WO2021011793A1 - Procédé amélioré de production d'isomalto-oligosaccharides - Google Patents

Procédé amélioré de production d'isomalto-oligosaccharides Download PDF

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WO2021011793A1
WO2021011793A1 PCT/US2020/042367 US2020042367W WO2021011793A1 WO 2021011793 A1 WO2021011793 A1 WO 2021011793A1 US 2020042367 W US2020042367 W US 2020042367W WO 2021011793 A1 WO2021011793 A1 WO 2021011793A1
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amylase
oligosaccharides
imo
malto
maltodextrins
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PCT/US2020/042367
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English (en)
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Bart C. Koops
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Danisco Us Inc
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Priority to EP20751017.3A priority Critical patent/EP3999654A1/fr
Priority to US17/628,094 priority patent/US20220259630A1/en
Priority to CA3147605A priority patent/CA3147605A1/fr
Priority to KR1020227004859A priority patent/KR20220034866A/ko
Priority to JP2022502972A priority patent/JP2022541526A/ja
Priority to CN202080064743.9A priority patent/CN114402081A/zh
Priority to BR112022000746A priority patent/BR112022000746A2/pt
Publication of WO2021011793A1 publication Critical patent/WO2021011793A1/fr

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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2451—Glucanases acting on alpha-1,6-glucosidic bonds
    • C12N9/2457—Pullulanase (3.2.1.41)
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    • C12P19/00—Preparation of compounds containing saccharide radicals
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01001—Alpha-amylase (3.2.1.1)
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    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/0102—Alpha-glucosidase (3.2.1.20)
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    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01041—Pullulanase (3.2.1.41)
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    • C13K—SACCHARIDES OBTAINED FROM NATURAL SOURCES OR BY HYDROLYSIS OF NATURALLY OCCURRING DISACCHARIDES, OLIGOSACCHARIDES OR POLYSACCHARIDES
    • C13K13/00—Sugars not otherwise provided for in this class

Definitions

  • the present method is for producing improved isomalto-oligosaccharides (IMO) from maltodextrins.
  • the improved method involves the complete or partial replacement of b-amylase, as used in a conventional method, with a selected a-amylase.
  • the resulting IMO have longer chain-length and reduced residual glucose content compared to IMO produced using a conventional method.
  • Isomalto-oligosaccharides 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 have a low glycemic index, making them desirable for consumption by diabetics, and are not metabolized by most oral bacteria, making the desirable for avoiding cavities.
  • 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 b-amylase and a transglucosidase.
  • the b-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.
  • the acceptor can be another maltose molecule, resulting in a tri saccharide.
  • 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.
  • IMO isomalto-oligosaccharides
  • an improved method for producing isomalto-oligosaccharides (IMO) from maltodextrins comprising the steps of: (i) contacting maltodextrins with an a- amylase to produce malto-oligosaccharides, and (ii) contacting the malto-oligosaccharides with a transglucosidase to produce IMO, wherein the method produces longer chain IMO and/or reduced amounts of glucose compared to a method for producing IMO from maltodextrins using b-amylase in step (i).
  • step (i) is performed in the presence of no more than 660 diastatic power (DP°) units b-amylase per kg dry weight maltodextrins. 3. In some embodiments of the method of paragraph 1, step (i) is performed in the presence of no more than 264 diastatic power (DP°) units per kg dry weight malto- oligosaccharides.
  • step (i) is performed in the presence of no more than 132 diastatic power (DP°) units per kg dry weight malto- oligosaccharides.
  • step (i) is performed in the presence of no more than 66 diastatic power (DP°) units per kg dry weight malto- oligosaccharides.
  • step (i) is performed in the absence of a b-amylase.
  • step (i) is performed using an a-amylase that produces malto-oligosaccharides that comprise at least 15% DP3.
  • step (i) is performed using an a-amylase that produces malto-oligosaccharides that comprise at least 10% DP4.
  • step (i) is performed using an a-amylase that produces malto-oligosaccharides that comprise at least 5% DP5.
  • step (i) is performed using an a-amylase that produces malto-oligosaccharides that comprise no more than 40% DP2.
  • step (i) is performed in the presence of a pullulanase.
  • steps (i) and (ii) are performed sequentially.
  • steps (i) and (ii) are performed simultaneously.
  • the maltodextrins are prepared from a starch-containing substrate using a liquefying a-amylase.
  • step (i) the liquefying a-amylase and the a-amylase used in step (i) are the same.
  • an improved method for producing isomalto-oligosaccharides comprising the steps of (i) contacting a starch-containing substrate with a liquifying a-amylase to produce maltodextrins, (ii) contacting the maltodextrins with a DP3+ generating a-amylase to produce malto-oligosaccharides and (iii) contacting the malto- oligosaccharides with a transglucosidase to produce IMO having longer chains compared to IMO produced using b-amylase instead of DP3+ generating a-amylase in step (ii).
  • the DP3+ generating a-amylase produces malto-oligosaccharides comprising at least 15% DP3, at least 10% DP4, at least 5% DP5, and/or no more than 40% DP2.
  • steps (i) and (ii), and/or steps (ii) and (iii), are sequential, overlapping or simultaneous.
  • step (ii) is performed in the presence of no more than 660, no more than 264, no more than 132, or no more than 66 diastatic power (DP°) units b-amylase per kg dry weight maltodextrins
  • step (ii) is performed in the absence of a b-amylase.
  • step (ii) is performed in the presence of a pullulanase.
  • step (ii) the liquefying a- amylase and the DP3+ generating a-amylase used in step (ii) are the same.
  • Figure 1 is a flowchart illustrating the steps and enzymes involved in a conventional process for preparing IMO.
  • Figure 2 is a flowchart illustrating the steps and enzymes involved in the present improved process for preparing IMO.
  • Figure 3 is a diagram illustrating transglucosidase reactions that occur in a conventional process for preparing IMO.
  • Figure 4 is a diagram illustrating reactions between a transglucosidase-glucose complex and malto-oligosacchade acceptor molecules to produce isomalto-oligosacchades.
  • Figure 5 is a diagram illustrating reactions between malto-oligosacchade donor molecules and transglucosidase to generate transglucosidase-glucose complexes that can react with malto- oligosacchade acceptor molecules to produce isomalto-oligosacchades.
  • starch refers to any material comprised of the complex polysaccharide carbohydrates of plants, comprised of amylose and/or amylopectin with the formula (C6HIO05) X , wherein X can be any number.
  • the term refers to any plant- based material including but not limited to grains, grasses, tubers and roots and more specifically wheat, barley, corn, 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.”
  • granular starch refers to uncooked (raw) starch, which has not been subject to gelatinization.
  • 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.
  • 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.
  • IMO isomalto-oligosaccharides
  • Exemplary isomalto-oligosaccharides 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- l,4)Glc) are often considered IMO as well.
  • IMO may include some a-D-1,4 bonds.
  • DP degree of polymerization
  • an“a-amylase” is an endo- acting enzyme having the systematic name a- D-(l 4)-glucan glucanohydrolase) and the Enzyme Commission designation EC 3.2.1.1.
  • a“starch processing enzyme” is an enzyme that depolymerizes a starch substrate (including maltodextrin).
  • Exemplary starch processing enzymes are a-amylase, glucoamylase, b-amylase, pullulanase, and a-glucosidase.
  • 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 endo- 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.
  • a“maltooligosaccharide producing enzyme” is an enzyme that produced mainly malto-oligsaccharides with a degree of polymerization of longer 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.
  • 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.
  • 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.
  • “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.”
  • “generating” refers to producing a reaction product as the result of an enzymatic process.
  • IMO isomalto-oligosaccharides
  • IMO is conventionally produced from maltodextrins by the sequential or simultaneuous action of a b-amylase and a transglucosidase.
  • a starch slurry is converted to maltodextrins in a liquefaction process and the maltodextrins are treated with b-amylase and pullulanase to produce a maltose syrup, which is then treated with transglucosidase to produce IMO.
  • a starch slurry is converted to maltodextrins in a liquefaction process and the maltodextrins are treated simultaneously with b- amylase, pullulanase and transglucosidase to produce IMO, without isolating or separating the maltose syrup.
  • the improved process may continue to begin with the conversion of starch slurry to maltodextrins in a liquefaction process.
  • matodextrins are now treated with an enzyme capable of generating malto-oligosaccharides longer than DP2 (z.e., maltose).
  • a DP3 (or longer malto-oligosaccharide)-producing enzyme and pullulanase are used to produce a 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).
  • the maltodextrins are treated simultaneously with a DP3 (or longer malto-oligosaccharide)-producing enzyme, pullulanase and transglucosidase to produce improved IMO, without isolating or separating maltotriose (or longer malto-oligosaccharide)- rich syrup in a one-step process (right side of flowchart).
  • DP3 or longer malto-oligosaccharide
  • pullulanase and transglucosidase
  • transglucosidase reaction produces no free glucose during the first part of the reaction which forms the transglucosidase-glucose complex.
  • Transglucosidase-glucose complex formed as in Figures 4B and 4C can interact with acceptor oligosaccharides of various lengths to produce IMO. No free glucose is generated in this part of the reaction ( Figure 5).
  • DP3+ generating (also called DP3+ producing) a-amylases suitable for producing malto- oligosaccharides for use in the improved process are those that produce malto-oligosaccharides longer than DP2 (z.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, a-amylases from Aspergilus, e.g., A. kawachi, A clavatus and A. oryzae.
  • Enzymes that produce significant amounts of DP4 include, but are not limited to, an amylase from Pseudomonas saccharophila.
  • Enzymes that produce significant amounts of DP5 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.
  • 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.
  • transglucosidase 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.
  • the enzyme exemplified herein is from Aspergillis niger and is 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 believed to be suitable for producing isomalto-oligosaccharides (IMO) from maltodextrins.
  • IMO isomalto-oligosaccharides
  • the exemplified enzyme is commercially-available as TRANSGLUCOSIDASE L2000 ® (DuPont Nutrition & Biosciences) with an activity of 1700 transglucosidase units (TGU)/g.
  • TGU transglucosidase units
  • 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 ® /MT of DS is needed. In all the work described herein, 1 kg/MT DS was used.
  • a-amylase for converting crude feedstocks, such as a starch from grains and other plant materials, to maltodextrins are well known in the art and include enzymes derived from numerous microorganisms. Exemplary enzymes are commercially available as, e.g. , FUELZYMETM (BASF Enzymes LLC, San Diego, CA), LPHERA®, AVANTEC® and
  • LIQUOZYME® products Novozymes
  • SPEZYME® products DuPont. More than one liquefying a-amylase can be used.
  • the liquefying a-amylase may additionally be useful as the DP3+ generating enzyme for use in the improved process, depending on the profile of malto-oligomers generated. Accordingly, the liquefying a-amylase(s) may be, or may include, the DP3+ generating enzyme(s).
  • the enzyme concentration needed to produce such a sugar profile 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.
  • Starch liquefaction can be performed above, at or below the gelatinization temperature of the starch substrate. Other enzymes may be present, e.g ., proteases.
  • Suitable enzyme blends are those that produce, from a 12 DE starch liquefact in the absence of transglucosidase, a syrup with a high content of DP3 - DP5 malto-oligosaccharides.
  • a high content means that a minimum of 15%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% of the total malto-oligosaccharides are DP3, DP4 and/or DP5.
  • Another way of defining a high content syrup is by individual sugar components, where DP3 is minimum 15%, DP4 is minimum 10%, DP5 is minimum 5% and/or DP2 is maximum 40%, 30%, 20%, 10% or even 5%.
  • Suitable enzyme blends are additionally or alternatively those that produce, from a 12 DE starch liquefact in the presence of transglucosidase, a syrup with content of more than 4% isomaltopentaose, more than 2% isomaltohexaose and/or more than 1% isomaltoheptaose as a percentage of total sugars, as measured as described in the Examples.
  • a key feature of the improved process and enzymatic compositions is that they are performed 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.
  • Substantially in the absence of b-amylase activity means that enzymes categorized as b-amylases or maltogenic amylases, and/or enzyme compositions (such as blends) having b-amylase activity, are not necessary or required to produce the improved IMO described herein. Accordingly, no b- amylase and/or beta-amylase activity need be applied to maltodextrins for the purpose of producing improved IMO as described.
  • b-amylase activity is typically expressed in degrees diastatic power (DP°).
  • DP° degrees diastatic power
  • One unit of diastase activity, expressed as degrees DP (DP°) is defined as the amount of enzyme, contained in 0.1 ml of a 5 % solution of the sample enzyme preparation, that will produce sufficient reducing sugars to reduce 5 mL of Fehling's solution when the sample is incubated with 100 mL of the substrate for 1 hour at 20°C.
  • the reducing sugar groups produced during the reaction are measured in a titrimetric procedure using alkaline ferricyanide. This enzyme assay measures the activity of both a-and b-amylases present in a given sample.
  • the amount of b-amylase activity that can be tolerated in the present improved methods is maximally about 660 DP° units b-amylase per kg DS starch hydrolysate, maximally about 264 DP° units b-amylase per kg DS starch hydrolysate, maximally about 132 DP° units b-amylase per kg DS starch hydrolysate, and maximally about 66 DP° units b-amylase per kg DS starch hydrolysate.
  • no measurable amount of b-amylase need be present.
  • GSHE granular starch hydrolyzing enzymes
  • 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.
  • enzymes that can liberate oligosaccharides from raw starch include, but are not limited to, SPEZYME® ALPHA PF, SPEZYME® XTRA, Aspergillus karwachi alpha-amylase, and OPTIMALT® 4G.
  • the improved process and enzymatic compositions allow the production of isomalto- oligosaccharides (IMO) from maltodextrins for any number of uses.
  • the IMO are 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 specifically, the IMO produced using the present
  • compositions and methods are longer than IMO produced using conventional methods, and the ratios of the content (% of total sugars) of longer IMO molecules over shorter molecules is increased. Longer IMO are likely to be more poorly metabolized, offering greater health benefits to consumers and more food ingredient options to food producers
  • Transglycosylation reactions were performed using reagent grade maltose and reagent grade maltotriose (both purchased from Sigma Aldrich) at 30% DS in water. Solutions of maltose or maltotriose at 30% DS were made and adjusted to pH 4.2. Approximately 2 g of the maltose or maltotriose solution was weighed into Eppendorf tubes. To each Eppendorf tube transglucosidase (TRANSGLUCOSIDASE® L-2000; DuPont) was added at a dose of 1 kg product per MT of substrate DS. The tubes were incubated in a thermostatic mixer
  • thermoblock for 48 hr at 60°C with a shaking speed of 750 rpm.
  • Example 1 Since it is not commercially attractive to perform the transglycosylation reaction with pure malto-oligosaccharides, the experiment in Example 1 was repeated using a starch hydrolysate rich in DP4.
  • This starch hydrolysate was prepared using a DP4-producing enzyme and was compared to a starch hydrolysate rich in maltose prepared using b-amylase.
  • Both starch hydrolysates were prepared from a com liquefact with a DE of 11.28 at 32.5% DS.
  • the liquefact was incubated with a DP4-producing a-amylase
  • Example 2 The experiment described in Example 2 was repeated by applying a more diverse panel of enzymes to maltodextrins to make DP2, DP3, DP4 and DP5-rich syrups from maltodextrins.
  • the enzyme used for the production of a DP2 rich syrup was either b-amylase
  • DP3-rich syrup was DP3-producing a-amylase from Aspergillus kawachi (GC626; DuPont) at 0.5 kg/MT DS, with or without pullulanase at 0.4 kg/MT DS.
  • the enzymes use for the production of DP4- rich syrups was the DP4-producing a-amylase (OPTIMALT® 4G, as above) at 0.9 kg/MT DS, with and without pullulanase at 0.4 kg/MT DS.
  • the enzymes used for DP5-rich syrups were either a Cytophaga sp. -based a-amylase at 6 pg purified protein/g DS, or a Bacillus stearothermophilus- based a-amylase (SPEZYME® ALPHA PF) at 0.2 kg/ MT DS, with or without pullulanase at 0.4 kg/MT DS.
  • amylase enzymes will be referred to as BBA, 2G, 626, 4G, CspAmy and PF, respectively, in this example and all further examples.
  • the pullulanse used in all further examples is OPTIMAX® L1000 and the transglucosidase is TRANSGLUCOSIDASE L-2000®, unless otherwise mentioned.
  • a 10 m ⁇ 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.
  • the column was equilibrated for 10 minutes with 10% 1 M NaOH and 10% 500 mM NaOAc and 80% MilliQ water. The separation of the sugars was accomplished by elution with constant 10% 1M sodium hydroxide and 90% MilliQ water for 5 minutes.
  • 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.
  • the DP2 content in the syrup is 10% (measured by conventional HPLC)
  • DPI, DP2 and DP3 isomers can be distinguished.
  • linear isomalto-oligosaccharides up to DP7 are distinguished, i.e., isomalto-tetraose, isomalto-pentaose, isomalto-hexaose and isomalto- hetpaose 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. The absence of commercially-available standards and methods for separating the longer isomers is understandable as the practical production of these IMO is made possible only in view of the present improved method.
  • the IMO content (z.e., the sum of isomaltose, isomaltotriose, pannose, isomaltotetraose, isomaltopentaose, isomaltohexaose and isomaltoheptaose) is higher when staring 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 content of DPn, possibly due to the lack of a debranching activity during the tranglycosylation reaction. If these non-available, presumably branched maltooligosaccharides are be made available by the addition of pullulanse at the time of TG treatment, the IMO content following transglucosidase treatment using longer donor molecules would probably be higher.
  • Table 9 IMO content of above syrups as measured by HPAE-PAD analysis.
  • DPI content decreases with increasing length of the donor molecule. Overall, more DPI is formed in the one-step process compared to the two-step process. Even during transglycosylation with longer donor molecules, glucose is eventually released.
  • DP2 - DP5 content is similar in reactions with the DP3+ generating enzymes and a little lower than the reactions using the enzymes that generate maltose.
  • DP6 - DP 10 content also increases with increasing length of the donor molecule in the one-step reaction, in the order: PF > CspAmy2 > 4G, 626 > BBA/2G.
  • the amount of shorter IMO generally appears to decrease with increasing length of the donor molecule.
  • the levels of isomaltotriose, isomaltotetraose and isomaltopentaose appear to remain approximately the same, while he levels of isomaltohexaose and isomaltoheptaose clearly increase with increasing length of the donor molecule.
  • the traditional one-step saccharification was performed with b-amylase (OPTIMALT ® BBA) at 0.9 kg/MT, pullulanase (OPTIMAX ® L2500) at 0.16 kg/MT and transglucosidase at 1.0 kg/MT at pH 5.0 and 60°C.
  • b-amylase OPTIMALT ® BBA
  • pullulanase OPTIMAX ® L2500
  • transglucosidase 1.0 kg/MT at pH 5.0 and 60°C.
  • OPTIMALT ® 4G produces a syrup having much lower DPI than the traditional reaction with OPTIMALT ® BBA. It also shows that a small amount of b-amylase can be present during the reaction with OPTIMALT ® 4G without influencing the results significantly. Specifically, when there is up to 0.05 kg/MT OPTIMALT ® BBA present, the DPI is as low as when no BBA is present. Only when 0.1 kg/MT OPTIMALT ® BBA is present, the DPI begins to increase, and further increases, in a dose-dependent manner, according to the amount of the b-amylase. At a dose of 0.5 kg/MT the DPI level is very close to that of the conventional reaction performed with only the b-amylase.
  • the amount of longer IMO is larger in the reaction with OPTIMALT ® 4G than in the reaction with OPTIMALT ® BBA. Also, in this example, less IM2 and more panose was measured. Overall, the total amount of IMO made in this experiment was higher than in previous experiments, which is likely due to the use of a different liquefact.
  • IM2 is higher than without. The amount does not change with the dose up to a b-amylase dose of 0.05 kg/MT. At higher dosages of OPTIMALT ® BBA the IM2 content increases to the same level as the conventional reaction with only OPTIMALT ® BBA. Similar effects are seen for other sugars.
  • enzyme dose is expressed in kg enzyme/MT substrate, which is convenient for commercial products, and is common practice in the industry.
  • dose of b-amylase should be expressed in terms of activity units present in the reaction.
  • OPTIMALT ® BBA has an average b-amylase activity of 1320 DP°/g product, where DP° refers to diastatic power.
  • DP° determination is based on a 30-min hydrolysis of a starch substrate at pH 4.6 and 20°C. The reducing sugar groups produced upon hydrolysis are measured in a titrimetric procedure using alkaline ferricyanide.
  • One unit of diastase activity, expressed as degrees DP (°DP) is defined as the amount of enzyme, contained in 0.1 ml of a 5 % solution of the sample enzyme preparation, that will produce sufficient reducing sugars to reduce 5 mL of Fehling's solution when the sample is incubated with 100 mL of the substrate for 1 hour at 20°C.
  • a dose of 0.05 kg/MT 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 equivalent to 66 DP° units/kg, or 66 DP° units per kg of dry solids in a starch hydrolysate.
  • Example 5 The results described in Example 5 indicate that the present improved method is not adversely affected by the presence of up to 66 DP° Units of b-amylase activity present per kg dry solids in the starch hydrolysate. With up to 66 DP° Units present, DPI is as low as without b- amylase, with the same, or even higher, IMO content. It is estimated that the presence of as little as 13.2 DP°/kg may even be beneficial. However, when an increased amount of b-amylase activity is present, the benefits of the improved method is eroded, in a dose-dependent manner, until the IMO profile resemble that obtained by conventional method.

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Abstract

L'invention concerne un procédé de production d'isomalto-oligosaccharides (IMO) améliorés à partir de maltodextrines. Le procédé amélioré implique le remplacement complet ou partiel de la β-amylase, telle qu'utilisée dans un procédé classique, par une α-amylase sélectionnée. Les IMO ainsi obtenus présentent une longueur de chaîne plus longue et une teneur en glucose résiduel réduite par rapport aux IMO produits à l'aide d'un procédé classique.
PCT/US2020/042367 2019-07-16 2020-07-16 Procédé amélioré de production d'isomalto-oligosaccharides WO2021011793A1 (fr)

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EP20751017.3A EP3999654A1 (fr) 2019-07-16 2020-07-16 Procédé amélioré de production d'isomalto-oligosaccharides
US17/628,094 US20220259630A1 (en) 2019-07-16 2020-07-16 Improved method for producing isomaltooligosaccharides
CA3147605A CA3147605A1 (fr) 2019-07-16 2020-07-16 Procede ameliore de production d'isomalto-oligosaccharides
KR1020227004859A KR20220034866A (ko) 2019-07-16 2020-07-16 이소말토올리고당을 생산하기 위한 개선된 방법
JP2022502972A JP2022541526A (ja) 2019-07-16 2020-07-16 イソマルト－オリゴ糖を生成するための改良された方法
CN202080064743.9A CN114402081A (zh) 2019-07-16 2020-07-16 用于产生低聚异麦芽糖的改进的方法
BR112022000746A BR112022000746A2 (pt) 2019-07-16 2020-07-16 Método melhorado para produzir isomalto-oligossacarídeos

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