CN1756769A - Grain compositions containing pre-biotic isomalto-oligosaccharides and methods of making and using same - Google Patents

Abstract

Methods for the production of substrate, tuber, and grain compositions containing isomalto-oligosaccharides are described. The methods comprise (a) contacting a substrate, tuber or grain containing ungelatinized starch with a maltogenic enzyme and a starch liquefying enzyme to produce maltose; (b) contacting said maltose with a transglucosidic enzyme, wherein said steps (a) and step (b) occur at a temperature less than or at a starch gelatinization temperature; and (c) obtaining a substrate, grain or tuber composition having an enzymatically produced isomalto-oligosaccharide, wherein the oligosaccharide is derived from the grain. The maltogenic enzyme can be either exogenous or endogenous to the grain. The contacting steps can be sequential or concurrent. The present invention also describes flour, oral rehydrating solutions, beer adjuncts, food, feed, beverage additives incorporating the grain compositions made as described.

Description

The grain compositions and the manufacturing and methods for using them thereof that contain the isomalto-oligosaccharides of useful life

Technical field

The invention describes the grain compositions and the manufacture method thereof that contain isomalto Oligosaccharide (isomalto-oligosaccharides).This method comprises the starch that the contains isomalto Oligosaccharide of deriving from cereal.

Background technology

Isomalto Oligosaccharide (" IMOs ") is blended keyed jointing oligosaccharides (mixed linkage oligosaccharides), contains the mixture of 1,4 α and/or 1,6 α glycosidic link.They are also referred to as irregular connection oligosaccharides (" ALOs ").Isomalto Oligosaccharide comprises a large amount of branch's oligosaccharides, as isomaltose, panose, Isomaltotriose, different maltotetrose, different panose and more hyperbranched oligosaccharides.

Existence is to the market requirement of the product that contains IMOs.The IMO product is sold with powder type or liquid form, and this depends on the purposes of being planned.The potential purposes is at field of food.The example of IMO product is: seasonings (mayonnaise, vinegar, at the bottom of the soup etc.), sweet food (candy, chewing gum, chocolate, ice cream, fruit syrup, syrup), fruits and vegetables processed food (jam, marmalade orange, fruity sauces, salted vegetables), meat or fish food (ham, sausage etc.), bakery product (bread, cake, cookies, short crust pastry), cook ripe food (salad in advance, the beans that boil etc.), canned and bottled food, ready-to-eat (soluble coffee, at the bottom of the instant cake wine (instant cake base) etc.), and beverage, both comprised alcopop (alcoholic beverage, seju, fruit wine, sake wine, beer [international publication number WO 02/20712 A1], etc.) comprise nonalcoholic drinks (coffee again, fruit juice, honey dew beverage, fizz or soda pop, lemonade, laughable).Isomalto Oligosaccharide can be further as the composition in animal-feed and the feed for pet.The Application Areas of non-food is makeup and medicine (cigarette, lipstick, toothpaste, medicine for oral administration etc.).

Isomalto Oligosaccharide belongs to a class oligosaccharides that is classified as functional health food oligosaccharides (" FHFO ").Representational IMOs comprises fructose-oligosaccharides, semi-lactosi-oligosaccharides, wood sugar-oligosaccharides and rough gentian-oligosaccharides (gentio-oligosaccharides).When IMOs when every day is oral regularly, what interrelate with it is the enhancement of human and animal's comprehensive health state, IMOs is classified as " benefit is given birth to matter (prebiotics) ".Benefit is given birth to matter and is defined as stodgy material (for example, foodstuff fibre), and beneficial microorganism growth or biological activity that it is present in enteron aisle by selective stimulating or is imported into enteron aisle in treatment apply some biological effects to the mankind.(Przemyslaw Jan Tomasik and Piotr Tomasik.2003 American Association of CerealChemists，Inc.80(2)：113-117)。" benefit living (prebiotic) " effect of oligosaccharides is to increase bifidus bacillus (bifidobacteria) and the quantity of lactobacillus (lactobacilli) (" benefit is given birth to matter ") and the concentration of reduction putrefactive bacterium (putrifactive bacteria) in the large intestine.Bifidus bacillus is relevant with some sanatory character, for example suppresses pathogenic growth, and this is by forming acid or realizing by antimicrobial acivity.They are also relevant with all effects of this class, as regulating immunity system (antitumor character), triglyceride reducing and cholesterol levels, generate VITAMIN (B family), reduce ammonia concentration, prevent transposition, recover normal intestinal microflora behind the antibacterial therapy, produce digestive ferment, reduce side effect (the Kohmoto T. relevant with microbiotic, Fukui F., Takaku H., Machida Y., et al., Bifidobacteria Microflora, 7 (2) (1988), 61-69; Kohmoto K., Tsuji K., Kaneko T., Shiota M., et al., Biosc.Biotech.Biochem., 56 (6) (1992), 937-940; KanekoT., Kohmoto T., Kikuchi H., Fukui F., et al., Nippon Nogeikagaku Kaishi, 66 (8) (1992), 1211-1220, Park J-H, Jin-Young Y., Ok-Ho S., Hyun-Kyung S., et al., Kor.J.Appl.Microbiol.Biotechnol., 20 (3) (1992), 237-242).Modler，H.W.，1992，″Compoundswhich enhance the growth of Prebiotic Bacteria″，presented at the InternationalRoundtable on Animal Feed Biotechnology，Ottawa，Ontario，Canada。

Isomalto Oligosaccharide is to use D-Transglucosylase (E.C.2.4.1.24, transglucosidase, alpha-glucosidase), by enzymatic commentaries on classics glucosyl reaction (transglucosylation reaction) synthetic.This enzyme with α-D-glucose-oligosaccharides incubation the time, catalytic hydrolysis reaction and shift reaction.Transfer to 6-OH (oh group 6 of glucose molecule) takes place the most frequently, produces isomaltose by D-glucose, perhaps produces panose from maltose.This enzyme also can catalysis to the transfer of the 2-OH or the 3-OH of D-glucose, form kojibiose (kojibiose) or black mould sugar, perhaps get back to 4-OH and form maltose again.The result of transglucosidase reaction is that Fructus Hordei Germinatus oligose changes isomalto Oligosaccharide into, has formed a class oligosaccharides that contains more a high proportion of glucose moiety, described glucose moiety is connected in the one-level oh group of the non-reduced end of glucose molecule, for example, by α-D-1,6 glycosidic links connect.Only act on oligosaccharides (McCleary B.V., Gibson T.S., Carbohydrate Research 185 (1989) 147-162 of low polymerization degree (DP) from the transglucosidase of aspergillus niger; Benson C.P., Kelly C.T., Fogarty W.M., J.Chem.Tech.Biotechnol., 32 (1982) 790-798; Pazur J.H., Tominaga Y, DeBrosse C.W., Jackman L.M., Carbohydrate Research, 61 (1978) 279-290).The polymerization degree is meant the number of glucose unit.For example, didextrose base molecule, for example the DP of maltose is 2.These sugar have received increasing concern as foodstuff additive, reason is that they help preventing dental caries (Oshima, et.al 1988.The caries inhibitory effects of gos-sugar in vitro and ratexperiments.Microbial Immunol.32,1093-1105), and can be used as the somatomedin (benefit is given birth to matter) of bifidus bacillus, the improvement mankind's enteric microorganism effect (Komoto, et.al 1988; Effect ofIsomalto-oligosaccharides on human fecal flora, Bifidobacteria Micro flora 7,61-69).

Can obtain isomalto Oligosaccharide with different methods.For example, handle high level cadre's solids concn with glucoamylase, i.e. the dextrose syrup of 60-80% causes DP to be mainly the formation of 2 isomalto Oligosaccharide.The existence of high solid level forces reaction to reverse from the normal direction that helps hydrolysis.

Cereal comprises wheat, barley etc., at many value-added nutraceutical compositions, in the economic production as wheat-flour, starch, starch hydrolysate (glucose, fructose, high malt sugar syrup etc.) and Wheat bran (wheat gluten), is fabulous starting material.At microbiotic, medicine, vaccine, biological chemistry goods, in the production as ethanol (both comprised beverage and also comprised fuel), amino acid, organic acid etc., and recently in the production of the functional health food oligosaccharides that is called as isomalto Oligosaccharide, the syrup that contains high-level maltose also is used as the carbon source in many microbial fermentations.Producing starch hydrolysate, in the traditional method as maltose syrups, before hydrolysis, by starch liquefacation and product maltogenic alpha-amylase, insoluble pearl starch is being separated with other cellular constituent of wheat usually.Maltose is by two disaccharides that the glucosyl residue is formed that connect by α 1-4D-glycosidic link, and it is a material minimum in the Fructus Hordei Germinatus oligose family.It is as other syrup of different purity level, powder and crystal and scale operation.Various maltose syrups have received tangible concern, and they can be used for brewageing, in the commercial use of baking, canned soft drink, candy and other food and drink industry.In Japan, ultrapure maltose is used as the intravenously nutrition.The catalytic reduction of maltose forms maltose alcohol, and it is considered to the sweeting agent of low calory.Recently, the high malt sugar syrup has become important source material (the J.K.Shitty and O.J.Lantero of industrial production isomalto Oligosaccharide, 1999 " Transglucosylation of Malto-oligosaccharides. " Paper presented at 50thStarch Convention, Detmold, Germany).

In producing starch hydrolysate such as the syrupy traditional method of high malt sugar, before hydrolysis, use heat-resisting liquefaction αDian Fenmei [EC3.2.1.2 from Bacillus licheniformis (Bacillus licheniformis) or bacstearothermophilus (Bacillusstearothermophilus), α (1,4)-dextran glucan hydrolase] insoluble starch is separated.The hydrolysis of purifying starch (purified) is by, suspension insoluble particle starch in water (30%-50% dissolved solids composition [dsb]) and is heated to temperature between 85 ℃ to 120 ℃ with dissolving starch with make it be vulnerable to enzymically hydrolyse to carry out.Use maltose to generate enzyme, the starch of further process liquefaction, has the starch hydrolysate that different carbohydrates is formed with generation, as fungi αDian Fenmei (Genencor International, Palo Alto, CA sells, trade mark is called CLARASE L) be used to contain the syrup that is less than 55% maltose, βDian Fenmei (Genencor International, Palo Alto, CA sells, and trade mark is called OPTIMAL BBA) be used to produce maltose composition that contains 55% to 62% and the syrup that is less than 1% glucose.Higher level maltose syrups for＞62%, it is useful uniting adding debranching factor (CA sells for GenencorInternational, Palo Alto, and trade mark is called OPTIMAX L-1000) and βDian Fenmei.(Faigh，J.；Duan，G.；Strohm，B.and Shetty，J.(2002)″Production of Maltose，High Maltose & Very High Maltose Syrups，″Technical Bulletin，Genencor InternationalInc.)。

Reported and a kind ofly changed granular starch (purified) method of solubility hydrolysate into, this is by being lower than the temperature of starch gelatinization temperature, with bacterial alpha amylase incubation (Leach et.al 1978; U.S. Patent number 4,113,509),, produce high malt sugar syrup (Leach et.al 1975 subsequently by the βDian Fenmei hydrolysis; U.S. Patent number 3,922,196) implement.Yet the maltose of the syrup formation that produces by this method only accounts for 55% of total sugar degree, and the trisaccharide maltose level that contains is very high.European patent application 0905256 (Christophersen, et.al 2000) and United States Patent (USP) 5,141,859 (Nimmi, et.al 1992), describe the starch (after the gelatinization, using heat-resisting αDian Fenmei hydrolysis) of using liquefaction and produced the syrupy method of high malt sugar.This process trouble, costliness, and starch need be separated from other cellular component, the maltose of interpolation generates the cost height of enzyme, needs pyroprocessing and long reaction times.European patent application 0350737 A2 (Shinke, et.al 1989) a kind of method that generates maltose syrups disclosed, method is the αDian Fenmei of using from bacstearothermophilus, at 60 ℃, hydrolysis is from particulate state (purifying) starch of corn, wheat, potato and sweet potato, there is not traditional liquefaction step (gelation, liquefaction at high temperature subsequently).Yet it is 50% to 55% that the starch of this hydrolysis causes the maltose concentration scope.The trisaccharide maltose level that this syrup contains also very high (30-36%).No matter the source of starch how, this method all causes the ratio of maltose and trisaccharide maltose to be less than 2.0.Because the difficulty of metabolism trisaccharide maltose is big, the maltose syrups that contains high-level trisaccharide maltose comprises in the zymic ethanol fermentation at many microbial fermentations, not preferred substrate (the J.K.Shetty and O.J.Lantero that carbon is supplied with, 1999 " Transglucosylation of Malto-oligosaccharides. ", paper is issued in the 50th starch conference of German Detmold).United States Patent (USP) 6,361,809 have described the method for producing maltose and limit dextrin, method is for using lytic enzyme, what be categorized as EC 3.2.1.133 handles the particulate state waxy corn starch of purifying from the maltogenase αDian Fenmei of bacstearothermophilus, uses hyperfiltration process subsequently and separates maltose.The evaporation that the dilution that contains maltose is seen through thing (dilute permeate) is expensive, because energy cost is high, and exists the excessive risk of microbial contamination.

Traditionally, cereal such as wheat, Fructus Hordei Germinatus, sorghum (milo), broomcorn millet (ragi), whole in particular cereal can be used as the carrier of magnanimity (micro-) element and trace element, protein, fiber and VITAMIN, is used in the nutritious prod.As if the great majority in the cereal cereal are digested too easily, to such an extent as to can not give birth to matter or even play effective function as nutritive substance (nutraceuticals) as benefit.Advise, the design genetic modification, more stodgy, as to be suitable for giving birth to matter cereal as benefit, to regulate intestinal microbiota (Gibson, G.R, and Roberfroid, M.B.1995, Dietary modulation of the human colonicmicrflora:Introducing the concept ofprebiotics.J.Nutr.125,1401-1412).

The method that production contains the grain compositions of isomalto Oligosaccharide receives publicity always, hope is being changeed glucoside reaction (transglucosidation action) before in such method, do not need the high temperature of separating and/or make the starch of substrate to suffer the steam pressurization starch and other grain component, just can from source substrate such as grain or stem tuber enzymatic derive isomalto Oligosaccharide.Be used for the low pH method of minimise microbiological contamination, also receive publicity always.The present invention promptly is at these concerns.

Summary of the invention

The invention describes the method that is used to produce the isomalto Oligosaccharide grain compositions, described method comprises: (a) contact the cereal that contains unpaste starch with producing maltase (maltogenic enzyme) with starch liquefacation enzyme (starch liquefying enzyme), to generate maltose; (b) contact described maltose with the enzyme (transglucosidic enzyme) that changes glucosides, the temperature of wherein said step (a) and described step (b) generation is to be lower than or to equal starch gelatinization temperature; (c) acquisition has the grain compositions of the isomalto Oligosaccharide of enzymatic generation, and wherein said oligosaccharides derives from described cereal.

Can be randomly, in one embodiment, step (a) and (b) take place simultaneously.In another embodiment, this method further comprises the step of dry described grain compositions.In another embodiment, described cereal is selected from wheat, rye, barley, Fructus Hordei Germinatus and paddy rice.In another embodiment, described cereal is selected from Chinese sorghum, broomcorn millet and paddy rice.In another embodiment, described product maltase is a βDian Fenmei.In another embodiment, described product maltase is endogenic concerning described cereal.In another embodiment, described product maltase is ectogenic concerning described cereal.In another embodiment, described starch liquefacation enzyme is the αDian Fenmei that is derived from bacillus (Bacillus).In another embodiment, described starch liquefacation enzyme is derived from Bacillus licheniformis or bacstearothermophilus.In another embodiment, the enzyme of described commentaries on classics glucoside is transglucosidase (transglucosidase).In another embodiment, transglucosidase is derived from aspergillus tubigensis (Aspergillus).In another embodiment, transglucosidase is derived from aspergillus niger (Aspergillusniger).Other embodiment of the present invention comprises the grain compositions that produces according to top institute described method.Another embodiment of the present invention comprises the foodstuff additive that contain the top grain compositions of describing.

The present invention has also described and produced the method that isomalto Oligosaccharide is strengthened flour when being equal to or less than gelatinization point, wherein make the ungelatinized cereal that has endogenic product maltase contact the lytic enzyme (solubilizing enzyme) that is selected from bacillus, to produce maltose syrups.Make maltose syrups contact transglucosidase, produce matrix (stem tuber or cereal) composition, described composition comprises the different Fructus Hordei Germinatus-carbohydrate that derives from them.

The present invention has also described and produced the method that isomalto Oligosaccharide is strengthened flour when being equal to or less than gelatinization point, the ungelatinized cereal (wheat, barley etc.) that wherein will have an endogenic product maltase and the ungelatinized cereal that does not have endogenic product maltase are (for example, Chinese sorghum, broomcorn millet or paddy rice) mix, this maslin is contacted the lytic enzyme that is selected from bacillus, to generate maltose syrups.This maltose syrups is contacted with transglucosidase, produces matrix (stem tuber or cereal) composition, described composition comprises the different Fructus Hordei Germinatus-carbohydrate that derives from them.

The present invention has also described and has been used to make the wheat grain method for compositions, and described method comprises: (a) will contain the ungelatinized wheat grain of endogenic product Fructus Hordei Germinatus βDian Fenmei and contact with starch liquefacation αDian Fenmei from bacillus, to produce maltose; (b) contact described maltose with transglucosidase, wherein said step (a) and step (b) occur in the temperature that is lower than the wheat gelatinization point; (c) acquisition contains the wheat grain composition of the isomalto Oligosaccharide of enzymatic generation, and wherein said oligosaccharides derives from described ungelatinized cereal.

Can be randomly, in another embodiment, present method is used aforesaid method and is made the grain compositions that is used to make foodstuff additive.Another embodiment comprises the grain compositions of making thus.Another embodiment comprises the flour that contains aforesaid grain compositions.Another embodiment comprises the isomalto Oligosaccharide of making according to top described method.Another embodiment comprises the oral rehydrated solution that contains above-mentioned isomalto Oligosaccharide.Another embodiment comprises the grain compositions that contains ungelatinized cereal and at least a isomalto Oligosaccharide, derives from described ungelatinized cereal wherein said isomalto Oligosaccharide enzymatic.In another embodiment, grain compositions contains weight greater than 1% at least a isomalto Oligosaccharide.

The accompanying drawing summary

Fig. 1 describes isomalto Oligosaccharide to strengthen the schema that flour is produced.

Fig. 2 describes another schema that the isomalto Oligosaccharide flour enrichment is produced.

Detailed Description Of The Invention

Definition

Term " cereal (grain) " refers to plant, and the monocotyledon that it is classified as cereal (cereal) or belongs to Poales especially belongs to monocotyledon gramineous. The example that belongs to this type of plant is to be selected from Triticum (wheat), Hordeum (barley); Secale (rye); Zea (corn (corn)) or maize (maize)); Avena (oat), Fagopyrum (buckwheat); Sorghum (Chinese sorghum or milo), Panicum or setaria (broomcorn millet or ragi); Perhaps Oryza (rice).

For example, in one embodiment, term " wheat (wheat) " refers to plant, and it is classified as or once was classified as wheat (Triticum aestivum) strain.

For example, in one embodiment, term " barley (barely) " refers to plant, and it is classified as or once was classified as barley (Hordeum vulgare) strain.

For example, in one embodiment, term " rye (rye) " refers to plant, and it is classified as or once was classified as rye (Secale cereal) strain.

For example, in one embodiment, term " corn (corn) " refers to plant, and it is classified as or once was classified as corn (Zea mays) strain.

For example, in one embodiment, term " oat (oats) " is meant plant, and it is classified as or once was classified as oat (Avena sativa) strain.

For example, in one embodiment, term " buckwheat (buckwheat) " is meant plant, and it is classified as or once was classified as buckwheat (Fagopyrum esculentum) strain.

For example, in one embodiment, term " Chinese sorghum (sorghum) " is meant plant, and it is classified as or once was classified as jowar (Sorghum bicolor) strain.

For example, in one embodiment, term " broomcorn millet (millet) " is meant plant, and it is classified as or once was classified as broomcorn millet (Panicum miliaceum) or grain (Setaria italic) strain.

For example, in one embodiment, term " rice (rice) " is meant plant, and it is classified as or once was classified as paddy rice (Orvza sativa) strain.

Term " stem tuber (tuber) " is meant starch depot organ (for example potato, sweet potato, Chinese yam, cassava etc.), and its far-end by subterraneous stem or root expands and forms.

For example, in one embodiment, term " potato (potato) " is meant plant, and it is classified as or once was classified as potato (Solanum tuberosum) strain.

For example, in one embodiment, term " sweet potato (sweet potato) " is meant plant, and it is classified as or once was classified as sweet potato (ipom[oe] a batatas) strain.

For example, in one embodiment, term " Chinese yam (yam) " is meant plant, and it is classified as or once was classified as Chinese yam (Dioscorea sativa, D.villosa, C.batatas) strain.

Term substrate (substrate is also referred to as " matrix " herein sometimes) thus be meant the material that can enzymatic ground change maltose into and change IMOs into.Term " substrate " comprises, for example, and cereal and stem tuber.Further, the term substrate comprises the form of ownership of cereal (decapsidate or not decapsidate) or stem tuber, as complete cereal, broken cereal, meal and flour and any plant part.

Term " starch (strach) " is meant any material of being made up of the complicated polysaccharide carbohydrate of plant, is made up of for amylose starch and the amylopectin of (C6H10O5) x formula, and wherein X can be any numeral.

That term " granular starch (granular strach) " is meant is uncooked (giving birth to) starch, it is not also by gelatinization.Term " gelatinization (gelatinization) " is meant the dissolving of starch molecule, forms the suspended substance of viscosity.

Term " " substrate ", " cereal " or " stem tuber " that contain unpaste starch " is meant substrate, cereal or the stem tuber of ungelatinized, it is higher than the temperature of starch gelatinization temperature, and described starch gelatinization temperature causes the starch pasting or the liquefaction that comprise in the substrate.

Term " maltose (maltose) " is meant the disaccharides with two glucosyl residues, and these two glucosyl residues are by α 1, and the 4D-glycosidic link connects.

Term " isomaltose (isomaltose) " is meant the disaccharides with two glucosyl residues, and these two glucosyl residues are by α 1, and the 6D-glycosidic link connects.

Term " isomalto Oligosaccharide (isomalto-oligosaccharide, IMO) " be meant sugar with at least two glucosyl residues, described residue is by connecting at the α of non-reducing end 1,6 glycosidic link.In addition, this term can refer to the oligosaccharides of irregular connection, and existing α 1,6 glycosidic link has the sugar of α 1,4 glycosidic link again.Representational isomalto Oligosaccharide comprises isomaltose, panose (panose) and Isomaltotriose (isomalto-triose).

Term " isomalto Oligosaccharide " grain compositions is meant grain compositions, it is characterized by different Fructus Hordei Germinatus-sugar (isomalto-sugars) level and accounts at least 1% (w/w%) of total sugar degree, as being measured by efficient liquid-phase chromatography method.

Term " produces maltase (maltogenic enzyme) " and is meant the enzyme that starch is changed into maltose.Representational product maltase comprises αDian Fenmei and the βDian Fenmei that is derived from fungi, bacterium and plant.

Term " amylase (amylases) " is meant the amylolytic enzyme of catalysis.

Term " α-Dian Fenmei (α-amylases) " is meant the enzyme of (E.C.) 3.2.1.1 classification and/or 3.2.1.133 classification, its catalysis α-1, the hydrolysis of 4-glycosidic link.These enzymes also are described to, and are containing 1, in the polysaccharide of the D-glucose unit that 4-α connects, and circumscribed hydrolysis or inscribe hydrolysis 1, those enzymes of 4-α-D-glycosidic link.In order to another term of describing these enzymes is glycogenase.Representational enzyme comprises α-1,4-Dextran 4-glucan hydrolase glucan hydrolase.

Term " beta-amylase (β-amylases) " is meant the enzyme of (E.C.) 3.2.1.2 classification, its catalysis α-1, and the hydrolysis of 4-glycosidic link discharges maltose unit.These enzymes also are described to, and make in the polysaccharide 1,4-α-D-glycosidic link hydrolysis, so that remove those enzymes of successive maltose unit from the non-reduced end of chain.

Term " changes (transglucosidic) of glucosides ", and enzyme is meant with α D-glucose-oligosaccharides (α D-gluco-oligosaccharides) incubation the time, the enzyme of catalytic hydrolysis and two kinds of reactions of transfer.Representational enzyme comprises transglucosidase (transglucosidases) and/or (E.G.) those enzymes of 2.4.1.24 classification, for example, and the D-Transglucosylase.These enzymes are also referred to as 1,4-alpha-glucan-alpha-glucosyl transferring enzyme and few dextran branch glycosyltransferase.

Term debranching factor (debranching enzyme) is meant catalysis α-1, the enzyme of 6-glycosidic link hydrolysis.In this connection, the enzyme of E.C.3.2.1.41 class is useful.This type of representative enzyme is Pullulanase (pullanase), is also referred to as Schardinger dextrin inscribe-1,6-α Polyglucosidase, limit dextrinase, debranching factor, amylopectin 1,6-glucan hydrolase.(E.C.) the representative enzyme of other of 3.2.1.41 class, for example, Starch debranching enzyme (pullulanases), [α-(1-6)-dextran 6-glucan hydrolase, be also referred to as α-(1,6)-Polyglucosidase]).

Term " starch gelatinization temperature (starch gelatinizing temperatures) " is meant sufficiently high so that can efficient liquefaction or the temperature of gelatinization granular starch.Heating starch causes that starch granules expands in water.When enough solids concns, the expansible particle has occupied most of space, and the viscous substance that is called paste (paste) produces.The dissolving of starch molecule is called as gelatinization.Gelatinization is accompanied by birefringent forfeiture.The term starch gelatinization temperature is meant the temperature when gelatinization takes place.

Term " starch liquefacation enzyme (starch liquefying enzyme) " is meant the fluidised enzyme of realizing granular starch.Representational starch liquefacation enzyme comprises the αDian Fenmei of (E.C.) 3.2.1.1. class.

Term " endogenous " is meant the enzyme that exists in cereal or stem tuber, add this product maltase and need not to seek help to cereal.

Term " exogenous " is meant non-existent enzyme in cereal.Representational exogenous enzymes comprises, for example, at the wild-type substrate for example, non-existent enzyme in rice, the broomcorn millet etc.

Term " total sugar degree (total sugar content) " is meant the total amount of the sugar that exists in starch, cereal or stem tuber composition.

Term " IMO number (IMO No.) " is calculated as, and isomaltose, panose, Isomaltotriose and DP are greater than the summation of branch's sugar of 3.The IMO number provides the indication of the amount of the IMO compound that exists in compound or solution.

Term " ratio of branch's sugar (ratio of branched sugars, RBS) " is meant, the ratio of trisaccharide maltose (DP3) level that exists in the maltose that exists in cereal (DP2) and the grain compositions that obtains.

Term " saccharogenic power degree (Degrees of Diastatic Power) " (DP °) unit is meant, the enzyme amount that in enzyme prepared product sample 5% solution of 0.10ml, contains, it was 20 ℃ (68) and 100ml substrate incubation 1 hour, can generate enough reducing sugars, with reduction 5ml Fehling ' s solution.

Term " DE " or " dextrose equivalent (dextrose equivalent) " are the industrial standardss that is used to measure the concentration of total reducing sugars, the concentration of described total reducing sugars is changed as D-glucose when being calculated and is calculated, and calculates (on a dry weight basis) by dry weight.The DE of unhydrolysed granular starch is 0 basically, and the DE of D-glucose is 100.

Term " total sugar degree (total sugar content) " is meant the total content of the sugar that exists in starch composites.

Term " dried solids component (dry solid basis) " and " dsb " are meant the compound total amount of flour (representing with %) for example in the slurry, calculate by dry weight.

Term " Brix " is meant the known hydrometer degree value (hydrometer scale) of the sugared content that is used to measure solution under the specified temp.Therefore, term " Brix " is meant measuring of dissolved sugar in the solution.The Brix scale value is measured is the gram number (total dissolved solids content) of the sucrose that exists in the aqueous sugar solutions of per 100 grams.For example, the measured value of 1.00Brix is meant that the sugar in the solution approximately is 10mg/ml.The measured value of Brix is normally finished by using liquid specific gravity hydrometer or refractometer.

Term " extent of polymerization (degree of polymerization, DP) " is meant the unitary number of glucopyranose acid anhydride (n) in certain sugars.The example of DP1 is a monose, as glucose and fructose.The example of DP2 is a disaccharides, for example maltose and sucrose.DP4 ⁺Mean that the polymerization degree is greater than 3 polymkeric substance.

Term " enzymatic generates (enzymatically produced) " is meant that substrate is IMO by enzyme-catalyzed change, and this chemosynthesis or organic chemistry that is different from IMO is synthetic.

Term " filamentous fungus (filamentous fungi) " be meant Eumycotina (subdivision Eumycotina) all thread forms (referring to, Alexopoulos, C.J. (1962), INTRODUCTORY MYCOLOGY, New York:Wiley).These fungies are characterised in that vegetative mycelium, and it has the cell walls of being made up of shell element, Mierocrystalline cellulose and other complex polysaccharide.Filamentous fungus of the present invention is different from yeast on morphology, physiology and genetics.Nourishing and growing of filamentous fungus is to prolong by means of the silk bacterium, and carbon katabolism is obligate aerobic.In the present invention, the filamentous fungus parental cell can be the cell of following dependent of dead military hero, but be not limited to these cells: trichoderma (Trichoderma), for example, Trichodermareesei (Trichoderma reesei) (be classified as long shoot wood mould (T.longibrachiatum) in the past, and be called as Hypocrea jecorina at present), viride (Trichodermaviride), healthy and free from worry wood mould (Trichoderma koningii), Kazakhstan wooden now mould (Trichoderma harzianum); Penicillium (Penicillium sp.); Humicola (Humicola sp.) comprises Humicola insolens and Humicola grisea; The gold mould Pseudomonas of spore (Chrysosporium sp.) comprises C.lucknowense; Gliocladium (Gliocladium sp.); Aspergillus (Aspergillus sp.) comprises aspergillus oryzae (A.oryzae), Aspergillus nidulans (A.nidulans), aspergillus niger (A.niger) and Aspergillus awamori (A.awamori); Fusarium (Fusariumsp.), arteries and veins are embraced Pseudomonas (Neurospora sp.), meat seat Pseudomonas (Hypocrea sp.) and Emericella (Emericellasp.).Also with reference to Innis et al., (1985) Sci.228:21-26.

Term " aspergillus tubigensis " or " Aspergillus " are meant any mould that is classified as aspergillus tubigensis in the past or is classified as aspergillus tubigensis at present.

Term " bacterium " is meant bacillus (Bacillus), includes but not limited to subtilis (B.subtilis), bacillus amyloliquefaciens (B.amyloliquefaciesn), slow genus bacillus (B.lentus), B.Carlsberg, Bacillus licheniformis (B.licheniformis) and bacstearothermophilus (B.stearothermophilus).

Term " plant origin (plant origin) " is meant that by the enzyme of deriving in the plant origin, extract, separate, expressing, described plant origin is barley germ, soybean, wheat or barley for example.

Term " contact " is meant each enzyme (one or more) is placed on the position that enough near-earths approach substrate separately, so that enzyme (one or more enzymes) can change substrate into required end product.Person of skill in the art will appreciate that, the solution of described enzyme or described plurality of enzymes is mixed with separately substrate, can realize contact.

Term " incubation (incubating) " is meant under given conditions, and the matrix that will contain substrate at the time durations that limits is mixed with dividing other enzyme, described enzyme for example, Ye Huamei or product Fructus Hordei Germinatus wheat or transglucosidase.

Term " Enzymatic transformation (enzymatic conversion) " is meant the rice substrate is modified, and produces soluble hydrolysis particulate state rice starch, preferably produces glucose.

Term " slurry (slurry) " is meant the aqueous mixture that contains insoluble particles starch.Sometimes the commutative herein use of term " slurry " and " suspension ".

Term " cultivation " is meant under conditions suitable, growth a group microorganism cells in liquid or solid dielectric.In one embodiment, cultivation is meant that the fermentable bio-transformation of granular starch substrates is dextrose syrup or other required end product (typically in container or reactor).

For example, in one embodiment, term αDian Fenmei unit is defined as, under the standard analysis condition of pH5.2 and 40 ℃, and the αDian Fenmei amount of hydrolysis 1 micromole's starch substrates in 1 minute.

For example, in one embodiment, term βDian Fenmei unit is defined as, under the standard analysis condition of pH4.6 and 20 ℃, and the βDian Fenmei amount of hydrolysis l micromole starch substrates in 1 minute.

For example, in one embodiment, term transglucosidase unit is defined as, and under the standard analysis condition of pH4.8 and 37 ℃, transforms the transglucosidase amount of 1 micromole's maltose substrate in 1 minute.

In another embodiment, term transglucosidase unit is defined as, and under the standard analysis condition of pH4.8 and 37 ℃, per minute generates the transglucosidase amount of 1 micromole's panose.

For example, in one embodiment, a Liquefon unit of term (LU) is that iodine solution produces measuring of the required digestion time of colour-change, and it has represented under the standard analysis condition of pH5.6 and 25 ℃, and the starch substrates dextrinization is phase regularly really.

" ATCC " is meant American type culture collection (American Type Culture Collection), is positioned at Manassas, and VA20108 (ATCC, www/atcc.org).

" NRRL " is meant farming research DSMZ, American National agricultural effectiveness research centre (Agricultural Research Service Culture Collection, National Center for AgriculturalUtilization Research) (was called as research laboratory, USDA North Area (USDA NorthernRegional Research Laboratory) in the past, Peoria, ILL.

" NCBI " is meant American National biotechnology information center (National Center for BiotechnologyInformation), Natl Library Med. (www.ncbi.nlm.nih.gov/).

" one (a) ", " one (an) " and " being somebody's turn to do (the) " comprise plural implication, unless context clearly indicates in addition.

The invention describes and be used to make isomalto Oligosaccharide matrix (substrate), cereal or stem tuber method for compositions, described method comprises: (a) contact the matrix that contains unpaste starch with producing maltase with the starch liquefacation enzyme, such as grain or stem tuber are to generate maltose; (b) contact described maltose with the enzyme that changes glucoside, the temperature of wherein said step (a) and described step (b) generation is lower than or equals starch gelatinization temperature; (c) acquisition has the matrix of the isomalto Oligosaccharide of enzymatic generation, cereal or stem tuber composition, and wherein said oligosaccharides derives from described matrix, cereal or stem tuber.Fig. 1 has described a kind of embodiment of the present invention.

The present invention has also described and has been equal to or less than the temperature of gelatinization point, the matrix of isomalto Oligosaccharide is rich in manufacturing, cereal or stem tuber composition, powder, oral rehydrated solution, and/or the method for foodstuff additive, wherein will have or contain unpaste starch and contact with the lytic enzyme that is selected from genus bacillus with the matrix that contains endogenous product maltase, produce maltose syrups.Then,, maltose syrups is contacted with transglucosidase, produce grain compositions with isomalto Oligosaccharide in the temperature that is equal to or less than gelatinization point or liquefaction temperature.In one embodiment, this grain compositions is characterised in that, in sugar is formed maltose more than 60% and the ratio of branch's sugar greater than 2.0 to 1.0.Conversion from the substrate to IMO can produce on enzymatic ground.

The present invention has also described and has been used to make isomalto Oligosaccharide matrix, cereal or stem tuber method for compositions, and this method comprises: (a) contact the matrix that contains starch with producing maltase with the starch liquefacation enzyme, cereal or stem tuber generate maltose; (b) with the enzyme contact maltose that changes glucoside, the temperature of wherein said step (a) and described step (b) generation is lower than or equals starch gelatinization temperature; (c) acquisition has the matrix of the isomalto Oligosaccharide of enzymatic generation, cereal or stem tuber composition, and wherein said oligosaccharides derives from matrix, cereal or stem tuber.The present invention can randomly further describe insoluble moiety and the isolating additional step of solubility moiety.The present invention has further described dry matrices, the additional step of cereal or stem tuber composition.In one embodiment, cereal is selected from wheat, rye, barley, Fructus Hordei Germinatus, buckwheat, jowar (milo), broomcorn millet (ragi) and rice.In another embodiment, producing maltase is βDian Fenmei.In another embodiment, producing maltase is endogenic for cereal.In another embodiment, the starch liquefacation enzyme is the αDian Fenmei that derives from bacillus.In another embodiment, the starch liquefacation enzyme source is in Bacillus licheniformis or bacstearothermophilus.In another embodiment, the enzyme that changes glucoside is a transglucosidase.In another embodiment, transglucosidase derives from aspergillus tubigensis.In another embodiment, aspergillus tubigensis is an aspergillus niger.The present invention has also described grain compositions, foodstuff additive, oral rehydrated solution and/or the powder that generates according to top described method.

In another embodiment, the invention describes and be used to make the wheat grain method for compositions, described method comprises: (a) will have endogenous and produce the ungelatinized wheat grain of Fructus Hordei Germinatus βDian Fenmei and contact generation maltose with starch liquefacation αDian Fenmei from bacillus; (b) contact maltose with transglucosidase, the temperature that wherein said step (a) and described step (b) take place is lower than or equals starch gelatinization temperature; (c) acquisition has the wheat grain composition of the isomalto Oligosaccharide of enzymatic generation, and wherein said oligosaccharides derives from described ungelatinized cereal.Fig. 2 has described a kind of embodiment of the present invention.

In another embodiment, above-described method can be used to make foodstuff additive, bakes product, oral rehydrated solution and/or powder.In another embodiment, producing maltase is βDian Fenmei.In another embodiment, producing maltase is endogenic for cereal.In another embodiment, the starch liquefacation enzyme is the αDian Fenmei that derives from bacterial origin.In another embodiment, bacterial origin is a bacillus.In another embodiment, the starch liquefacation enzyme source is in Bacillus licheniformis or bacstearothermophilus.In another embodiment, the enzyme that changes glucoside is a transglucosidase.In another embodiment, transglucosidase derives from originated from fungus.In another embodiment, originated from fungus is an Eurotium.In another embodiment, aspergillus tubigensis is an aspergillus niger.The present invention has also described grain compositions, foodstuff additive, oral rehydrated solution and/or the powder that generates according to top described method.Grain compositions can contain weight greater than 1% at least a isomalto Oligosaccharide.Described at least a isomalto Oligosaccharide can be selected from isomaltose, panose, Isomaltotriose.In further embodiment of the present invention, endogenous produces maltase and is selected from βDian Fenmei or αDian Fenmei.In further again embodiment of the present invention, lytic enzyme is the liquefaction αDian Fenmei that derives from genus bacillus.In further again embodiment of the present invention, liquefying amylase derives from Bacillus licheniformis or bacstearothermophilus.

Matrix (perhaps substrate) (substrates)

The present invention includes the matrix that contains starch, for example contain the cereal or the stem tuber of starch, it contacts with the starch liquefacation enzyme with producing maltase, produces maltose.Thereby term matrix is meant and can changes the material that maltose also changes MIO into in enzymatic ground.Representational matrix can be at least a matrix that is selected from cereal and stem tuber.Maltose can be the form that is rich in the syrup or the slurries of maltose.

Starch exists with two kinds of forms, amylose starch, a kind of linear chain polysaccharide, and amylopectin, a kind of branched chain polysaccharide.Amylose starch contains long unbranched chain, and wherein all D-glucose units are by α-1, and 4-key (" α-1,4-key " or " 1,4-α-D-glucose base key ") connects.Amylopectin is hyperbranched, and the glycosidic link of skeleton is α-1,4 key, but the glycosidic link of tapping point is α-1,6 key.The main component of starch can be with two kinds of different modes by the hydrolysis of enzymatic ground.Amylose starch can be by αDian Fenmei (E.C.3.2.1.1), for example, and α-(1-4)-Dextran 4-glucan hydrolase (α-(1-4)-glucan 4-glucanohydrolase) hydrolysis.αDian Fenmei hydrolyzing alpha-(1,4) key produces glucose, maltose, trisaccharide maltose and the mixture of high-grade sugar more.Amylose starch also can be by beta-amylase (E.C.3.2.1.2) [α (1,4)-dextran Fructus Hordei Germinatus lytic enzyme, 1,4-α-D-dextran Fructus Hordei Germinatus lytic enzyme] hydrolysis.This enzyme begins to cut away successive maltose unit from non-reduced end, produces maltose quantitatively.αDian Fenmei and βDian Fenmei be the hydrolysis amylopectin also.αDian Fenmei and βDian Fenmei all can not hydrolysis amylopectin branch location α (1-6) key.Beta-amylase is big, hyperbranched core texture or β limit dextrin to the end product that amylopectin thoroughly acts on.Debranching factor (E.C.3.2.1.41, for example, pullulanases, [α-(1-6)-dextran 6-glucan hydrolase is also referred to as α-(1,6)-Polyglucosidase]) can the hydrolysis branch location the key of α-(1-6).Therefore, beta-amylase and α 1, the 6 glucuroide combined action amylopectin of can thoroughly degrading is maltose and glucose, cause maltose content up to total sugar degree 60%, 65%, 79%, 75%, 80% or higher.

For purpose of the present invention, the matrix that contains starch can be cereal or stem tuber or its mixture.Cereal can be any cereal crop or the seed that contains starch.Matrix can be pulverized, be ground or otherwise reduce its size, to increase the surface area that matrix contacts with separately enzyme.For example, according to needs, matrix can be by wet-milling or dry grinding.In one embodiment of the present invention, starch is granular starch.The cereal that expection can be used in the present invention comprises, is used at present to cure, any cereal of dough or other purposes.The representative cereal of expection includes but not limited to, be selected from following at least a: wheat (Triticum, include but not limited to one grained wheat (T.monococcum), duckbill wheat (T.turgidum), spelt (T.spelta) and/or common wheat (T.aestivum)), barley (for example, barley (Hordeum vulgare) and United States Patent (USP) 6,492, the mutation of describing in 576 tables 1), rye (Secale (Secale sp.), include but not limited to rye (S.cereal)), corn (Zea, include but not limited to corn (Zea mays)), buckwheat (Fagopyrum, include but not limited to sweet buckwheat (F.esculentum)), Fructus Hordei Germinatus (for example, the barley that germinates), (jowar belongs to jowar, include but not limited to jowar (Sorghum bicolor) or be called as milo in addition, (Panicum and setaria include but not limited to broomcorn millet (P.milaceum) to broomcorn millet (ragi); Setaria includes but not limited to chestnut (S.italica)) and rice (Oryza includes but not limited to rice (Oryza sativa)).Can expect that wild-type plant and the transgenic plant with beneficial characteristics all can be used as amyloid matrix, for example the endogenous enzyme level increases described beneficial characteristics or the existence of exogenous enzymes.

Germination corniness, for example, Fructus Hordei Germinatus is used as one of key component of numerous food product and healthy beverage prescription, and reason is that they have high nutritive value, for example, contains Fructus Hordei Germinatus food (Table A).Germination has caused the synthetic and activation of endogenic product maltase and proteolytic ferment.Therefore, germination corniness is to contain the good source that endogenous produces the cereal of maltase.Malt meal and malt extract also are used as the source of brewageing and baking and banking up with earth the digestive ferment in the application.Yet, the germination of barley causes too readily digested of grain component, so that can not be as the living matter (prebiotics) of benefit or even as nutritive health-care material (nutraceuticals) useful effect, because they trend towards being digested all sidedly before arriving lower gastrointestinal tract.Unfortunately, the beneficial effect of the living materialization compound of benefit obtains approval most at lower gastrointestinal tract.Therefore, highly digestible Fructus Hordei Germinatus-carbohydrate is converted into more stodgy different Fructus Hordei Germinatus-carbohydrate, makes the Fructus Hordei Germinatus of modification can play the effect that benefit is given birth to matter, make Fructus Hordei Germinatus can arrive lower gastrointestinal tract and extra function and health advantages is provided.For example, Table A provides the suitable commercially available food that contains malt extract.

Table A

The commercial food product that contains malt extract

Trade name	Manufacturers	The place
			HORLICKS	Glaxosmithkline	Punjab, India
MALTOVA	Glaxosmithkline	Punjab, India
			VIVA	Glaxosmithkline	Punjab, India
BOURNAVITA	Cadbury	Bombay, India
			BOOST	Jagjit Industries	Punjab, India
MILO	Nestle	New Delhi, India

Therefore, use Fructus Hordei Germinatus, change in the granular starch that is included in this matrix some other useful form of oligosaccharides into as amyloid matrix, for example, IMO.

In addition, amyloid matrix can be stem tuber.The stem tuber that can expect comprises potato (Solanum (Solanumsp.), include but not limited to potato (S.tuberosum)), (sweet potato belongs to (Ipomoea sp.) to sweet potato, include but not limited to sweet potato (Ipomoea batatas)), cassava [tapioca, cassava] (cassava (Manihot sp.), include but not limited to cassava (Manihot esculenta, Manihot aipi and Manihot utilissima)) and/or taro root (taro belongs to (Colocasia sp.), includes but not limited to C.esculenta or C.macrorhiza).

Amyloid matrix can be aqueous slurry, and feature is to have 10% to 50% dissolved solids (DS) concentration.In another embodiment, the feature of starch-containing matrix is to have 2% to 90% DS concentration.In another embodiment, the feature of starch-containing matrix is to have 5% to 70% DS concentration.In another embodiment, the feature of starch-containing matrix is to have 10% to 60% DS concentration.In another embodiment, the feature of starch-containing matrix is to have 20% to 40% DS concentration.In another embodiment, the feature of starch-containing matrix is to have 25% to 35% DS concentration.

In another embodiment of the invention, the pH of starch-containing matrix is between 1.00 to 9.00.In another embodiment of the invention, the pH of starch-containing matrix is between 2.00 to 8.00.In another embodiment of the invention, the pH of starch-containing matrix is between 3.00 to 7.50.In another embodiment of the invention, the pH of starch-containing matrix is between 4.00 to 6.50.In another embodiment of the invention, the pH of starch-containing matrix is between 4.25 to 5.75.

Enzyme

The present invention includes, contact amyloid matrix with the starch liquefacation enzyme, generate maltose with producing maltase." product Fructus Hordei Germinatus " means that enzyme can enzymatic ground convert starch be a maltose.Representational product maltase comprises αDian Fenmei and βDian Fenmei.As described previously, amylose starch can be by α-Dian Fenmei (E.C.3.2.1.1), for example, and α-(1-4)-Dextran 4-glucan hydrolase hydrolysis.αDian Fenmei hydrolyzing alpha-(1,4)-key produces glucose, maltose, trisaccharide maltose and the mixture of high-grade sugar more.Amylose starch also can be by βDian Fenmei (E.C.3.2.1.2) [α (1,4)-dextran maltose lytic enzyme, 1,4-α-D-dextran maltose lytic enzyme] hydrolysis.This enzyme begins to cut away successive maltose from non-reduced end, produces the maltose of some amount.αDian Fenmei and βDian Fenmei be the hydrolysis amylopectin also.

αDian Fenmei

In some included embodiments of the present invention, αDian Fenmei is enzyme in the fungi or the enzyme in the microorganism, has the E.C. numbering, E.C.3.2.1.1-3, particularly E.C.3.2.1.1.In some embodiments, αDian Fenmei is heat-stable fungi αDian Fenmei.Suitable αDian Fenmei can be natural generation, also can be the αDian Fenmei of reorganization and sudden change.In some embodiments, αDian Fenmei derives from genus bacillus.Preferred genus bacillus comprises bacillus amyloliquefaciens, slow genus bacillus, Bacillus licheniformis and bacstearothermophilus.In particularly preferred embodiments, αDian Fenmei derives from aspergillus tubigensis.Preferred aspergillus tubigensis comprises aspergillus niger and aspergillus oryzae.Also with reference to the bacterial strain of NCIB 11837.

The commercial available αDian Fenmei that can be used for the inventive method comprises CLARASE L ([aspergillus oryzae] Genencor International Inc.) and NOVAMYL ([bacstearothermophilus] NovozymeBiotech.).

It will be appreciated by those skilled in the art that the amount of the αDian Fenmei that is used for the inventive method, depend on the enzymic activity of αDian Fenmei.In general, αDian Fenmei is added in 1 tonne of (MT) amyloid substrate with about amount of 0.01 to 5.0kg.In some embodiments, αDian Fenmei is added into about 0.05 to 4.0kg the amount of every MT.In other embodiment, αDian Fenmei, also can about 0.5 to 1.5kg the amount of every MT be added into to 2.5kg with every MT about 0.1.In further embodiment, other amount is employed.For example, normally, the CLARASE L of the amount between about 0.01 to 1.5kg (Genencor International Inc.) is added in the 1MT starch.In other embodiment, the amount that enzyme adds is, between every MT starch CLARASEL about 0.05 to 1.0kg; Between about 0.1 to 0.6kg; Between about 0.2 to 0.6kg and between about 0.4 to 0.6kg.

βDian Fenmei

In some embodiments that the present invention includes, producing maltase is βDian Fenmei.Though, from being contacted with αDian Fenmei, amyloid matrix can provide on this meaning of maltose, αDian Fenmei is to produce Fructus Hordei Germinatus, but the application of βDian Fenmei is useful, this is because they contact with granular starch more maltose rather than other carbohydrate, for example glucose of volume can be provided.In some embodiments, βDian Fenmei is plant enzyme or microbial enzyme, has E.C. numbering, E.C.3.2.1.2 (for example, United States Patent (USP) 4,970, those βDian Fenmeis of describing in 158 and 4,647,538).In some embodiments, βDian Fenmei is that heat-stable bacterium βDian Fenmei is.Suitable βDian Fenmei can be natural generation, also can be the βDian Fenmei of reorganization and sudden change.Term " bacterium " is meant and derives from bacillus, for example subtilis, Bacillus licheniformis, bacstearothermophilus, Bacillus coagulans (B.coagulans), bacillus amyloliquefaciens and/or the slowly enzyme of genus bacillus.Particularly preferred βDian Fenmei derives from Bacillus strain bacstearothermophilus, bacillus amyloliquefaciens and Bacillus licheniformis.Also with reference to NCIB 11608 bacterial strains.Term " plant origin " is meant that plant origin is derived, extracts, separates, is expressed in to enzyme, for example, and barley germ, soybean, wheat or barley.

The βDian Fenmei that commercial available expection can be used for the inventive method comprises OPTIMAL BBA, Spezyme DBA and OPTIMAL ME (Genencor International Inc.).Other commercial available wheat βDian Fenmei also is useful in the method for the invention.

In some embodiments, amyloid matrix, for example, wheat, rye, barley, Fructus Hordei Germinatus comprise endogenic product maltase, and its level is enough to produce enough maltose, is used to change into isomalto Oligosaccharide.Term " endogenic " is meant the enzyme that exists in cereal or the stem tuber, need not to add the product maltase or cereal is carried out genetic modification so that the product maltase to be provided to cereal.

Do not comprise in starch-containing matrix that endogenous produces maltase or endogenous produces in the low embodiment of maltase level, for example, matrix is rice, broomcorn millet, jowar and/or corn, and the equivalent of any exogenous product maltase adds is also expected by the contriver.Ectogenic product maltase can be added into, and for example, handles host cell expressing the product maltase of enough levels by genetics, and/or provides from the product maltase enriched material in another source or produce the maltase material.The exogenous product maltase of term is meant non-existent product maltase in the cereal.In this embodiment, produce maltase and be contacted with substrate, to produce maltose with enough amounts.

In one embodiment, the amount of the exogenous product maltase that contacts with starch-containing matrix is the product maltase of 0.050 to 5.000 saccharogenic power degree (" DP ° ") units/gram.In another embodiment of the invention, the product maltase with 0.100 to 2.000DP ° of units/gram contacts with starch-containing cereal.In another embodiment, the product maltase of 0.100 to 3.000DP ° of units/gram is contacted with starch-containing cereal.

In another embodiment, the amount that is contacted with the exogenous product maltase of starch-containing matrix is to show with the kilogram numerical table that the PMT (Per metric ton) substrate produces maltase.In one embodiment, the amount that is contacted with the exogenous product maltase of matrix is to produce the about 0.05kg of maltase (" kg/mt dsb ") in the dried solids component of PMT (Per metric ton) (dry solid basis).In another embodiment, the amount of exogenous product maltase is to produce the about 0.1kg of maltase (" kg/mt dsb ") in the dried solids component of PMT (Per metric ton).In other embodiment, 0.2,0.4,0.6,0.8 and/or 1.0kg/mt dsb the product maltase of capacity is provided, for example, beta-amylase.

In another embodiment, the amount that is contacted with the exogenous product maltase of starch-containing matrix is to show with the kilogram numerical table that PMT (Per metric ton) matrix is produced maltase.In one embodiment, the amount that is contacted with the exogenous product maltase of matrix is to produce the about 0.05kg of maltase (" kg/mt dsb ") in the PMT (Per metric ton) dissolving starch component (dissolved starch basis).In another embodiment, the amount of exogenous product maltase is to produce the about 0.1kg of maltase (" kg/mt dsb ") in the PMT (Per metric ton) dissolving starch component.In other embodiment, 0.2,0.4,0.6,0.8 and/or 1.0kg/mt dissolving starch component the product maltase of capacity is provided, for example, beta-amylase.

In another embodiment, the product maltase amount that contact with cereal is represented with producing maltase unit.Being used to measure the active analysis of product Fructus Hordei Germinatus comprises those analyses of describing in an embodiment and describes active those analyses of βDian Fenmei.Term DP ° unit is meant the enzyme amount that is included among 5% the sample enzyme prepared product solution 0.10ml, when producing enough reducing sugar at 20 ℃ of (68F °) incubations in the time of 1 hour with the 100ml substrate, with reduction Fehling ' s solution 5ml.

In another embodiment, the cereal (barley, wheat etc.) with endogenous product maltase can mix with those cereal of the exogenous product maltase of needs.The contriver has expected, ratio with the endogenous cereal that produces maltase and the cereal that utilizes exogenous product maltase is 30: 70,60: 40,50: 50,60: 40,70: 30 a mixture, and condition is the product maltase (endogenous source or exogenous source) that has q.s in the mixture.Use the product maltase in endogenous source, can reduce the amount of the exogenous enzymes that joins maslin or contact with maslin.

Starch liquefacation enzyme (starch liquefying enzymes)

The starch liquefacation enzyme is contacted with starch, with the starch of reduction liquefaction or the viscosity of dissolved starch.In one embodiment of the present invention, the starch liquefacation enzyme is the enzyme that is selected from E.C.3.2.1.1, for example, and αDian Fenmei.Representational αDian Fenmei can derive, separates or extract from bacterial origin.In one embodiment, bacterial origin is a bacillus.In another embodiment, the αDian Fenmei that derives from bacillus comprises that those are derived from the enzyme that is selected from following at least a bacterial origin: subtilis, Bacillus licheniformis, bacstearothermophilus, Bacillus coagulans, bacillus amyloliquefaciens and slow genus bacillus.Those enzymes in Bacillus licheniformis and the bacstearothermophilus are particularly useful.The contriver has expected other amylase, such as but not limited to those of EC3.2.1.133 (United States Patent (USP) 6,361, No. 809).Other is comprised that by the amylase that the contriver expects feature is the enzyme of oxidation or thermally-stabilised increase, is included in United States Patent (USP) 5,763,385; 5,824,532; Those mutant in 5,958,739 and/or 6,008,026 or the amylase of genetic modification or the amylase of variation.Useful αDian Fenmei is those enzymes that come from Bacillus licheniformis strain NCIB 8059, ATCC 6598, ATCC 6634, ATCC8480, ATCC 9945A, ATCC 11945.Useful αDian Fenmei is those enzymes that come from bacstearothermophilus strain ATCC 39709.Such enzyme comprises that trade mark is called the enzyme of " SPEZYME AA " or " SPEZYME FRED ", " SPEZYME LT300 " and " SPEZYME LT75 ", can (Palo Also, California USA) obtains from Genencor International.Other such enzyme comprises the αDian Fenmei from bacstearothermophilus, it is sold with trade name GZYME G997, GC007, with the αDian Fenmei from Bacillus licheniformis, it is sold with trade name GC262SP, also can obtain from GenencorInternational.

To produce maltase and contact with amyloid cereal, produce maltose with the starch liquefacation enzyme.As the skilled personnel to understand, be used for the amount of the starch liquefacation enzyme of the inventive method, will depend on the enzymic activity of starch liquefacation enzyme.In one embodiment, the starch liquefacation enzyme is contacted with amyloid cereal with 0.01 to 25Liquefon unit/gm amount.In another embodiment, the starch liquefacation enzyme of l to 10Liquefon unit/gm is contacted with amyloid cereal.A Liquefon unit (LU) makes iodine solution produce measuring of the needed digestion time of colour-change, this expression be the specific period of starch substrates dextrinization under given conditions.

In one embodiment, the cereal dissolved solids composition (grain dissolved solid basis) (kg/mt dsb) that 0.1kg starch liquefacation enzyme is added PMT (Per metric ton).In other embodiment, with 0.2,0.4,0.6,0.8 or 1.0kg starch liquefacation enzyme add PMT (Per metric ton) cereal (kg/mt dissolves starch component).In one embodiment, the cereal dissolving starch component (kg/mt dissolves starch component) that 0.1kg starch liquefacation enzyme is added PMT (Per metric ton).In other embodiment, with 0.2,0.4,0.6,0.8 or 1.0kg starch liquefacation enzyme add PMT (Per metric ton) cereal (kg/mt dissolves starch component).Be used for determining that the active analysis of starch liquefacation comprises those that describe among the embodiment herein.The representational analysis that is used for alpha-amylase activity mensuration is also in United States Patent (USP) 5,763,385; 5,824,532; Describe in 5,958,739 and/or 6,008,026, these patents are incorporated as reference at this.

Change the enzyme (Transglucosidic enzyme) of glucoside

Maltose is contacted with the enzyme that changes glucoside, obtain to have the grain compositions of the isomalto Oligosaccharide of the enzymatic generation that derives from starch-containing cereal.When with α-D-glucose-oligosaccharides incubation, change the enzymatic hydrolysis reaction and the shift reaction of glucoside, produce isomaltose, panose, kojibiose or Nigerose (nigerose).The increasing amount of DP2 disaccharides has been pointed out the existence of these sugar and the conversion that is therefore mediated by the enzyme that changes glucoside.The enzyme (E.C.2.4.1.24) that changes glucoside can be transglucosidase (transglucosidase).Representational transglucosidase have TRANSGLUCOSIDASE L-1000 (Genencor International, Inc.) and AmanoEnzymes, Inc., (Nagoya, TRANSGLUCOSIDE L Japan).In one embodiment, change the enzyme source of glucoside and originate in filamentous fungus, for example, Eurotium.The enzyme that derives from the commentaries on classics glucoside of aspergillus tubigensis can derive from aspergillus niger.In one embodiment, Aspergillus niger strain is ATCC14916.

In one embodiment, with the enzyme and the matrix of the commentaries on classics glucoside of q.s, for example amyloid cereal contact produces maltose.As skilled in the art to understand, be used for the amount of enzyme of the commentaries on classics glucoside of the inventive method, will depend on the enzymic activity of αDian Fenmei.In one embodiment, the transglucosidase with 0.01 to 25.00 transglucosidase unit (" TGU ")/gm contacts with amyloid cereal.In another embodiment of the invention, the transglucosidase with 0.05TGU to 10.00TGU/gm contacts with amyloid cereal.In another embodiment of the present invention, the transglucosidase of 0.10TGU to 5.00TGU/gm is contacted with amyloid cereal.Term TGU is meant that under analysis condition per minute produces the required enzymic activity of 1 micromole's panose.

In one embodiment, 0.05 to the 6.00kg enzyme that changes glucoside is added PMT (Per metric ton) cereal (kg/mtdsb).In another embodiment, 0.10 to the 5.00kg enzyme that changes glucoside is added PMT (Per metric ton) cereal (kg/mtdsb).In another embodiment, 0.25 to the 3.00kg enzyme that changes glucoside is added PMT (Per metric ton) cereal (kg/mtdsb).In another embodiment, 0.50 to the 1.50kg enzyme that changes glucoside is added PMT (Per metric ton) cereal (kg/mtdsb).Being used for determining to change active other analysis of glucoside comprises, those are described and in an embodiment at Shetty, J., et al (United States Patent (USP) 4,575,487 (1986), exercise question is " Method for determination oftransglucosidase ") the middle analysis of describing, they are incorporated as reference at this.

In one embodiment, 0.05 to the 6.00kg enzyme that changes glucoside is added PMT (Per metric ton) dissolving starch (kg/mt starch dsb).In another embodiment, 0.10 to the 5.00kg enzyme that changes glucoside is added PMT (Per metric ton) cereal (kg/mt starch dsb).In another embodiment, 0.25 to the 3.00kg enzyme that changes glucoside is added PMT (Per metric ton) cereal (kg/mt dsb).In another embodiment, 0.50 to the 1.50kg enzyme that changes glucoside is added PMT (Per metric ton) cereal (kg/mt starch dsb).

As the result of transglucosidase effect, Fructus Hordei Germinatus-oligosaccharides is changed into isomalto-oligosaccharides, forms the polysaccharide of new classification, and it contains the more a high proportion of glucosyl residue that is connected with the one-level oh group of glucose molecule at non-reduced end.The isomalto-oligosaccharides that this method produces comprises isomaltose, panose, different Fructus Hordei Germinatus-trisaccharide, different Fructus Hordei Germinatus-tetrose, different Fructus Hordei Germinatus-pentasaccharides, different Fructus Hordei Germinatus-hexose and different Fructus Hordei Germinatus-heptose.These sugar have obtained increasing concern as foodstuff additive, because they help preventing dental caries (Oshima, et.al 1988, and improve people's intestinal microflora Thecaries inhibitory effects of gos-sugar in vitro and rat experiments.Microbial Immunol.32.1093-1105)), work as the somatomedin of bifidus bacillus (benefit is given birth to matter) (Komoto, et.al 1988; Effect of Isomalto-oligosaccharides on human fecal flora, Bifidobacteria Micro flora 7,61-69).

In order to determine the generation of IMO, can application test and/or other analytical procedure, determine the amount of the IMO that produces.A kind of method of the level of definite IMO that generates comprises high performance liquid chromatography (HPLC).For example, the analysis of mixture can provide the prompting to the level of the various sugar that produced by this method.Useful evaluation is the extent of polymerization (DP) of mixture.The term extent of polymerization is the measuring of relative quantity of glucosyl residue number in the molecule.For example, glucose (a glucosyl unit, minimum polymerization level) is considered to DP1 usually.Isomalto-oligosaccharides is considered to DP2 (two glucose units) usually.In one embodiment, grain compositions contains weight greater than at least 1%, at least 5%, at least 25%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70% at least a isomalto-oligosaccharides.In one embodiment, described at least a isomalto-oligosaccharides is selected from isomaltose, panose and/or different Fructus Hordei Germinatus-trisaccharide.In one embodiment, the amount of the isomalto-oligosaccharides that generates in the grain compositions account for grain compositions 1% to 99% between.In one embodiment, the amount of the isomalto-oligosaccharides that generates in the grain compositions account for grain compositions 1% to 90% between.In one embodiment, the amount of the isomalto-oligosaccharides that generates in the grain compositions account for grain compositions 1% to 80% between.In one embodiment, the amount of the isomalto-oligosaccharides that generates in the grain compositions account for grain compositions 1% to 70% between.In one embodiment of the present invention, the total reducing sugar that exists in the grain compositions after the said procedure, comprise account for total sugar content greater than 50%, greater than 60%, greater than 70% or greater than 80% maltose level.Maltose level greater than 50% comprises from 50% to 85%, from 55% to 80% and/or from 60% to 75% scope.Term RBS is than the ratio that is meant trisaccharide maltose (DP3) level that exists in maltose (DP2) that exists in the cereal and the grain compositions that obtains.The prompting of higher PBS value, exist the maltose of a large amount more and therefore starch change maltose more up hill and dale into, other more unwanted finished product of this and existence is different as trisaccharide maltose.In one embodiment, the RBS ratio is greater than 2.0.In one embodiment, RBS is than greater than 3.0, greater than 4.0.Representational scope comprises, 2.0 to 50.00,2.0 to 30.00 and/or 2.0 to 10.00 RBS ratio.Various RBS ratios have been described among the embodiment.Point out that liquefying starch is by coml βDian Fenmei (barley or wheat) hydrolysis, the maltose content that produces is between 55% to 65% usually.Obtain maltose content in order to use liquefying starch, need to add the low-down initial DE of debranching factor and/or liquefying starch before greater than 50%.Can randomly add the generation that debranching factor can be used to increase maltose.The term debranching factor is meant catalysis α-1, the enzyme of the hydrolysis of 6-key.With regard to this point, the enzyme of E.C.3.2.1.41 class is useful.This type of representative enzyme is pullanase, is also referred to as Schardinger dextrin inscribe-1,6-α Polyglucosidase, limit dextrinase, debranching factor, amylopectin 1,6-glucan hydrolase.

Amyloid cereal is contacted with producing maltase, generate maltose, and maltose is contacted with the enzyme that changes glucoside, the temperature when it takes place is lower than the gelatinization point of the starch of employed cereal.Matrix is contacted or incubation with dividing other enzyme, and the incubation time is at least 12 hours, at least 18 hours, at least 24 hours, at least 30 hours and/or at least 36 hours.Be meant during the time that at least one is mentioned 12-80 hour, at least 18-60 hour and/or at least 24-48 hour during.The term incubation time is meant that maltose or substrate change the time durations of IMO into.Can with the enzyme that changes glucosides with for example amyloid cereal of matrix, produce maltase for example αDian Fenmei or βDian Fenmei, and/or Ye Huamei, for example αDian Fenmei respectively or contact simultaneously or adding.In one embodiment, the enzyme and the Ye Huamei of commentaries on classics glucosides add simultaneously.Therefore in one embodiment, step (a) and step (b) are carried out simultaneously.In another embodiment, step (a) and step (b) are carried out in order or respectively.In another embodiment, step (a) is carried out early than step (b).The term gelatinization point is meant that the starch that contains in the cereal changes mutually or form gelatinous temperature.Although concrete temperature is different and different according to cereal, be enough to make the temperature of starch pasting comprise greater than 45 ℃, greater than 50 ℃, greater than 60 ℃, greater than 70 ℃, greater than 80 ℃ and/or greater than 90 ℃ temperature.Representative temperature greater than the gelatinization point of pointing out comprises 45 ℃ to 120 ℃, 50 ℃ to 110 ℃, 50 ° to 100 ℃.In one embodiment, for example, wheat, gelatinization point are that cereal is maintained at and is lower than, and for example, are selected from and are lower than 50 ℃ to 70 ℃ temperature, in another embodiment, are lower than 55 ℃ to 65 ℃, in another embodiment, are lower than 60 ℃.For example, the gelatinization point of corn, potato, wheat, cassava, waxy corn, jowar, rice, Saigon's rice, arrowroot, starch maize (arrowroot amylomaize) and/or sweet potato has been described, as (Beynum as shown in the table 1, G.M.A and Roels, J.A., Starch Conversion Technology (Marcel Dekker, Inc., NewYork, NewYork (1985), pp.15-45):

Table 1

Starch	The Kolfler gelatinization temperature range (℃)	The Brabenber gelatinization point (℃)
			Corn	62-67-72	75-80
Potato	58-63-68	60-65
			Wheat	58-61-64	80-85
Cassava	59-64-69	65-70
			Waxy corn	63-68-72	65-70
Jowar	68-74-78	75-80
			Rice	68-74-78	70-75

Saigon's rice	60-66-72	65-70
			Arrowroot	62-66-70
Starch maize	67-80-92	90-95
			Sweet potato	58-65-72	65-70

In another embodiment of the invention, incubation is after the time, and slurry can carry out the rapid heating in one period, and it is enough to end further enzymic activity, but does not make slurry gelatinization or liquefaction.For example, slurry can be heated to the temperature of 80 ℃, 85 ℃, 90 ℃, 95 ℃ or 100 ℃, the time length is 5-60 minute, 10.0 to 40.0 minutes or 30.0 minutes.

Another embodiment of the present invention further comprises, slurry is separated into the step of insolubles and solvend.Separating step can be any chromatogram analysis method as known in the art, such as but not limited to HPLC, and size exclusion and/or electric charge chromatography.Filtration can be used to solvend is separated with insolubles.Insolubles or all slurry can carry out the drying step described subsequently among the application.In another embodiment, the solvend that obtains from separating step can rotary evaporation, disc type drying etc. concentrate by for example evaporating.Enriched material after the evaporation can carry out charcoal and handle (filtering by carbon granule) and/or further chromatography, so that isolating IMO liquid concentrate to be provided.Isolating IMO enriched material can have greater than 75%, greater than 80%, greater than 90%, greater than 95%, greater than 97% and/or greater than 99% IMO concentration.

Another embodiment of the present invention is to use or be integrated into this syrup (deriving from the isomalto-oligosaccharides of the matrix with unpaste starch) in oral rehydrated solution enzymatic.The amount of isomalto-oligosaccharides can be a United States Patent (USP) 4,981,687; Amount or the prescription described in 5,096,894 and/or 5,733,579.

Another embodiment of the present invention is, dry aforesaid isomalto-oligosaccharides matrix, and cereal or stem tuber composition produce the powder that contains grain compositions.Typically, drying step quickens by heating.By using suitable drying means, such as but not limited to jet drying device, disc type heat drier, tumble dryer, rotary drum dryer or compartment dryer, grain compositions can be dried and be required level of wetness.Can use other drying means and learn, for example jet drying, decompression evaporation drying.

By dried grain composition, slurry, isolating insolubles and/or isolating solvend, therefrom obtain powder or other dried powder.Can incorporate the powder or the meal that obtain into need isomalto-oligosaccharides composition, for example, in food (breakfast cereal, dry bread), foodstuff additive and the baking goods.The term foodstuff additive are meant isomalto-oligosaccharides are interspersed among on the material, the batching during used as other food of manufacturing, and/or as the local composition application that joins food.

In another embodiment, the exsiccant powder can be merged in dietary supplement (food supplements).Can be with any acceptable augment or form is incorporated the exsiccant powder into dietary supplement.Dietary supplement can be mixed with the Orally administered form that is suitable in matrix, such as but not limited to, medicinal powder, crystal, saccharoid, particulate (comprises the particle that is of a size of micron level, as microsphere and microcapsule), particle (comprising the particle that is of a size of micron level), pearl, microballon, bead, pill, micro-tablet, compressed tablets or grinding tablet (tablettriturates), molded tablet or tablet abrasive material (tablet triturates), can also be formulated in the capsule, it is hard or soft, and to contain form be powder, particle, pearl, the composition of solution or suspended substance.Dietary supplement also can be mixed with in liquid, aqueous and be used for oral solution or suspension, can be the liquid of putting in order in the capsule, perhaps can be mixed with any other form easily that is suitable for using or is suitable for rectal administration, for example suppository, enema or other form easily.The isomalto-oligosaccharides composition also can be used as controllable release system and is provided.

The dietary supplement preparation also can comprise acceptable vehicle, additive or the carrier of any kind.For example, but not in order to limit, thinner or weighting agent, as dextrates (dextrates), Lin Suanergai, calcium sulfate, lactose, Mierocrystalline cellulose, kaolin, N.F,USP MANNITOL, sodium-chlor, dry starch, sorbyl alcohol, sucrose, inositol, Icing Sugar, wilkinite, Microcrystalline Cellulose or Vltra tears, can be added into the isomalto-oligosaccharides composition, to increase the volume of composition.Equally, tackiness agent such as but not limited to, starch, gelatin, sucrose, glucose, dextrose, molasses, lactose, Sudan Gum-arabic, sodiun alginate, sea moss (carrageen) extract, panwar glue, eggplant are for (ghatti) glue, isapgol husks gluing liquid, carboxymethyl cellulose, methylcellulose gum, polyvinylpyrrolidone, neusilin (modification) (veegum) and starch arabogalactan, polyoxyethylene glycol, methylcellulose gum, glyceryl monostearate and wax, can be added into preparation, stick character to increase it.

Additionally, lubricant, such as but not limited to, Zerol, talcum, Magnesium Stearate 15, calcium stearate, stearic acid, hydrogenated vegetable oil, polyoxyethylene glycol, Sodium Benzoate, sodium-acetate, sodium-chlor, leucine, carbowax (carbowax), Sulfuric acid,monododecyl ester, sodium salt (sodium laurylsulfate) and Magnesium Laurylsulfate can be added into preparation.Equally, glidant can be added into such as but not limited to silicon dioxide colloid, manganous silicate or mica, to improve the flowing property of powder formulation.At last, disintegrating agent, such as but not limited to, starch, clay, Mierocrystalline cellulose, alginic acid, resin, crosslinked polymkeric substance are (for example, croscarmellose, Crospovidone and Explotab), neusilin (modification), methylcellulose gum, agar, wilkinite, Mierocrystalline cellulose and timber-work, natural sponge (natural sponge), Zeo-karb, alginic acid, guar gum, oranges and tangerines pulp (citruspulp), carboxymethyl cellulose or have the Sulfuric acid,monododecyl ester, sodium salt of starch, also can be added into, in stomach or intestines, decompose to assist preparation.

Another embodiment of the present invention is, with new matrix described herein, stem tuber or grain compositions are applied to the production of flour, and described flour is used for various baking goodses.The term baking goods relates to the goods and the unfermentable goods of fermentation.Term " fermentation " is meant has used the zymic baking goods in the process of curing.And term " fermentation " is meant in the process of curing the not baking goods of using yeast.Representational goods comprise bread, biscuit, cake, pie, dessert, naan, bagel, pasta, crispbread, Wound product, doughnut, pitta and pastry.The representational article that do not ferment comprise matzo, chapati, breakfast cereal food and tortilla.Another embodiment of the present invention is new grain compositions to be applied to wheaten food, for example noodles (short macaroni, solid slim noodles, lasagne, corn noodles etc.).Another embodiment of the present invention is, according to the matrix that aforesaid method is made, and stem tuber or grain compositions.Another embodiment of the present invention is to comprise the matrix of making according to aforesaid method, the flour of stem tuber or grain compositions.Another embodiment of the present invention is to comprise the oral rehydrated solution of above-mentioned isomalto-oligosaccharides.According to aforesaid method, can make and comprise matrix, the powder of stem tuber or grain compositions.Another embodiment of the present invention is, matrix, stem tuber or the grain compositions made according to aforesaid method.

Another embodiment of the present invention is, according to the matrix that aforesaid method is made, and stem tuber or grain compositions.Another embodiment of the present invention is that new grain compositions is applied in fermentation/brewer's wort or the matrix.For example, new grain compositions can be used for beer fermentation as described, and described in international publication number WO02/20712 A1, it is incorporated as reference at this.New grain compositions also can be put in order into beer additive.

The matrix that contains isomalto-oligosaccharides can also be carried out extra step, by the maltose that generates for example malt syrup extraction with separate, reclaim maltose.The method that can be familiar with by this area is extracted syrup and/or is separated from grain compositions, for example at United States Patent (USP) 3,922, and the method in 196 and 4,113,509, it is incorporated as reference at this.

Another approach that increases sugariness or isomalto-oligosaccharides content is, handle the isomalto-oligosaccharides syrup that produces with lytic enzyme (with soluble form or immobilized form), lytic enzyme preferably or or even single-minded ground hydrolysis Fructus Hordei Germinatus-oligosaccharides, and isomalto-oligosaccharides only had little avidity or do not had avidity.The example of this kind of enzyme is to originate as the glucoamylase of Eurotium or Rhizopus from aspergillus niger or other, it is hydrolysis Fructus Hordei Germinatus-oligosaccharides (Manjunath P. preferably, Shenoy B.C., Raghavendra Rao M.R., Journal of AppliedBiochemistry, 5 (1983), 235-260; Meagher M.M., et al., Biotechnology andBioengineering, 34 (1989), 681-693; Pazur J.H., Kleppe K., The Journal of BiologicalChemistry, 237 (4) (1962), 1002-1006; Hiromi K., Nitta Y., et al., Biochimica etBiophysica Acta, 302 (1973), 362-37).

Also can use as enzyme from α-D-glycopyranoside enzyme of bacstearothermophilus.This enzyme can not the hydrolysis isomalto-oligosaccharides, only degraded be rich in Fructus Hordei Germinatus-oligosaccharides of existing in the syrup of isomalto-oligosaccharides (Suzuki Y., Shinji M., Nobuyuki E., Biochimica et Biophysica Acta, 787 (1984), 281-289).Also can use other the α-D-Polyglucosidase that is called as maltin.For example, from zymic maltin only can hydrolysis maltose and less degree ground hydrolysis trisaccharide maltose (Kelly C.T., Fogarty W.M., ProcessBiochemistry, May/June (1983), 6-12).Fructus Hordei Germinatus-oligosaccharides is hydrolyzed to after the glucose, and by chromatographic technique or by nanofiltration or ultra-filtration technique, syrup can the enrichment isomalto-oligosaccharides.

The following examples are for main embodiment of the present invention is described.

Embodiment

Following specific embodiment further illustrates the compositions and methods of the invention.Should be appreciated that these embodiment only are for illustration purpose, they can be applied to rich in starch and contain other suitable substance that endogenic maltose generates enzyme, for example, and wheat, rice, barley, Fructus Hordei Germinatus, potato, sweet potato etc.

Enzyme assay

The transglycosylase activity is by Shetty, J., and et al, the method for 1986 (United States Patent (USP) 4,575,487) is measured.By pH4.6 and 20 ℃, hydrolyzed starch substrate 30 minutes is measured the activity of βDian Fenmei.The reducing sugar group that hydrolysis produces is determined in the titration program of the using basic hexacyanoferrate.Amylase activity is expressed as the DP degree, and an one unit is meant the enzyme amount among the 5% solution 0.1ml that is included in sample enzyme prepared product, when itself and 100ml substrate at 20 ℃ of incubations in the time of 1 hour, can generate is enough to reduce the reducing sugar of 5ml Fehlings ' solution.

Based on the terminating point assay kit (end-point assay kit) that Megazyme (Aust.) Pty.Ltd provides, measure alpha-amylase activity.(p-nitrophenyl Fructus Hordei Germinatus seven glucosides BPNPG7) are dissolved in the 10ml sterilized water, carry out 1: 4 dilution in analysis buffer (50mM toxilic acid damping fluid, pH6.7,5mM calcium chloride, 0.002% polysorbas20) thereupon with a bottle substrate.At 25 ℃, add the amylase of 10 μ l in the 790 μ l substrates in transparent vessel, analyze.The speed of hydrolysis is measured as the rate of change of the absorbancy at 410nm place after 75 seconds the delay.This analysis is linear, until 0.2 absorbance units/minute speed.

Use the bovine serum albumin standard, based on Bradford, Anal.Biochem., Vol.72, p.248 the method for (1976) with the Bio-RadAssay (Bio-Rad Laboratories) of standard, is measured the protein concentration of αDian Fenmei.

Substrate (matrix)

Be used as the wheat-flour of substrate (matrix) in all embodiments, bought from retail shop.Other stem tuber or cereal substrate, for example, as the rice and the barley of substrate can buy from commercial source (Huai An LiujunFood processing company, Jiangshu province, China).

Oligosaccharides is analyzed

By being furnished with HPLC post (Rezex 8u8%H, monose (Monosaccharides)) HPLC (Agilent1010, Palo Alto, California, USA), it is maintained at 60 ℃ and be furnished with specific refractory power (RI) detector (ERC-7515A, RI Detector, from The Anspec Company, Inc.), measure the composition of oligosaccharides reaction product.The sulfuric acid (0.01N) of dilution is used as moving phase, and flow velocity is per minute 0.6ml.20 microlitres, 4.0% solution is annotated on the post.This post separates according to the molecular weight of sugar.For example, the meaning of DP1 is a monose, as glucose; The meaning of DP2 is a disaccharides, as maltose; The meaning of DP3 is a trisaccharide, as trisaccharide maltose; DP4 ⁺Meaning be that extent of polymerization (DP) is 4 or more oligosaccharides.Term high-grade sugar (higher sugar, " Hr.Sugar ") is meant that DP is greater than 3 sugar.

For heterosugar (iso-saccharides) or branch's sugar (branched sugars), (Agilent 1010 for HPLC by being furnished with HPLC post (Shodex Rspak Oligosaccharide Column #DC-613), PaloAlto, California, USA), it is maintained at 50 ℃ and be furnished with specific refractory power (RI) detector (ERC-7515A, RI Detector, from The Anspec Company, Inc.), reaction product is measured.70 (acetonitriles): 25 (methyl alcohol): 5 water are as moving phase, and flow velocity is per minute 2.5ml.20 microlitres, 4.0% solution is annotated on post.This post separates according to the molecular weight of sugar.The sugar of standard, (Missouri USA) is used to coupled columns and calibrates for Sigma Chemicals, St.Louis for glucose, maltose, trisaccharide maltose, isomaltose, panose and different Fructus Hordei Germinatus-trisaccharide.

Embodiment 1

Relatively from the αDian Fenmei of Bacillus licheniformis (by Genencor International, Palo Alto, a kind of αDian Fenmei that CA sells, trade name is GC262SP) and from the αDian Fenmei of bacstearothermophilus (by Genencor International, Palo Alto, a kind of αDian Fenmei that CA sells, trade name is GC007) the maltose production that utilizes wheat-flour to carry out.To be suspended in the 450ml deionized water from wheat-flour 150 grams in retailing source.Stirring at room suspension 15 minutes is with uniform mixing (pH5.5).With 6.0N sulfuric acid (H ₂SO ₄) adjustment pH value.The suspension that obtains is remained in the water-bath, maintain 60 ℃, and before enzyme adds, stir with uniform mixing.Continue under the stirring at 60 ℃, add respectively from the about 6000LU/g of the amylase of bacstearothermophilus (GC007[of 0.6kg is from Genencor International.Inc.]/metric ton (Mt.) starch dsb) with from the amylase 15 of Bacillus licheniformis, 100 LU/g (GC262SP[of 0.6kg is from GenencorInternational.Inc.]/Mt. starch dsb), and incubation.Collect sample at interval and use high pressure liquid chromatography (HPLC) analyzing total sugar component at predetermined different time.In the predetermined timed interval, from each container, take out the 2ml sample with plastic suction pipet, be transferred in the centrifuge tube.With sample centrifugal 3 minutes at 8000prm.From centrifuge tube collect supernatant liquor and with several Lecia of being added dropwise to AR200 (Leica Microsystems, Inc., Buffalo, NY is USA) in the sample well of digital handheld refractometer, and note down (table 2).Measure the Brix (as the measured value of dissolved sugar) (table 2) of solution.

Table 2

Between the wheat-flour incubation period, the liquefaction αDian Fenmei is to the glycogenetic comparison of Fructus Hordei Germinatus, pH5.5,60 ℃

Enzyme ﹠ dosage	Sugar ﹠ BRIX	The incubation time, 1 hour, 60 ℃ of sugared percentage ratios			80 ℃ the heating 30 minutes after
						2 hours	4 hours	6 hours	Gelatinization
		Do not add αDian Fenmei	DP1	3.86	3.70					3.74
DP2	54.87					56.24	56.78
			DP3	2.83	3.44			3.96
High-grade sugar	38.44					36.62	35.52
			BRIX	11.00	12.70			14.10
Bacstearothermophilus [GC007] 0.6kg/MT starch .dsb	DP1					2.67	2.89			3.23	3.17
		DP2	65.10	67.44	69.08			65.83
	DP3					10.62	12.18		13.07	13.42
		High-grade sugar	21.61	17.49	14.62			17.58
	BRIX					18.50	19.40		20.00	22.70
		Bacillus licheniformis [GC262SP] 0.6kg/MT starch .dsb	DP1	2.86	3.59			3.96			4.03
DP2	62.43					64.36	65.88		58.14
			DP3	13.19	14.82			15.93		16.69
High-grade sugar	21.52					17.23	14.23		21.14
			BRIX	17.40	18.40			19.10		22.60

Result in the table 2 shows, compares with the maltose content with from the αDian Fenmei incubation of Bacillus licheniformis the time, and wheat-flour has generated higher maltose content with from the αDian Fenmei incubation of bacstearothermophilus the time.The incubation of wheat-flour is compared when not adding αDian Fenmei, and the incubation of wheat-flour and αDian Fenmei causes significantly increasing owing to the dissolved solids that granular starch hydrolyzing obtains.Point out with interest at this, compare, cause the ratio of maltose and glucose and maltose and trisaccharide maltose higher from the reaction product of the αDian Fenmei of bacstearothermophilus with αDian Fenmei from Bacillus licheniformis.These results suggest are useful especially enzyme from the αDian Fenmei of Bacillus licheniformis producing aspect the syrup of high-content maltose very.

Embodiment 2

With between the wheat-flour incubation period, bacillus licheniformis alpha amylase (by Genencor international, the αDian Fenmei that PaloAlto, CA sell, trade name is GC 007) concentration is to the glycogenetic influence of Fructus Hordei Germinatus.Illustrated consistent among experiment condition and the embodiment 1.In addition, bacstearothermophilus (6,000 units/gram) is added into 0.1Kg, 0.2Kg and 0.6Kg/MT starch dsb.The results are summarized in the table 3.

Table 3

Between the wheat-flour incubation period, αDian Fenmei [GC007] concentration is to the glycogenetic influence of Fructus Hordei Germinatus, pH5.5,60 ℃

GC007 concentration	DP& BRIX	Incubation time, 1 hour, 60 ℃ of sugared percentage ratios
						2 hours	4 hours	6 hours	24 hours
		0.1kg/MT starch .dsb	DP1	1.87	2.10					2.27	3.70
DP2	65.56					64.25	65.63	70.22
			DP3	5.92	7.15				8.03	11.68
High-grade sugar	29.65					26.50	24.07	14.40
			BRIX	17.80	19.20				19.70	21.80
0.2kg/MT starch .dsb	DP1					1.71	2.13	2.37			3.90
		DP2	62.47	64.34	65.44				69.30
	DP3					6.75	7.78	8.58		12.27
		High-grade sugar	29.07	25.75	23.61				14.53
	BRIX					18.00	19.20	19.80		21.80
		0.6kg/MT starch .dsb	DP1	2.67	2.89				3.23		3.17
DP2	65.10					67.44	69.08	65.83
			DP3	10.62	12.18				13.03	13.42
High-grade sugar	21.61					17.49	14.62	17.58
			BRIX	18.50	19.40				20.00	22.70

Between the wheat-flour incubation period, the different levels of not observing αDian Fenmei has remarkably influenced to maltose content or to dissolved solids.Therefore, for further optimization research, use 0.1Kg GC007/MT starch, dsb.

Embodiment 3

Wheat-flour 150 grams are suspended among the deionized water 450ml, and use 6.0N H ₂SO ₄The pH value is adjusted to pH5.00,4.50 and 4.00.Fully the vibration slurries make uniform mixing, regulate pH until being stabilized in specific pH value.Amount with 0.1Kgs/MT starch dsb adds each experimental group with GC007, and at 60 ℃ of incubations.At predetermined different time interval draw samples, measure the composition and the brix of sugar, as described in Example 1.(table 4).

Table 4

Between wheat-flour and GC007 incubation period, pH is to the influence of maltose output

PH	DP& BRIX	The incubation time, 1 hour, 60 ℃ of sugared percentage ratios
						2 hours	4 hours	6 hours	24 hours
		5.0	DP1	2.59	3.22					3.77	5.17
DP2	63.87					66.89	67.41	65.77
			DP3	10.90	13.23				14.25	16.87
High-grade sugar	22.64					16.66	14.57	12.19
			BRIX	17.70	18.80				19.20	20.90
4.5	DP1					2.29	2.80	3.31			4.37
		DP2	63.86	67.44	68.03				65.97
	DP3					10.93	13.30	14.44		17.56
		High-grade sugar	22.92	16.46	14.22				19.10
	BRIX					17.60	18.60	19.10		20.70
		4.0	DP1	1.71	1.79				2.11		3.17
DP2	58.19					60.23	61.82	62.95
			DP3	11.16	13.47				14.87	18.67
High-grade sugar	28.93					24.51	21.74	15.21
			BRIX	17.60	18.60				19.00	19.50

PH value when the wheat-flour incubation reduces from 5.5, and maltose content increases, and reaches maximum when pH4.5, and is about 68%, then reduces when pH4.0.The appliable plant βDian Fenmei carry out amylolytic during, do not add debranching factor and produced maltose content greater than 60%, this is a unexpected result.The starch of liquefaction produces the maltose of content between 55% and 60% usually by the hydrolysis of commercial βDian Fenmei (barley or wheat).For the starch of using liquefaction obtains maltose content greater than 60%, the extremely low initial DE of starch that needs to add debranching factor or need liquefaction.Importantly, to point out that method described in the invention allows maltose manufacturers to produce pH4.5 and 60 ℃, with the excessive risk of the microbial contamination that reduces present method at this.

Embodiment 4

Wheat-flour 150 grams are suspended among the deionized water 450ml, and the pH value of slurries is adjusted to pH4.5.Fully the vibration slurries make uniform mixing and use 6.0N H ₂SO ₄Regulate pH until the pH value stabilization.The suspension that obtains is remained in the water-bath, maintain 60 ℃, vibration makes uniform mixing before enzyme adds.Add the starch liquefacation enzyme, for example, the bacstearothermophilus αDian Fenmei, the trade name when it is sold is that " GC 007 " (GenencorInternational Inc.), measures and is 0.1Kg/MT, dsb.Then, add debranching factor with the amount of 0.25Kg, 0.5Kg and 1.0Kg/MT dsb, this debranching factor is with trade name OPTIMAX L-1000 (Genencor International, the Inc.) pullulanase of Chu Shouing, and at 60 ℃ of incubations.At predetermined different time interval (2,4,6 and 24 hours) draw samples, measure the composition and the brix of sugar, as described in Example 1.Record result's (table 5).

Table 5

(0.1Kg/MT, dsb) between the incubation period, debranching factor (OPTIMAX L-1000) is to wheat for wheat-flour and GC007

The influence of bud candy output, pH4.5,60 ℃

OPTIMAX concentration	DP& BRIX	The incubation time, 1 hour, 60 ℃ of sugared percentage ratios
						2 hours	4 hours	6 hours	24 hours
		0.0	DP1	1.21	1.38					1.47	1.99
DP2	62.77					64.69	66.24	67.16
			DP3	6.29	7.32				8.64	10.97
High-grade sugar	29.73					26.61	23.65	19.90
			BRIX	17.5	18.6				19.4	20.40
0.25Kg/MT dbs	DP1					1.28	1.33	1.90			1.94
		DP2	66.61	69.97	72.01				73.64
	DP3					8.18	10.03	10.68		13.58
		High-grade sugar	23.93	18.67	15.41				10.86
	BRIX					17.70	18.60	19.40		20.80
		0.50Kg/MT dbs	DP1	1.41	1.41				1.64		2.01
DP2	70.50					73.35	74.46	74.32
			DP3	9.62	11.26				11.94	14.31
High-grade sugar	18.47					13.98	11.96	9.36
			BRIX	17.70	18.70				19.80	20.80
1.00Kg/MT dbs	DP1					1.47	1.56	1.58			2.02
		DP2	72.90	75.54	76.08				75.21
	DP3					10.05	11.35	11.95		13.53
		High-grade sugar	15.48	11.55	10.39				9.24
	BRIX					17.60	18.80	19.70		21.70

Producing maltase (as βDian Fenmei) or starch liquefacation αDian Fenmei (as GC007) can not hydrolyzing alpha 1-6 glycosidic link, and it is the tapping point in branch's starch starch substrate.Therefore, in common practice, between starch substrates and βDian Fenmei incubation period, add debranching factor, pullulanase (OPTIMAX L-1000 is from GenencorInternational Inc) is used to produce the maltose greater than 65%.Wheat-flour and GC007 are between incubation period, and the influence of OPTIMAX L-1000 concentration is studied, and the result is presented in the table 5.Compared with the control, the adding of OPTIMAXL-1000 has caused significantly higher maltose (DP2) level (＞75%).

Embodiment 5

Industrial, use the high malt sugar syrup that enzymatic means produced by the starch substrates that utilizes high temperature (＞90 ℃) enzyme liquefaction, handle to produce isomalto-oligosaccharides syrup, normally common practice with glucanotransferase then.Present embodiment has illustrated that the granular starch in the single step transfer diminishes flour is the method for isomalto-oligosaccharides.In the present embodiment, be placed on wheat-flour 275 gram in the flask and add deionized water 688ml.Then, vibrate 15 minutes, then, use 6.0N H with uniform mixing ₂SO ₄With pH regulator to 4.5.The suspension that obtains is remained in the water-bath, maintain 60 ℃, vibration is with uniform mixing before adding enzyme.Add the starch liquefacation enzyme, bacstearothermophilus αDian Fenmei ([GC007 that provides by Genencor International] (0.1Kgs/MT dsb) and debranching factor, for example pullulanase (the OPTIMAX L-1000 that provides by Genencor International) (0.5kgs/MT dsb) for example.Then, suspension is divided into two equal portions.For the portion in these two parts, add the aspergillus niger transglucosidase (Genencor International) of selling with trade name " TRANSGLUCOSIDASE L-500 ", measure dsb, and remain in the water-bath into 1.0Kg/MT, maintain 60 ℃ (samples 1).Another part elder generation was 60 ℃ of incubations four hours, add the aspergillus niger transglucosidase (GenencorInternational) of selling subsequently with trade name " TRANSGLUCOSIDASE L-500 ", amount is 1.0Kg/MT dsb, and remains in the water-bath, maintains 60 ℃ (samples 2).Result displayed prompting in the table 6, substrate takes place to the transformation of IMO, and it follows or is not accompanied by the preincubation of substrate (wheat-flour) before of adding transglucosidase.

Table 6

At wheat-flour and GC007 and OPTIMAXL-1000 between incubation period, the formation of isomalto-oligosaccharides,

PH4.5, utilizes TRANSGLUCOSIDASE L-500 by 60 ℃

Sample	Reaction times	Glucose	Maltose	Isomaltose	Trisaccharide maltose	4-α-glucosyl maltose	Isomaltotriose	The sugar G3+ of branch	The IMO number
										1	17	14.74	30.27	2.73	6.82	23.05	7.13	14.65	47.5610
2	17	13.92	33.17	2.32	7.70	23.69	4.88	14.32	45.21
										1	48	16.90	20.04	7.98	10.29	22.35	3.95	17.95	52.23
2	48	14.96	23.59	6.85	12.17	16.65	5.57	19.66	48.73
										1	72	16.78	21.21	5.78	8.51	22.83	4.17	20.24	53.02
2	72	15.24	25.44	4.99	8.88	18.48	7.52	19.03	50.0115

IMO number: be calculated as isomaltose, panose, Isomaltotriose and greater than the summation of branch's sugar of DP3.

The modification wheat-flour and the transglucosidase incubation that contain high-content maltose produce and the identical isomalto-oligosaccharides of composition that generates by traditional method.Simple, the economic and mass-producing easily of this method is to be used for commercial production.

Embodiment 6

Cereal such as wheat, barley and rye contain high-caliber βDian Fenmei, are common general knowledge.These cereal are at 55 ℃-60 ℃, and the pH5.5 incubation forms the syrup that contains greater than 50% maltose usually.Join in the 720gm deionized water by each powder, prepare 28% slurries of wheat, barley and rye meal respectively 280gm.The amount of bacstearothermophilus αDian Fenmei (for example, the bacstearothermophilus αDian Fenmei of being sold down with trade name " GC007 " by Genencor International) with the 0.2kg/MT powder joined in each prepared product.Then, use 6.0N H ₂SO ₄With pH regulator to 5.5,60 ℃ of incubations 4.5 hours.Then, use 6.0NH ₂SO ₄With the pH regulator to 4.5 of incubation sample, with the amount adding transglucosidase of 1.25kg transglucosidase (for example, the transglucosidase of selling with trade name " TRANSGLUCOSIDASE L-500 ")/MT powder by GenencorInternational.Afterwards, in 60 ℃ of water-bath incubation slurries 48 hours.Then, centrifugal sample is also analyzed IMO and is formed (table 7), such as among the embodiment 1 elaboration.

Table 7

After GC007 and " TRANSGLUCOSIDASE L-500 " incubation, wheat, barley and rye can

The dissolubility carbohydrate components

Cereal/cereal	Handle	Time (hour)	Soluble saccharide component %							IMO counts .10
											Glucose	Maltose	Isomaltose	Maltose	4-α-glucosyl maltose	Isomaltotriose	The sugar G3+ of branch
			Wheat	Tg L-500 *	48	21.92	19.37	9.97	3.67									26.52	3.12	14.85	54.46
Barley	Tg L-500 *	48								25.43	6.68	16.50	7.72	12.82	0.30	30.54	60.16 15
			Rye	Tg L-500 *	48	22.18	10.39	11.15	3.21									22.93	0.01	30.14	64.22 20

Tg L-500 ^*Refer to " TRANSGLUCOSIDASE L-500 "

Embodiment 7

In an experiment, (Cargill Malt/Schreier-Malting Company, Wisconsin USA) mix with 360 gram distilled water with 140 gram Fructus Hordei Germinatus.Then, the acetate of using dilution was with pH regulator to 4.5 with uniform mixing in 15.0 minutes for room temperature vibration slurries.After pH is stable, slurries are remained in the water-bath, maintain 60 ℃.Continue incubation 30 minutes, sustained oscillation simultaneously, and extraction 2ml sample is used for Brix and HPLC analyzes (0, the time).Amount with 1.5kg/MT Fructus Hordei Germinatus adds TRANSGLUCOSIDASE L-500, and at 60 ℃ of incubations.At predetermined different time at interval, for example, 2,4,6,12 and 24 hours, draw samples was determined Brix and IMO component (table 8), as described in Example 1 between incubation period.

Table 8

After hatching with TRANSGLUCOSIDASE L-500, the soluble sugar based composition of Fructus Hordei Germinatus

Cereal/cereal	Time (hour)	Brix	Solvable carbohydrate components %							The IMO number
											Glucose	Maltose	Isomaltose	Trisaccharide maltose	4-α-glucosyl maltose	Isomaltotriose	The G3+ of branch
			Fructus Hordei Germinatus	0	15.70	16.42	33.87	-	14.04									-	-	35.66	-
Fructus Hordei Germinatus	2	16.50								19.59	21.91	0.75	7.70	9.38	2.87	31.40	44.40
			Fructus Hordei Germinatus	4	16.70	21.82	19.70	0.90	6.16									12.35	1.42	30.94	45.62
Fructus Hordei Germinatus	6	16.90								24.43	18.29	1.00	4.63	11.50	2.51	30.73	45.84
			Fructus Hordei Germinatus	12	17.00	28.37	15.47	3.93	4.32									8.36	2.68	29.10	43.98
Fructus Hordei Germinatus	24	17.00								33.24	14.94	5.96	2.34	5.50	3.34	26.15	40.95

Result in the table 8 shows that commercially available malt extract can be used as suitable substrate, is used to produce the malt extract that contains isomalto-oligosaccharides.Can be by the dosage of control enzyme, the IMO of adjusting reaction time and malt extract sugar is formed.The adding of producing maltase can increase the IMO content of the composition that obtains.

Embodiment 8: jowar, broomcorn millet and rice (exogenous product maltase)

In another experiment, get 280 gram jowar, broomcorn millet and rice powder respectively, mix with deionized water 720 grams respectively.The pH value of suspension is adjusted to 5.5, and adds the bacstearothermophilus αDian Fenmei of selling with trade name " GC007 " (Genencor International) with the amount of 0.5kg/mt powder.Homogeneous remains on suspension in the water-bath after mixing, and maintains 75 ℃.Between 6 hours incubation period, continue stirred reaction mixture.Then, reduce temperature to 60 ℃ and add βDian Fenmei (selling with trade name OPTIMALBBA) by GenencorInternational with the amount of 1.0kg/mt powder.Continue the other 10-15 of incubation hour (sample thief carries out Brix and HPLC).At the appointed time, use 6.0N H ₂SO ₄PH is reduced to 4.5, and adds aspergillus niger transglucosidase (selling down with trade name " TRANSGLUCOSIDASE L-500 ") by Genencor International with the amount of 1.0kg/mt powder.At 24 hours and 48 hours, sample thief was analyzed (table 9).

Table 9

After " TRANSGLUCOSIDIDASE L-500 " handles, the solvable carbohydrate components of jowar, broomcorn millet and rice

Cereal/cereal	Handle	Time (hour)	Brix	Solvable carbohydrate components %
												Glucose	Maltose	Isomaltose	Trisaccharide maltose	4-α-glucosyl maltose	Isomaltotriose	The sugar G3+ of branch	The IMO number
				Jowar	Tg L-500 *	24	20.20	18.70	23.50	0.60	5.00									15.90	0.00	35.70	52.20
48	21.40	19.17	22.45									0.66	4.56	16.34	0.29	36.01	53.03
						Broomcorn millet	Tg L-500 *	24	23.50	38.00	16.40							5.00	1.70	8.30	2.50	28.0	43.90
48	23.9	40.20	17.13	5.36	0.14							6.08	5.49	24.26	41.19
								Rice	Tg L-500 *	24	25.50					15.90	22.90	0.60	6.80	18.90	1.70	33.20	54.40
48	26.00	17.83	20.30	0.54	4.58	22.13	0.38					34.21	51.25

As shown in Figure 9, obtained the IMO numerical value of 41 to 54% (52.20,53.03,43.90,41.19,54.40 and 51.25).

Embodiment 9: blended cereal/cereal composition

Among the embodiment 5 among the data of wheat and the embodiment 6 data presentation of barley and rye the endogenous of a great deal of produce the maltase activity, this causes forming and contains the very syrup of high malt sugar.On the other hand, the known cereal that does not contain endogenous product maltase, for example, jowar, broomcorn millet and rice need to add exogenous product maltase, are suitable for the substrate that transglucosidase is handled with generation.In this experiment, we have studied cereal for example wheat or additional jowar of barley and the rice that produces maltase with containing, and starch are changed into the matrix that contains the high malt sugar level.In typical experiment,, prepare 15% suspension of jowar and rice by the 140 gram powder that in 720 gram deionized waters, suspend.Use 6.0N H ₂SO ₄With pH regulator to 5.5, and add the bacstearothermophilus αDian Fenmei with the amount of 0.5/MT powder, the trade name when it is sold is " GC007 " (Genencor International).Afterwards, the suspension that obtains is maintained 75 ℃ in water-bath.Continue stirred suspension 6 hours.Then, temperature is reduced to 60 ℃.By adding barley meal, the solids content of for example pretreated rice powder of powder increases to 30% from 15%.Similarly, wheat is added pretreated jowar, make final concentration reach 30%.Then, continued incubation other 10-12 hour at 60 ℃.Reducing pH is 4.5, with the amount adding TRANSGLUCOSIDASE L-500 of 1.0kg/mt powder.60 ℃ were continued incubation 24 hours and 48 hours.Sample thief carries out HPLC and analyzes and brix; The results are shown in the table 10.

Table 10

Broomcorn millet and barley; The solvable carbohydrate components of rice and wheat

Cereal/cereal

Time (hour)

Brix

The IMO number

Glucose

Maltose

Isomaltose

4-α-glucosyl maltose

Trisaccharide maltose

Isomaltotriose

The sugar G3+ of branch

Wheat and jowar (50: 50)	24	16.1	48.70	29.50	18.60	2.90	1.00	13.00	3.40	29.50
											48	16.5	45.27	34.68	17.09	5.03	0.51	6.20	6.42	27.63
	Rice and barley (50: 50)	24	21.00	51.00	19.50	18.40	1.00	4.20	20.20	1.10											34.7
48											21.4	56.20	21.60	18.21	1.3	2.92	17.96	2.08	37.86

Show as top table 10, the mixture of broomcorn millet and barley and rice and wheat, caused described incubation period in the above after, IMO is 45% to 56% in the suspended substance that obtains.

Should be appreciated that, embodiment described herein and embodiment only are for illustrative purposes, to its various modifications or change to those skilled in the art, point out, and they are included in the application's the spirit and scope and in the scope of claim.All publications cited herein, patent and patent application are incorporated herein by reference at this with its integral body, are used for all propositions.

Claims (17)

1. be used to make the method for isomalto Oligosaccharide grain compositions, described method comprises:

(a) the ungelatinized cereal that will contain starch contact with the starch liquefacation enzyme with producing maltase, generation maltose;

(b) described maltose is contacted with the enzyme that changes glucosides, wherein said step (a) and step (b) are in the temperature generation that is lower than or equals starch gelatinization temperature; With

(c) acquisition has the grain compositions of the isomalto Oligosaccharide of enzymatic generation, and wherein said oligosaccharides derives from described cereal.

2. method according to claim 1, wherein said step (a) and (b) generation simultaneously.

3. method according to claim 1 further comprises the step of dry described grain compositions.

4. method according to claim 1, wherein said cereal is selected from wheat, rye, barley and Fructus Hordei Germinatus.

5. method according to claim 1, wherein said cereal is selected from broomcorn millet, jowar and rice.

6. method according to claim 1, wherein said product maltase is a βDian Fenmei.

7. method according to claim 1, wherein said product maltase is endogenic for described cereal.

8. method according to claim 1, wherein said starch liquefacation enzyme are the αDian Fenmeis from genus bacillus.

9. method according to claim 8, wherein said starch liquefacation enzyme is from Bacillus licheniformis (Bacilluslicheniformis) or bacstearothermophilus (Bacillus stearothermophilus).

10. method according to claim 1, the enzyme of wherein said commentaries on classics glucosides is a transglucosidase.

11. method according to claim 10, wherein said transglucosidase derives from aspergillus tubigensis.

12. method according to claim 11, wherein said aspergillus tubigensis are aspergillus niger (Aspergillus niger).

13. according to the grain compositions that claim 1 produced.

14. food additive, it contains grain compositions according to claim 13.

15. flour, it contains grain compositions according to claim 13.

16. isomalto Oligosaccharide according to claim 1 manufacturing.

17. oral rehydrated solution, it contains the isomalto Oligosaccharide of with good grounds claim 16.

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