as IMO in the compositional analyses describe herein and unfortunately affect IMO quantitation.

[0164]

[0086] To further characterize the branched IMO, and to roughly simulate digestibility in an animal gut, the branched IMO were treated with alpha-amylase and glucoamylase. Incubations were done with 0.4g of the branched IMO reaction mixture with an alpha-amylase (SPEZYME® SL; IFF) at l.Okg / MT.ds and glucoamylase (OPTIDEX® L400; IFF) at 1.0 kg / MT.ds at pH 5.0 and 60°C for 48 hours. At appropriate time points, samples were taken for HPLC analysis as described above. Table E6 shows the results of alpha-amylase hydrolysis of IMO produced in the presence of AMI. Table E7 below the results of glucoamylase hydrolysis of IMO produced in the presence of AMI .

[0165] Table E6. Sugar composition following hydrolysis by alpha-amylase

[0166] Table E7. Sugar composition following hydrolysis by glucoamylase

[0167]

[0087] Longer sugars (DP8 and above), which are more abundant in IMO produced in the presence of AMI, were hydrolysed to a much lower extend by alpha-amylase, so long as the amount of AMI was high (5.31 mg / 1). At lower amounts of AMI, the longer sugars were hydrolysed to the same level as the benchmark. It is postulated that amylomaltase elongates IMO during transglycosylation to form structures with longer chains containing alpha 1 -4 linkages and some alpha 1-6 linkages. Only in the presence of high AMI dosages are structures formed that are more difficult to hydrolyse by alpha-amylase.

[0168]

[0088] Furthermore, most, if not all, the sugars longer than glucose in IMO produced in the presence of AMI are hydrolysed faster and to a lower content by glucoamylase. Although the content of most of these sugars is higher in the IMO where AMI was present during production, glucoamylase is able to hydrolyse them more efficiently. It is hypothesized that this is a result of the lower content of alpha 1-6 linkages in these IMO. AMI elongates reaction intermediates by adding a linear alpha 1-4 chain to them. Where transglucosidase produces more consecutive alpha 1-6 linkages, the combination of amylomaltase and transglucosidase is likely to produce larger molecules with both alpha 1-4 and alpha 1-6 bonds in. Since consecutive alpha 1-6 linkages are more difficult to hydrolyse by glucoamylases, this may be the reason why the IMO produced in the presence of AMI are more susceptible to hydrolysis by glucoamylase.

[0169] Example 3. IMO Production in the presence of different branching enzymes

[0170]

[0089] In Example 1, IMO were produced from a com liquefact (DE of 12.8 at 32% DS) using an a-amylase (SPEZYME® ALPHA PF; IFF) at 0.65 kg / MT DS, a pullulanase (OPTIMAX® L- 1000; IFF) at 0.5 kg / MT DS and a transglucosidase (TRANSGLUCOSIDASE® L2000) at 0.9 kg / MT DS at pH 5.0 and 60°C for 48 hr. To test the hypothesis that other branching enzymes can improve the IMO production, the above reactions were done in the presence and absence of different branching enzymes, designated branching enzyme 5 (BR5) and branching enzyme 10 (BRIO). BR5 is GH57 UF1957 domain -containing protein from Anaerolineaceae bacterium. BRIO is GH13 1,4-alpha-glucan branching enzyme GlgB from Pontibacter akesuensis.

[0171] [0901 Branching enzymes were dosed at 5, 15, 30 and 50 pl in 2-g reactions. For BR5 the 5, 15, 30 and 50 pl volumes added are equal to 1.51, 4.54, 9.08 and 16.13 mg branching enzyme protein per liter reaction mixture respectively. For BRIO the 5, 15, 30 and 50 pl volumes added are equal to 1.34, 4.03, 8.06 and 13.43 mg branching enzyme protein per liter reaction mixture respectively.

[0172]

[0091] At appropriate time points, samples were taken for HPLC and HPAEC-PAD analysis as described in Example 1. The sugar compositions of the resulting syrups, following 48 hr of reaction time, are summarized in Tables E8 and E9. Results of IMO analysis are summarized in Tables E10 and El l.

[0173] Table E8. Sugar composition of syrups produced in the presence or absence of BR5 Table E9. Sugar composition of syrups produced in the presence or absence of BRIO

[0174]

[0092] Adding either branching enzyme to the transglycosylation reaction results in decreased DPI and DP2 and increased long oligosaccharides, especially DP5 and above. More longer oligosaccharides are formed using BRIO.

[0175] Table E10. IMO content of syrups as measured by HPAE-PAD analysis.

[0176]

[0093] Addition of either branching enzyme increased total IMO content, so long as the concentration of the branching enzyme was low (i.e., 4.54 mg / 1 or less for BR5 and 4.03 mg / 1 or less for BRIO). There appeared to be an increase in isomaltose, isomaltotriose, isomaltotetraose and isomaltopentaose. Panose was reduced when small amounts of branching enzyme were added. At higher concentration of branching enzyme, all IMO’s seem to be decreasing in content, with the exception of panose.

[0177] Table Ell. IMO content of syrups as measured by HPAE-PAD analysis.

[0178]

[0094] Superficially, BRIO appears to reduce IMO production compared to BR5. However, this is likely a consequence of its higher level of branching activity producing such a large number of branched oligosaccharides, which are as noted not affirmatively accounted for by IMO analysis. In this case, only IM2 - IM7 and panose were included in total IMO content. Accordingly, the present compositional analysis of reactions performed in the presence of branching enzymes may underestimate the actual amount of IMO produced.

[0095] The branched IMO made in presence of BRIO were treated with alpha-amylase and glucoamylase as described in Example 2. Table E12 shows the results of alpha-amylase hydrolysis of IMO produced in the presence of BRIO. Table El 3 shows the results of glucoamylase hydrolysis of IMO produced in the presence of BRIO.

[0179] Table E12. Sugar composition following hydrolysis by alpha-amylase

[0180] Table E13. Sugar composition following hydrolysis by glucoamylase

[0181]

[0096] Longer sugars (DP8 and above), which are more abundant in IMO produced in the presence of BRIO, were hydrolysed to the same extend by alpha-amylase as the benchmark. Difference before and after alpha-amylase treatment is similar in all reactions, despite the fact that total amount is different for the benchmark and IMO made with BRIO. It is hypothesized that the branching enzymes introduces branchpoints in all sugars, including oligosaccharides. During the IMO production an alpha-amylase is also present to hydrolyse oligosaccharides to shorter sugars (creating substrates for the transglucosidase reaction). Due to having additional branchpoints these oligosaccharide are less susceptible to the alpha-amylase during IMO production. As a result, the IMO made in presence of BRIO are hydrolysed as poor as the benchmark.

[0182]

[0097] Most, if not all, the sugars longer than glucose in IMO produced in the presence of BRIO were hydrolysed faster and / or to a lower compositional amount by glucoamylase. Although the amounts of most of these sugars was higher in IMO produced when BRIO was present, glucoamylase was able to hydrolyse them more efficiently. This may be a result of the type of linkages that are formed during the IMO produced by BRIO and transglucosidase. Where transglucosidase produces more consecutive alpha 1-6 linkages, the combination of branching enzymes and transglucosidase is likely to produce larger molecules with both alpha 1-4 and alpha 1-6 linkages. The branching enzyme introduces branches during the IMO production and some of the oligosaccharides present early on in that process cannot be hydrolysed by the alphaamylase that is present in that process. As a result, some complex branched IMO still have relative long alpha 1-4 parts better hydrolysable by Glucoamylase. Consecutive alpha 1-6 linkages are more likely to be present in the benchmark, which makes it a poor substrate for glucoamylase.

Claims

CLAIMSWhat is claimed is:

1. A method for producing complex branched isomalto-oligosaccharides from maltodextrins, comprising:(i) contacting maltodextrins with an enzyme to produce malto-oligosaccharides, and(ii) contacting the malto-oligosaccharides with one or more of an enzyme having transglucosidase activity, an enzyme having pullulanase activity and an enzyme having a-1,6- branching activity to produce complex branched IMO, wherein the method produces more complex branched IMO compared to an otherwise identical method for producing IMO from maltodextrins in the substantial absence of an enzyme having a-l,6-branching activity.

2. The method of claim 1, wherein the enzyme in (i) comprises an enzyme with a- amylase and / or P-amylase activity.

3. The method of claim 1 or claim 2, wherein the method further comprises contacting the maltodextrins in (i) or the malto-oligosaccharides in (ii) with an enzyme having amylomaltase activity.

4. The method of any one of claims 1-3, wherein the enzyme having a-l,6-branching activity comprises a dose enzyme concentration between about 0.5 -20 mg branching enzyme / liter reaction medium.

5. The method of any one of claims 1-4, wherein the enzyme having a-l,6-branching activity is glycosyl hydrolase.

6. The method of claim 5, wherein the glycosyl hydrolase is a GH13 or a GH57 enzyme.

7. The method of claim 5 or claim 6, wherein the glycosyl hydrolase comprises a sequence that is at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 1 or SEQ ID NO:2.

8. The method of claim 1 or claim 2, wherein step (i) is performed in the substantial absence of a P-amylase.

9. The method of any of claims 1-8, wherein steps (i) and (ii) are performed sequentially.

10. The method of any of claims 1-8, wherein steps (i) and (ii) are performed simultaneously.

11. The method of any of claims 1-10, wherein the maltodextrins are prepared from a starch-containing substrate using a liquefying a-amylase.

12. The method of claim 11, wherein the liquefying a-amylase and the a-amylase used in step (i) are the same.

13. A method for producing complex branched isomalto-oligosaccharides from maltodextrins, comprising:(i) contacting maltodextrins with an enzyme to produce malto-oligosaccharides, and(ii) contacting the malto-oligosaccharides with one or of an enzyme having transglucosidase activity, an enzyme having pullulanase activity and an enzyme having amylomaltase activity to produce complex branched IMO, wherein the method produces more complex branched IMO compared to an otherwise identical method for producing IMO from maltodextrins in the substantial absence of an enzyme having amylomaltase activity.

14. The method of claim 13, wherein the enzyme in (i) comprises an enzyme with a- amylase and / or P-amylase activity.

15. The method of claim 13 or claim 14, wherein the method further comprises contacting the maltodextrins in (i) or the malto-oligosaccharides in (ii) with an enzyme having a-l,6-branching activity.

16. The method of any one of claims 13-15, wherein the enzyme having amylomaltase activity comprises a dose enzyme concentration between about 0.1-20 mg amylomaltase / L reaction medium.

17. The method of any one of claims 13-16, wherein the enzyme having amylomaltase activity is glycosyl hydrolase.

18. The method of claim 17, wherein the glycosyl hydrolase is a GH77 enzyme.

19. The method of claim 17 or claim 18, wherein the glycosyl hydrolase comprises a sequence that is at or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO:3 or SEQ ID NO:4.

20. The method of claim 13 or claim 14, wherein step (i) is performed in the substantial absence of a P-amylase.

21. The method of any of claims 13-20, wherein steps (i) and (ii) are performed sequentially.

22. The method of any of claims 13-20, wherein steps (i) and (ii) are performed simultaneously.

23. The method of any of claims 1-22, wherein the maltodextrins are prepared from a starch-containing substrate using a liquefying a-amylase.

24. The method of claim 23, wherein the liquefying a-amylase and the a-amylase used in step (i) are the same.

25. The method of any one of claims 1-24, wherein the method produces less glucose compared to an otherwise identical method for producing IMO from maltodextrins in the substantial absence of an enzyme having amylomaltase and / or a-l,6-branching activity.

26. An IMO produced by the method of any of claims 1-25.

27. The IMO of claim 26, wherein the resistance to digestibility is increased relative to an IMO not produced by the method of any one of claims 1-26.

28. The IMO of claim 26 or claim 27, wherein the IMO is more branched, longer, and the content of glucose in the syrup is lower, compared to an IMO produced from maltodextrins by an otherwise identical method but in the substantial absence of an enzyme having amylomaltase and / or a-l,6-branching activity.

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