JP7725444B2 - Improved methods for producing isomalto-oligosaccharides - Google Patents

Description

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.

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 textural 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. IMOs 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 dental caries.

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 the acceptor.

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 form isomaltose, 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 form.

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.

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 maltodextrins, comprising the steps of (i) contacting the maltodextrins with α-amylase to produce malto-oligosaccharides, and (ii) contacting the malto-oligosaccharides with 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 maltodextrins in step (i).
2. In some embodiments of the method of paragraph 1, step (i) is carried out in the presence of a β-amylase having no more than 660 saccharifying power (DP°) units per kg dry weight of maltodextrin.
3. In some embodiments of the method of paragraph 1, step (i) is carried out in the presence of β-amylase having no more than 264 saccharifying power (DP°) units 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 a 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 having no more than 66 saccharifying power (DP°) units 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 through 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 through 13, the maltodextrin is prepared from a starch-containing substrate using a liquefying α-amylase.
15. In some embodiments of the process 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 chain lengths 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 liquefaction 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.

These and other aspects and embodiments of the present compositions and methods will become apparent from the description and accompanying drawings herein.

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

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

FIG. 1 shows the transglucosidase reaction occurring in a conventional process for preparing IMOs.

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

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.

I. Definitions 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.

As used herein, the term "starch" refers to any substance composed of a complex polysaccharide carbohydrate of plants, composed of amylose and/or amylopectin, having _the formula _{( C6H10O5} ) _x , where X can be any number. In particular, the term refers to any plant-based substance, including, but not limited to, grains, grasses, tubers, and roots, more particularly 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."

The term "granular starch" refers to uncooked (raw) starch that has not been gelatinized.

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.

As used herein, "malto-oligosaccharide" refers to an oligosaccharide of glucose linked via α-D-1,4 linkages. 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(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).

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.

As used herein, the term "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 a DP2 are the disaccharides maltose and isomaltose.

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

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.

As used herein, a "maltogenic enzyme" is an enzyme that produces primarily maltose as a product. Such enzymes include extracellular-acting enzymes in the EC 3.2.1.2 classification. Some primarily intracellular-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.

As used herein, the term "malto-oligosaccharide-forming 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.

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.

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 purposes of the present invention.

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."

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

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:
°C Celsius temperature BBA Barley β-amylase DE Dextrose equivalent DP Degree of polymerization DP° Saccharification power (unit of β-amylase activity)
DP3+ DP3 or more DPn DP with unknown value
DS dry solids g or gm grams HPAE high performance liquid anion exchange chromatography HPLC high performance liquid chromatography hr hours IM2 isomaltose IM3 isomaltotriose IM4 isomaltotetraose IM5 isomaltopentaose IM6 isomaltohexaose IM7 isomaltoheptaose IMO isomalto-oligosaccharides IUPAC International Union of Pure and Applied Chemistry kg kilogram M mole mg milligram min minute mL and ml milliliter mm millimeter mM millimole 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 microliter μm micrometer μM micromole

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

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

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 an improved IMO in a two-step process (left side of the flowchart). Alternatively, the maltodextrins are simultaneously treated with DP3 (or longer-chain malto-oligosaccharide)-producing enzymes, pullulanase, and transglucosidase to produce an improved IMO without isolating or separating the maltotriose (or longer-chain malto-oligosaccharide)-rich syrup in a one-step process (right side of the flowchart).

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.

In conventional processes, especially early in the transglycosidation reaction, maltose produced from starch hydrolysis 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 significantly more abundant, producing longer-chain IMOs along with free glucose (Figure 3B). 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).

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, which forms 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).

III. Enzyme Compositions A. DP3+ Producing Alpha-Amylases DP3+ generating (also referred to as DP3+ producing) alpha-amylases suitable for producing malto-oligosaccharides for use in the improved process are alpha-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, alpha-amylases from Aspergillus species, such as A. kawachi, A. clavatus, and A. oryzae. Maltotriose-producing amylases have been identified in Streptomyces griseus, Bacillus subtilis, Mycobacterium imperiale, 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, amylases from B. stearothermophilus and B. spp. Examples of suitable α-amylases include, but are not limited to, α-amylases from several Bacillus spp., including B. licheniformis, as well as Cytophaga spp.

Generally, a DP3+ producing α-amylase suitable for use in accordance with the present method is any α-amylase that produces a sugar profile having at most 15% DP3, at most 10% DP4, or at most 5% DP5, along with at most 40%, at most 30%, at most 20%, at most 10%, or even at most 5% DP2 (when the reaction is allowed to proceed for a sufficient period of time). Two or more DP3+ producing α-amylases can be used, in which case the combination of DP3+ producing α-amylases will produce malto-oligosaccharides with the profile described above.

B. Transglucosidases A 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.

The enzyme exemplified herein is derived from Aspergillus niger and expressed in Trichoderma reesei. While this enzyme is expressed at high levels, it is not otherwise recognized as having unique properties compared to other transglucosidases studied. Therefore, numerous transglucosidases from numerous organisms are believed to be suitable for producing isomalto-oligosaccharides (IMOs) derived from maltodextrin.

An exemplary 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 assay conditions. 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.

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

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. Thus, the liquefying α-amylase may be or include a DP3+ producing enzyme.

The enzyme concentration required to produce such a sugar profile depends on the type of reaction product it produces, the reaction conditions, and the reaction time. A trained individual 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 over 20% in approximately 7 hours.

Starch liquefaction can be carried out above, at, or below the gelatinization temperature of the starch substrate. Other enzymes, such as proteases, may be present.

D. Enzyme Blends for Implementing the Improved Process A preferred enzyme blend is one that produces a syrup with a high content of DP3 to DP5 malto-oligosaccharides from a 12 DE starch liquefact in the absence of a transglucosidase. High in this context means that at least 15%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% of the total malto-oligosaccharides are DP3, DP4, and/or DP5. Another way to define a high content 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.

A suitable enzyme blend is one that, in addition or alternatively, produces a syrup from a 12 DE starch liquefact in the presence of a transglucosidase having 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.

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 intent 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 maltodextrin to produce the improved IMOs described herein.

β-Amylase activity is typically expressed in degrees of diastase power (DP°). One unit of diastase activity, expressed as DP° (degrees DP), is defined as the amount of enzyme contained in 0.1 ml of a 5% solution of 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.

Amounts of β-amylase activity that may be tolerated in the improved methods 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 (DS) of starch hydrolysate, up to about 264 DP° units of β-amylase per kg (DS) of starch hydrolysate, up to about 132 DP° units of β-amylase per kg (DS) of starch hydrolysate, and up to about 66 DP° units of β-amylase per kg (DS) of starch hydrolysate. As noted above, measurable amounts of β-amylase need not be present.

F. Raw Starch Hydrolyzing Enzymes Numerous 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 amylolytic 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.

IV. Improved IMO Characteristics 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 susceptible to metabolism, providing greater health benefits to consumers and more food ingredient options for food producers.

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 examples further illustrate, but do not limit, the compositions and methods.

Example 1. Transglycosylation Reactions with Maltose and Maltotriose. Transglycosylation reactions were performed 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 using a shaking speed of 750 rpm.

At 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 x 7.8 mm). The mobile phase was HPLC-grade distilled water, flowing 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.

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 released after 48 hours when the reaction was carried out 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.

Example 2. Transglycosylation Reactions Using DP2 and DP4 Syrups Because performing 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 synthase and compared to a maltose-rich starch hydrolysate prepared using β-amylase. Both starch hydrolysates were prepared from corn liquefacts with a DS of 32.5% and a DE of 11.28. To produce a DP4-rich hydrolysate, the liquefact was incubated for 48 hours at pH 5.0 and 60°C with 0.9 kg/MT (DS) of DP4-producing α-amylase (OPTIMALT® 4G; DuPont) plus 0.4 kg/MT (DS) of pullulanase (OPTIMAX® L-1000; DuPont). To produce a maltose-rich hydrolysate, the same liquefact was treated with 0.9 kg/MT (DS) of β-amylase (OPTIMALT® BBA; DuPont) plus 0.4 kg/MT (DS) of pullulanase.

At 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 x 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.

These syrups were further reacted with transglucosidase to produce IMOs. The above syrups enriched in DP2 or DP4 were adjusted to pH 4.2 and 30% DS. A 2-g sample of either the DP4-enriched syrup or the DP2-enriched syrup was treated with 1 kg/MT (DS) of transglucosidase at 60°C for 24 hours. 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 the 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 the sugar profiles obtained from the transglucosidase reaction after 24 hours are shown in Table 3.

IMO syrups prepared with DP4-rich hydrolysates have a distinctly different composition compared to syrups prepared with DP2-rich hydrolysates. DP1 levels are much lower after TG reactions with DP4-rich syrups (17%) compared to DP2-rich syrups (28%). DP2 is also lower after TG reactions with DP4-rich syrups, which are more enriched in longer-chain oligosaccharides, including DP5, DP6, DP7, D8, and DP9.

Example 3. Selection of enzymes to produce 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.

The enzymes used to produce DP2-rich syrup were either 0.9 kg/MT (DS) β-amylase (OPTIMALT® BBA, as described above) plus 0.4 kg/MT (DS) pullulanase (OPTIMAX® L1000, as described 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 the 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 the 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.

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 noted.

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.

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.

In the second step, the above syrup was adjusted to pH 4.2 and DS 30%, and 2 g of each was further reacted with 1 kg/MT (DS) transglucosidase at 60°C for 24 hours. As described above, a 100 μl aliquot was removed from the reaction medium and used to perform 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 sample was injected onto a Carbopac PA200 column (3 mm × 250 mm) equipped with a guard column set 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 chromatographic run: (i) Prior to sample injection, the column was equilibrated with 10% 1 M NaOH, 10% 500 mM NaOAc, and 80% MilliQ water for 10 minutes. Sugar separation was achieved by elution with a constant 10% 1 M sodium hydroxide and 90% MilliQ water for 5 minutes. During the next 5 minutes, a gradient was initiated using 500 mM NaOAc, increasing the percentage of NaOAc in the mobile phase from 0% to 8% and decreasing the percentage of MilliQ water from 90% to 82%. Over the next 50 minutes, the gradient was changed and the % of MilliQ water in the mobile phase was decreased from 82% to 0% and the % of NaOAc was increased from 8% to 90%. The gradient is shown in Table 5.

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 areas 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 DP2 peak is present in the final sugar composition, etc. For the purposes of Table 5, DPn refers to ≥ DP11. As noted above, this analysis does not provide information regarding the isomers present. However, isomers present within the DP2 peak, for example, 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% wt/vol maltose, 7.4% wt/vol isomaltose, 2.4% wt/vol kojibiose, and 1.6% wt/vol 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 manner, DP1, DP2, and DP3 isomers can be distinguished.

For DP1, DP2, and DP3 sugars, the majority of isomers likely to be formed can be identified conclusively 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 of DP4 and above, only linear malto-oligosaccharides up to DP10, namely, malto-tetraose, malto-pentaose, malto-hexaose, malto-heptaose, malto-octaose, malto-nanoose, and malto-decaose, are identified. Additionally, linear isomalto-oligosaccharides up to DP7, namely, isomalto-tetraose, isomalto-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.

To identify the percentage of DP4 that is isomalto-tetraose, it was hypothesized that DP4 contains only malto-tetraose and isomalto-tetraose. Other unidentified branched-chain tetramers were not taken into account 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 isomers DP8-DP11, it is assumed that all of them are linear malto-oligosaccharides. This again results in a small underestimation of the total IMO content.

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.

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.

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

When starting from syrups rich in DP4 or DP5, there is little DP2 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.

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

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.

Samples were removed at appropriate times during the one-step reaction for HPLC and HPAE-PAD analysis 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 determined by HPAE-PAD and using the same calculations described in Example 3. Panose is listed separately in Table 9 and subsequent tables.

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. Degradation of higher sugars clearly continues during the one-stage reaction, resulting in better utilization of available oligosaccharides.

As above, the content of DP1 decreases as the length of the donor molecule increases. 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 to DP5 is similar to that in reactions using DP3+-synthesizing enzymes but slightly lower than that in reactions using maltose-producing enzymes. The content of DP6 to DP10 also increases with increasing donor molecule length in the one-step reaction, in the following order: PF > CspAmy2 > 4G, 626 > BBA/2G.

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

When 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.

Example 5. Desirable Amount of β-Amylase Activity In the above example, 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 dosages of β-amylase present.

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

Samples were removed at appropriate time points during saccharification for HPLC and HPAE-PAD analysis as described in 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 described in Example 3.

From Table 11, it is clear that, as seen in the previous examples, the reaction with OPTIMALT® 4G 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 performed using only β-amylase.

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, there is little change in the sugar profile compared to reactions without OPTIMALT® BBA. Further increases in dosage result in smaller differences between the improved and conventional methods. The amount of DP11+ was lower for reactions using both OPTIMALT® 4G and OPTIMALT® BBA compared to the conventional reaction using only OPTIMALT® BBA. Clearly, the longer-chain starch fractions are better hydrolyzed when both enzymes are present.

As shown in Table 12, the amount of longer-chain IMOs (see, e.g., IM6 and IM7) was higher in the reaction using OPTIMALT® 4G than in the reaction using 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 a different liquefact.

When small amounts of β-amylase (OPTIMALT® BBA) are 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, IM2 content increases to levels similar to those in conventional reactions using OPTIMALT® BBA alone. Similar effects are observed with other sugars.

One surprising observation is that total IMO content increases with 0.01 kg/MT of OPTIMALT® BBA present in the OPTIMALT® 4G reaction. This may be substantially beneficial 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.

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 the improved method, the dosage of β-amylase should be expressed in terms of activity units present in the reaction.

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 at 20°C for 1 hour. 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.

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. It is estimated that as little as 13.2 DP°/kg may be even 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 (13)

1. A method for producing isomalto-oligosaccharides (IMO) from maltodextrin, comprising:
(i) contacting maltodextrin with DP3+ producing α-amylase in the presence of β-amylase having no more than 66 saccharifying power (DP°) units per kg dry weight of maltodextrin, or in the absence of β-amylase, to produce malto-oligosaccharides; and (ii) contacting the malto-oligosaccharides with transglucosidase to produce IMOs, which produces longer chain IMOs and/or reduced amounts of glucose compared to a process for producing IMOs from maltodextrin under the same conditions, except that β-amylase is used 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 . The method of claim 1, wherein the DP3+-producing α-amylase produces malto-oligosaccharides containing at least 15% DP3. The method according to claim 1 or 2 , wherein the DP3+ producing α-amylase produces malto-oligosaccharides containing 40% or less DP2. The method according to any one of claims 1 to 3 , wherein step (i) is carried out in the presence of pullulanase. The method of any one of claims 1 to 4 , wherein steps (i) and (ii) are carried out sequentially. The method of any one of claims 1 to 4 , wherein steps (i) and (ii) are carried out simultaneously. 7. The method of claim 1 , wherein the maltodextrin is prepared from a starch-containing substrate using a liquefying α-amylase. 8. The method of claim 7 , wherein the liquefying α-amylase and the DP3+ producing α-amylase used in step (i) are the same. 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, which produces longer chain IMOs and/or reduced amounts of glucose compared to a method of 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. 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. The method of claim 9 or 10 , wherein steps (i) and (ii) and/or steps (ii) and (iii) are sequential, overlapping or simultaneous. 12. The method according to any one of claims 9 to 11 , wherein step (ii) is carried out in the presence of pullulanase. 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.