CN112553270A - Method for preparing alpha-1, 3 glycosidic bond modified low-calorie dextrin - Google Patents

Method for preparing alpha-1, 3 glycosidic bond modified low-calorie dextrin Download PDF

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CN112553270A
CN112553270A CN202011475893.7A CN202011475893A CN112553270A CN 112553270 A CN112553270 A CN 112553270A CN 202011475893 A CN202011475893 A CN 202011475893A CN 112553270 A CN112553270 A CN 112553270A
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吴敬
夏伟
魏贝贝
王蕾
陈晟
杨卫康
金雪薇
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Jiangnan University
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Abstract

The invention discloses a method for preparing alpha-1, 3 glycosidic bond modified low-calorie dextrin, and belongs to the field of functional foods. The low-calorie dextrin has the alpha-1, 3 glycosidic bond content of 2-40%, the alpha-1, 4 glycosidic bond content of 10-30%, the alpha-1, 6 glycosidic bond content of 45-80%, the average molecular weight of about 1000-5000Da and the polymerization degree of about 5-30. The preparation method of the low-calorie dextrin comprises the following steps: starch is used as a substrate, enzymatic reaction is carried out by using 4, 6-alpha-glucosyltransferase Gtf B and branching sucrase BSR-B-delta 1, and the low-calorie dextrin modified by alpha-1, 3 glycosidic bonds is prepared by separating and drying after thermal stability alpha-amylase and amyloglucosidase enzymolysis. The method has the advantages of mild conditions, high content of alpha-1, 3 glycosidic bonds and low cost, and lays a foundation for the realization of the industrial production of the complex bond type low-calorie dextrin.

Description

Method for preparing alpha-1, 3 glycosidic bond modified low-calorie dextrin
Technical Field
The invention relates to a method for preparing alpha-1, 3 glycosidic bond modified low-calorie dextrin, belonging to the field of functional foods.
Background
The low-calorie dextrin is a carbohydrate which cannot be digested by a human body, is used as a novel food additive auxiliary material and nutrient, has various effects of reducing energy value, improving taste, enhancing texture and the like, has a good prevention and treatment effect on 'rich diseases' (diabetes, obesity, intestinal cancer, constipation and the like) caused by improving diet delicacy by living standard, and is an essential important nutrient in modern life.
So far, high-temperature acidolysis chemical method is mainly adopted to mediate glycosidic bond reconstruction to prepare low-calorie dextrin, starch substrates are treated by different concentrations of acid and temperature to hydrolyze alpha-1, 4 glycosidic bonds in the starch substrates, and different generated sugar chains are subjected to dehydration condensation again to form anti-digestible glycosidic bonds. However, the high-temperature acidolysis chemical method has the outstanding problems of high energy consumption, low yield, complex separation and purification process, easy generation of harmful substances such as furfural and the like, so that the enzymatic conversion method serving as a green, energy-saving, efficient and safe alternative method can obviously reduce the production cost of low-calorie dextrin, improve the product quality, meet the mass consumption demand, and is a prime motive power for promoting the sustainable development of the low-calorie dextrin industry. The key point for promoting the sustainable development of the industry is to prepare the low-calorie dextrin by using the starch as the raw material and adopting the high-efficiency enzyme method.
We have used 4, 6-alpha-glucosyltransferase Gtf B as a substrate to produce a low molecular weight resistant dextrin which is rich in alpha-1, 6 glycosidic linkages, low in molecular weight and high in yield, and which is disclosed in Chinese patent publication No. CN202010288282.5, but which contains only alpha-1, 4 and alpha-1, 6 glycosidic linkages. How to further enrich bond types in the resistant dextrin and improve the application value of the resistant dextrin is always a problem to be solved in the field.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a method for preparing low-calorie dextrin modified by alpha-1, 3 glycosidic bonds, which can introduce alpha-1, 3 bonds into linear alpha-1, 6 glycosidic bond sugar chains or isomalto/malt-polysaccharide chains to enable the product to have- (1 → 3,6) -alpha-D-Glcp- (1 → 6) -branch points. The low-calorie dextrin comprises the following components in percentage by mass: the content of alpha-1, 3 glycosidic bonds is 2-40%, the content of alpha-1, 4 glycosidic bonds is 10-30%, and the content of alpha-1, 6 glycosidic bonds is 45-80%. A new process technology is developed by using biochemical characteristics of the branched sucrase BSR-B-Delta 1 and the 4, 6-alpha-glucosyltransferase disclosed in Chinese patent document with the publication number of CN202010288282.5, a series of products meeting the physicochemical indexes of the low-calorie dextrin can be obtained, the product has good solubility, high alpha-1, 3 glycosidic bond content and resistance content of more than 60 percent, can form a novel prebiotic and provides a powerful guarantee for maintaining and improving the health of human beings, and the invention lays a solid theoretical foundation for the industrial production of the multi-bond type low-calorie dextrin prepared by the enzyme method.
The invention provides a method for preparing low-calorie dextrin, which utilizes 4, 6-alpha-glucosyltransferase and branching sucrase BSR-B-delta 1 to convert starch to generate the low-calorie dextrin.
In one embodiment of the invention, the 4, 6-alpha-glucosyltransferase is derived from Lactobacillus fermentum.
In one embodiment of the present invention, the amino acid sequence of said 4, 6-alpha-glucosyltransferase is disclosed in the patent application No. CN 202010288282.5.
In one embodiment of the invention, the branched sucrase BSR-B-Delta 1 is derived from Leuconostoc citreum (Leuconostoc ccitreum) NRRL B-742.
In one embodiment of the invention, the amino acid sequence of the branched sucrase BSR-B-Delta 1 is shown in SEQ ID NO. 1.
In one embodiment of the present invention, the amount of the branched sucrase added to the reaction system is 0.5 to 5U/mL.
In one embodiment of the present invention, the 4,6- α -glucosyltransferase is added to the reaction system in an amount of 2500-.
In one embodiment of the invention, the starch is a cereal starch, or a potato starch, a starch derivative, or a starch-containing material.
In one embodiment of the invention, the cereal starch is corn starch, wheat starch, rice starch, mung bean starch, pea starch; the potato starch is cassava starch, potato starch, sweet potato starch and other potato starches; the starch derivatives are maltodextrin, dextrin, soluble starch and other starch derivatives; the substance containing starch is rice protein peptide.
In one embodiment of the invention, starch is prepared into suspension, and the suspension is gelatinized and liquefied to prepare reaction liquid; catalyzing reaction liquid by using pullulanase and 4, 6-alpha-glucosyltransferase; adding sucrose and branched sucrase BSR-B-Delta 1 after the catalysis is finished, and reacting for 20-30h at 35-40 ℃; after the reaction is finished, inactivating enzyme in the reaction liquid, adding alpha-amylase for reaction for 20-40min, adding amyloglucosidase for reaction for 20-40 min; digesting the prepared reaction solution, filtering and drying to obtain a finished product.
In one embodiment of the invention, the final concentration of sucrose in the reaction system is 2-40 g/100 mL.
In one embodiment of the invention, the concentration of the suspension prepared from the starch is 10-30 g/100 mL.
In one embodiment of the invention, the pullulanase is added in an amount of 10-80U/g substrate.
In one embodiment of the invention, the temperature is adjusted to 90-100 ℃, thermostable alpha-amylase is added, and the reaction is carried out for 30 min; the dosage of the alpha-amylase to the enzyme is not less than 1000U/g substrate.
In one embodiment of the invention, the temperature is adjusted to 50-70 ℃, the pH is adjusted to 4.0-5.0, amyloglucosidase is added, and the reaction is carried out for 25-35 min; purifying the product after the reaction, and removing glucose, fructose, maltose and sucrose in the system; the acting dosage of the amyloglucosidase to the enzyme is not less than 660U/g substrate.
The invention provides resistant dextrin prepared by the method.
The invention provides the use of said method, or of said resistant dextrin, in the food or cosmetic field.
In one embodiment of the invention, the use comprises preparing a low calorie dextrin containing alpha-1, 3 glycosidic linkages, and/or using an oligosaccharide, and/or producing a dextrin containing alpha-1, 3 glycosidic linkages.
In one embodiment of the invention, the food product comprises dairy products, infant formula, rice-flour products, meat products, confectionery, beverages.
In one embodiment of the invention, the dairy product comprises yoghurt, cheese, ice cream, vegetable protein dairy products.
In one embodiment of the invention, the beverage comprises a sports drink, a low-sugar drink, a dietary fiber drink, a cereal drink.
In one embodiment of the invention, the rice and flour product comprises a baked product, noodles, rice flour, instant noodles, instant rice.
In one embodiment of the invention, the baked product comprises bread, cookies, cakes.
The invention has the beneficial effects that: compared with the prior art, the method provided by the invention has the following characteristics:
(1) the branched sucrase BSR-B-Delta 1 used by the invention can not only take linear alpha-1, 6 bond dextran as a receptor, but also isomalto-malt-polysaccharide (IMMP) as a receptor, and sucrose with different concentrations as donors, and introduces a (1 → 3,6) -alpha-D-Glcp- (1 → 6) -branch point to generate a series of low-calorie dextrins with controllable alpha-1, 3 glycosidic bond content;
(2) the invention utilizes a double-enzyme method to prepare the low-calorie dextrin containing alpha-1, 3 glycosidic bonds, the content of the alpha-1, 3 glycosidic bonds is 2-40%, the content of the alpha-1, 4 glycosidic bonds is 10-30%, the content of the alpha-1, 6 glycosidic bonds is 45-80%, the average molecular weight is 1000-5000Da, the average polymerization degree is 5-30, and the content of dietary fibers is more than 60%;
(3) compared with the high-temperature acidolysis method for preparing the low-calorie dextrin, the method has the advantages of high yield, simple preparation process, mild reaction conditions, cleanness, safety and no toxic product generation;
(4) the low-calorie dextrin prepared by the invention can be used as a food additive to enter the colon, for example, as a prebiotic, can be used in various food applications from dairy products to baked foods, candies and beverages, can improve the texture and mouthfeel of foods, and is convenient to be widely applied in the fields of foods, health-care products, particularly high-end foods and health-care products.
Drawings
FIG. 1 is a ratio diagram of low-calorie dextrin bonds containing alpha-1, 3 glycosidic bonds, and the addition amount of sucrose donor is 25-30%.
FIG. 2 is a ratio chart of low-calorie dextrin bonds containing alpha-1, 3 glycosidic bonds, wherein the addition amount of sucrose donor is 7-10%.
FIG. 3 is a ratio chart of low-calorie dextrin bonds containing alpha-1, 3 glycosidic bonds, and the addition amount of sucrose donor is 2-4%.
FIG. 4 is a graph showing the bond type ratio of the 4, 6-alpha-glucosyltransferase product.
FIG. 5 shows the molecular weight distributions of low-calorie dextrins with different contents of alpha-1, 3 glycosidic bonds.
Detailed Description
The technical solution of the present invention is further described below with reference to the specific embodiments, but the scope of the present invention is not limited thereto.
The medium formulation described in the example (one) is as follows:
LB medium (g/L): peptone 10, yeast extract 5, NaCl 10.
TB medium (g/L): peptone 10, yeast powder 24, glycerol 5, K2HPO4·3H2O 16.43，KH2PO4 2.31。
The high-temperature resistant alpha-amylase, the amyloglucosidase and the pullulanase in the example are all purchased from Shandong Kete enzyme preparation Co, and the enzyme activities are respectively 40kU/mL, 100kU/mL and 2 kU/mL.
(III) enzyme activity determination:
the total enzyme activity of 4, 6-alpha-glucosyltransferase GtfB used in examples 3 to 5 is determined by an iodine method, and the specific determination method and the definition of the enzyme activity are disclosed in Chinese patent document with the application number of CN 202010288282.5;
the enzyme activity of the branched sucrase BSR-B-Delta 1 used in examples 3 to 5 was measured by a modified 3, 5-dinitrosalicylic acid method (DNS method):
(1) 20g/L of sucrose is prepared by 50mM acetic acid buffer solution (pH5.5) as a substrate, 1mL of the substrate and 0.9mL of 50mM acetic acid buffer solution (pH5.5) are added into a 15mL test tube with a plug, the mixture is uniformly shaken and mixed, and then the mixture is placed into a water bath kettle at 40 ℃ to be preheated for 10 min. Adding 100 μ L enzyme solution/blank, reacting at 40 deg.C for 30min, adding 3mL DNS, boiling for 7min, rapidly cooling, adding distilled water to desired volume of 15mL, and measuring absorbance at 540nm (using inactivated enzyme solution or buffer solution as blank control);
(2) definition of enzyme activity: under the above enzyme reaction conditions, the amount of enzyme required to produce 1. mu. mol of fructose per minute was 1 enzyme activity unit (U).
Figure BDA0002835392870000041
S: reducing the sugar content; 1000: conversion between mg and mug; n: dilution factor of enzyme activity
T: reaction time; v: the amount of enzyme solution; m: relative molecular mass
(3) Preparation of DNS reagent: 6.5g of DNS is dissolved in a 500mL beaker by using a small amount of distilled water, 262mL of 2mol/L sodium hydroxide solution is added, then the solution is added into 500mL of hot water solution containing 185.0g of potassium sodium tartrate, then 5.0g of phenol and 5.0g of anhydrous sodium sulfite are respectively added, the solution is stirred and dissolved, after cooling, the distilled water is added to the solution with a constant volume of 1L, and the solution is fully, uniformly mixed and stored in a brown bottle.
(IV) the method for measuring the content of the dietary fiber comprises the following steps:
the determination method refers to national standard GB/T22224-2008 "determination of dietary fiber in food-enzyme gravimetric method", and the determination result is mass ratio and is expressed by percentage, for example, 60g/L is expressed as 60%.
(1) Calculation of IDF + SDF
Figure BDA0002835392870000042
In the formula:
IDF + SDF — the percentage (in mass fraction) of the total content of Insoluble Dietary Fiber (IDF) and high molecular mass Soluble Dietary Fiber (SDF) precipitated in ethanol in the sample,%;
mSR1and mSR2The mass of residue in crucibles 1 and 2 in duplicate samples, in milligrams (mg);
mPS-mass of protein in milligrams (mg) in the residue;
mAS-mass of ash in milligrams (mg) in the residue;
mBR-mass of residue in milligrams (mg) in the blank crucible;
mS1and mS2Mass in milligrams (mg) of duplicate samples 1 and 2.
(2) Calculation of RMD
Figure BDA0002835392870000051
In the formula:
RMD-the percentage (by mass fraction) of low molecular mass ethanol-soluble dietary fiber in the sample,%;
mRMD1and mRMD2-the mass in milligrams (mg) of the low molecular mass ethanol-soluble dietary fiber with a DP > 3 in both samples 1 and 2;
mS1and mS2Mass in milligrams (mg) of duplicate samples 1 and 2.
Figure BDA0002835392870000052
In the formula:
PARMD-the chromatographic peak area of low molecular mass ethanol soluble dietary fiber with DP > 3;
PAglyIS-chromatographic peak area of glycerol internal standard;
mglyIS-the mass of glycerol internal standard added to the suction filtrate in milligrams (mg);
RF-response factor for dextroglucose.
(3) Calculation of TDF
TDF＝(IDF+SDF)+RMD
In the formula:
TDF-percentage of total dietary fiber in the sample,%.
(4) Reference conditions for chromatography
A chromatographic column: agilent Hi-Plex Ca, the mobile phase is ultrapure water, the flow rate is 0.5mL/min, the column temperature is 80 ℃, the detector temperature is 40 ℃, and the sample injection amount is 10 muL.
Example 1: synthesis of branched sucrase BSR-B-delta 1 gene and transformation of escherichia coli
Chemically synthesizing a target gene branched sucrase gene (brs-b-delta 1) fragment with a nucleotide sequence shown as SEQ ID NO.2, fermenting in a shake flask, extracting a recombinant plasmid pET-24 a-brs-b-delta 1, and transforming to E.coli BL21(DE 3).
(1) Placing prepared E.coliBL21(DE3) on ice for 5min, adding 5 μ L recombinant plasmid pET-24a-brs-b- Δ 1, blowing and sucking gently, and standing on ice for 30 min;
(2) heating in 42 deg.C water bath for 90s, quickly taking out from ice, and cooling for 2 min;
(3) adding 200 μ L LB liquid culture medium preheated at 37 deg.C, mixing, and slightly shaking culturing at 37 deg.C for about 40min to allow cells to recover;
(4) coating a proper amount of recovered competent cells on an LB solid culture medium containing kanamycin (100 mu g/mL), carrying out inverted culture at 37 ℃ for 12h, and observing the growth condition of a single colony;
(5) single colonies were picked into 10mL of liquid LB medium containing kanamycin resistance (100. mu.g/mL), cultured at 37 ℃ for 8 hours, stored in a glycerin tube, and stored in a refrigerator at-80 ℃. The positive transformant which is verified to be correct by sequencing is the genetic engineering escherichia coli containing the plasmid pET-24 a-brs-b-delta 1.
Example 2: shake flask fermentation for producing enzyme
The recombinant Escherichia coli obtained in example 1 was inoculated into LB medium, cultured at 37 ℃ for 8 hours, transferred into 50mL TB fermentation medium at a constant temperature of 37 ℃ and 200rpm for 1-2 hours in an inoculum size of 5% of the volume of the fermentation medium, and cultured in OD cells600When the concentration is 0.5 to 0.7, IPTG (final concentration is 0.4mM) is added and fermentation is induced at 25 ℃ and 200rpm for 12 hours. After the fermentation is finished, the fermentation liquor is homogenized and crushed under high pressure (the crushing condition is 4 ℃, 800Bar), then is centrifuged (8000rpm, 20min, 4 ℃), and the supernatant is the branched sucrase BSR-B-Delta 1 enzyme solution produced by the recombinant escherichia coli.
And (3) measuring the enzyme activity of the enzyme solution, wherein the enzyme activity of the branched sucrase is 1U/mL through measurement.
Example 3: application of 4, 6-alpha-glucosyltransferase and branching sucrase BSR-B-delta 1 in preparation of low-calorie dextrin
(1) Taking starch as a substrate, adding water into the starch to prepare 20% (20g/100mL) of suspension, heating to 60-80 ℃ for gelatinization, adding alpha-amylase for liquefaction for 10-30min, and controlling the DE value to be 3-7;
(2) cooling to 30-45 ℃, adjusting the pH value to 5.0-7.5, simultaneously adding 10-50U/g substrate of pullulanase and 4000U/g substrate of 4, 6-alpha-glucosyltransferase, putting into a constant-temperature water bath shaker, and reacting for 24h at 37 ℃;
(3) adjusting pH value to 5.0-6.0, adding sucrose with final concentration of 25-30% (25-30g/100mL), adding 1-2U/mL branched sucrase BSR-B-Delta 1, placing in a constant temperature water bath shaker, and reacting at 45 deg.C for 24 h;
(4) heating to 95 ℃ to inactivate enzyme, adding thermostable alpha-amylase until the acting dose of the enzyme is not less than 1000U/g substrate, placing in 95 ℃ water bath for continuous oscillation, and reacting for 30 min;
(5) taking out the reactant, cooling to room temperature, adjusting pH to 4.0-5.0, adding amyloglucosidase until the action dosage of the enzyme is not less than 660U/g substrate, placing in a water bath shaking table at 60 ℃ and continuously oscillating for reaction for 30 min;
(6) after the reaction is finished, inactivating enzyme at 95 ℃ for 20min, adding 10g/L of food-grade dry yeast powder for digestion, putting the mixture into a yeast shaker, reacting for 12h at 30 ℃ and 200rpm to purify the product and remove glucose, fructose, maltose and sucrose in the system;
(7) centrifuging the reaction product at 8000rpm for 20min, collecting supernatant, filtering with 0.45 μm filter membrane, and spray drying to obtain light yellow powder.
The low-calorie dextrin yield was determined to be 52.52%, and after yeast digestion, the low-calorie dextrin content was 89.97%, the alpha-1, 3 linkage content was 21.68%, the alpha-1, 4 linkage content was 14.45%, the alpha-1, 6 linkage content was 63.87% (see fig. 1), the average molecular weight was about 3852Da, and the degree of polymerization was about 24 glucose.
Example 4: application of 4, 6-alpha-glucosyltransferase and branching sucrase BSR-B-delta 1 in preparation of low-calorie dextrin
(1) Taking starch as a substrate, adding water into the starch to prepare 20% (20g/100mL) of suspension, heating to 60-80 ℃ for gelatinization, adding alpha-amylase for liquefaction for 10-30min, and controlling the DE value to be 3-7;
(2) cooling to 30-45 ℃, adjusting the pH value to 5.0-7.5, simultaneously adding 10-50U/g substrate of pullulanase and 4000U/g substrate of 4, 6-alpha-glucosyltransferase, putting into a constant-temperature water bath shaker, and reacting for 24h at 37 ℃;
(3) adjusting pH to 5.0-6.0, adding 7-10% (7-10g/100mL) sucrose, simultaneously adding 1-2U/mL branched sucrase, placing in a constant temperature water bath shaker, and reacting at 45 deg.C for 24 hr;
(3) heating to 95 ℃ to inactivate enzyme, adding thermostable alpha-amylase until the acting dose of the enzyme is not less than 1000U/g substrate, placing in 95 ℃ water bath for continuous oscillation, and reacting for 30 min;
(4) taking out the reactant, cooling to room temperature, adjusting pH to 4.0-5.0, adding amyloglucosidase until the action dosage of the enzyme is not less than 660U/g substrate, placing in a water bath shaking table at 60 ℃ and continuously oscillating for reaction for 30 min;
(5) after the reaction is finished, inactivating enzyme at 95 ℃ for 20min, adding 10g/L of food-grade dry yeast powder for digestion, putting the mixture into a yeast shaker, reacting for 12h at 30 ℃ and 200rpm to purify the product and remove glucose, fructose, maltose and sucrose in the system;
(6) centrifuging the reaction product at 8000rpm for 20min, collecting supernatant, filtering with 0.45 μm filter membrane, and spray drying to obtain light yellow powder.
The low-calorie dextrin yield was determined to be 60.18%, and after yeast digestion, the low-calorie dextrin content was 92.1%, the alpha-1, 3 linkage content was 14.66%, the alpha-1, 4 linkage content was 16.47%, the alpha-1, 6 linkage content was 68.89% (see fig. 2), the average molecular weight was about 3234Da, and the degree of polymerization was about 20 glucose.
Example 5: application of 4, 6-alpha-glucosyltransferase and branching sucrase BSR-B-delta 1 in preparation of low-calorie dextrin
(1) Taking starch as a substrate, adding water into the starch to prepare 20% (20g/100mL) of suspension, heating to 60-80 ℃ for gelatinization, adding alpha-amylase for liquefaction for 10-30min, and controlling the DE value to be 3-7;
(2) cooling to 30-45 ℃, adjusting the pH value to 5.0-7.5, simultaneously adding 10-50U/g substrate of pullulanase and 4000U/g substrate of 4, 6-alpha-glucosyltransferase, putting into a constant-temperature water bath shaker, and reacting for 24h at 37 ℃;
(3) adjusting pH to 5.0-6.0, adding 2-4% (2-4g/100mL) sucrose, simultaneously adding 1-2U/g branched sucrase, placing in a constant temperature water bath shaker, and reacting at 45 deg.C for 24 hr;
(4) heating to 95 ℃ to inactivate enzyme, adding thermostable alpha-amylase until the acting dose of the enzyme is not less than 1000U/g substrate, placing in 95 ℃ water bath for continuous oscillation, and reacting for 30 min;
(5) taking out the reactant, cooling to room temperature, adjusting pH to 4.0-5.0, adding amyloglucosidase until the action dosage of the enzyme is not less than 660U/g substrate, placing in a water bath shaking table at 60 ℃ and continuously oscillating for reaction for 30 min;
(6) after the reaction is finished, inactivating enzyme at 95 ℃ for 20min, adding 10g/L of food-grade dry yeast powder for digestion, putting the mixture into a yeast shaker, reacting for 12h at 30 ℃ and 200rpm to purify the product and remove glucose, fructose, maltose and sucrose in the system;
(7) centrifuging the reaction product at 8000rpm for 20min, collecting supernatant, filtering with 0.45 μm filter membrane, and spray drying to obtain light yellow powder.
The low-calorie dextrin yield was determined to be 63.25%, after yeast digestion, the resistant content was 90.32%, the α -1,3 linkage content was 7.55%, the α -1,4 linkage content was 17.15%, the α -1,6 linkage content was 75.3% (see fig. 3), the average molecular weight was about 3097Da, and glucose was about 19 degrees of polymerization.
Comparative example 1
See example 3 for a difference that no sucrose and branched sucrase were added during the preparation, and after completion of the reaction, the resulting low-calorie dextrin was determined to have a yield of 49.91%, a resistance content of 89% (percentage in the prepared low-calorie dextrin) after yeast digestion, an α -1,4 linkage ratio of 12.19%, an α -1,6 linkage ratio of 87.81%, no α -1,3 glycosidic linkages, an average molecular weight of about 3057Da, and a degree of polymerization of about 19 glucose.
(1) Adding water into starch as substrate to obtain 20% suspension, heating to 60-80 deg.C for gelatinization, adding alpha-amylase for liquefaction for 10-30min, and controlling DE value to 3-7;
(2) cooling to 30-45 ℃, adjusting the pH value to 5.0-7.5, simultaneously adding 10-50U/g substrate of pullulanase and 4000U/g substrate of 4, 6-alpha-glucosyltransferase, putting into a constant-temperature water bath shaker, and reacting for 24h at 37 ℃;
(3) heating to 95 ℃ to inactivate enzyme, adding thermostable alpha-amylase until the acting dose of the enzyme is not less than 1000U/g substrate, placing in 95 ℃ water bath for continuous oscillation, and reacting for 30 min;
(4) taking out the reactant, cooling to room temperature, adjusting pH to 4.0-5.0, adding amyloglucosidase until the action dosage of the enzyme is not less than 660U/g substrate, placing in a water bath shaking table at 60 ℃ and continuously oscillating for reaction for 30 min;
(5) after the reaction is finished, inactivating enzyme at 95 ℃ for 20min, adding 10g/L of food-grade dry yeast powder for digestion, putting the mixture into a yeast shaker, reacting for 12h at 30 ℃ and 200rpm to purify the product and remove micromolecular sugar such as glucose, maltose and the like in the system;
(6) centrifuging the reaction product at 8000rpm for 20min, collecting supernatant, filtering with 0.45 μm filter membrane, and spray drying to obtain light yellow powder.
Because the addition of the amyloglucosidase in the reaction product after the enzyme reaction is less than 660U/g substrate (66U/g substrate is actually added), part of alpha-1, 4 glycosidic bonds and alpha-1, 6 glycosidic bonds in the conversion product are not completely hydrolyzed by the glucoamylase, so that the content of dietary fibers is higher than 75.32 percent, and the actual yield of the conversion product is 49.91 percent after the amyloglucosidase is treated according to the national standard GB/T22224-2008 'determination of dietary fibers in food-enzyme gravimetric method'.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> a process for producing alpha-1, 3-glycosidically modified low calorie dextrins
<130> BAA200815A
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 1282
<212> PRT
<213> Leuconostoc citreum
<400> 1
Met Ala Asp Thr Gln Thr Pro Val Gly Thr Thr Gln Ser Gln Gln Asp
1 5 10 15
Leu Thr Gly Gln Thr Gly Gln Asp Lys Pro Thr Thr Lys Glu Val Ile
20 25 30
Asp Lys Lys Glu Pro Val Pro Gln Val Ser Ala Gln Asn Val Gly Asp
35 40 45
Leu Ser Ala Asp Ala Lys Thr Pro Lys Ala Asp Asp Lys Gln Asp Thr
50 55 60
Gln Pro Thr Asn Ala Gln Leu Pro Asp Gln Gly Asn Lys Gln Thr Asn
65 70 75 80
Ser Asn Ser Asp Lys Gly Val Lys Glu Ser Thr Thr Ala Pro Val Lys
85 90 95
Thr Thr Asp Val Pro Ser Lys Ser Val Ala Pro Glu Thr Asn Thr Ser
100 105 110
Ile Asn Gly Gly Gln Tyr Val Glu Lys Asp Gly Gln Phe Val Tyr Ile
115 120 125
Asp Gln Ser Gly Lys Gln Val Ser Gly Leu Gln Asn Ile Glu Gly His
130 135 140
Thr Gln Tyr Phe Asp Pro Lys Thr Gly Tyr Gln Thr Lys Gly Glu Leu
145 150 155 160
Lys Asn Ile Asp Asp Asn Ala Tyr Tyr Phe Asp Lys Asn Ser Gly Asn
165 170 175
Gly Arg Thr Phe Thr Lys Ile Ser Asn Gly Ser Tyr Ser Glu Lys Asp
180 185 190
Gly Met Trp Gln Tyr Val Asp Ser His Asp Lys Gln Pro Val Lys Gly
195 200 205
Leu Tyr Asp Val Glu Gly Asn Leu Gln Tyr Phe Asp Leu Ser Thr Gly
210 215 220
Asn Gln Ala Lys His Gln Ile Arg Ser Val Asp Gly Val Thr Tyr Tyr
225 230 235 240
Phe Asp Ala Asp Ser Gly Asn Ala Thr Ala Phe Lys Ala Val Thr Asn
245 250 255
Gly Arg Tyr Ala Glu Gln Thr Thr Lys Asp Lys Asp Gly Asn Glu Thr
260 265 270
Ser Tyr Trp Ala Tyr Leu Asp Asn Gln Gly Asn Ala Ile Lys Gly Leu
275 280 285
Asn Asp Val Asn Gly Glu Ile Gln Tyr Phe Asp Glu His Thr Gly Glu
290 295 300
Gln Leu Lys Gly His Thr Ala Thr Val Asp Gly Thr Thr Tyr Tyr Phe
305 310 315 320
Glu Gly Asn Lys Gly Asn Leu Val Ser Val Val Asn Thr Ala Pro Thr
325 330 335
Gly Gln Tyr Lys Ile Asn Gly Asp Asn Val Tyr Tyr Leu Asp Asn Asn
340 345 350
Asn Glu Ala Ile Lys Gly Leu Tyr Gly Ile Asn Gly Asn Leu Asn Tyr
355 360 365
Phe Asp Leu Ala Thr Gly Ile Gln Leu Lys Gly Gln Ala Lys Asn Ile
370 375 380
Asp Gly Ile Gly Tyr Tyr Phe Asp Gln Asn Asn Gly Asn Gly Glu Tyr
385 390 395 400
Arg Tyr Ser Leu Thr Gly Pro Val Val Lys Asp Val Tyr Ser Gln His
405 410 415
Asn Ala Val Asn Asn Leu Ser Ala Asn Asn Phe Lys Asn Leu Val Asp
420 425 430
Gly Phe Leu Thr Ala Glu Thr Trp Tyr Arg Pro Ala Gln Ile Leu Ser
435 440 445
His Gly Thr Asp Trp Val Ala Ser Thr Asp Lys Asp Phe Arg Pro Leu
450 455 460
Ile Thr Val Trp Trp Pro Asn Lys Asp Ile Gln Val Asn Tyr Leu Lys
465 470 475 480
Leu Met Gln Gln Ile Gly Ile Leu Asp Asn Ser Val Val Phe Asp Thr
485 490 495
Asn Asn Asp Gln Leu Val Leu Asn Lys Gly Ala Glu Ser Ala Gln Ile
500 505 510
Gly Ile Glu Lys Lys Val Ser Glu Thr Gly Asn Thr Asp Trp Leu Asn
515 520 525
Glu Leu Leu Phe Ala Pro Asn Gly Asn Gln Pro Ser Phe Ile Lys Gln
530 535 540
Gln Tyr Leu Trp Asn Val Asp Ser Glu Tyr Pro Gly Gly Trp Phe Gln
545 550 555 560
Gly Gly Tyr Leu Ala Tyr Gln Asn Ser Asp Leu Thr Pro Tyr Ala Asn
565 570 575
Thr Asn Pro Asp Tyr Arg Thr His Asn Gly Leu Glu Phe Leu Leu Ala
580 585 590
Asn Asp Val Asp Asn Ser Asn Pro Val Val Gln Ala Glu Gln Leu Asn
595 600 605
Trp Leu Tyr Tyr Leu Met Asn Phe Gly Gln Ile Thr Ala Asn Asp Ser
610 615 620
Asn Ala Asn Phe Asp Ser Met Arg Ile Asp Ala Ile Ser Phe Val Asp
625 630 635 640
Pro Gln Ile Ala Lys Lys Ala Tyr Asp Leu Leu Asp Lys Met Tyr Gly
645 650 655
Leu Thr Asp Asn Glu Ala Val Ala Asn Gln His Ile Ser Ile Val Glu
660 665 670
Ala Pro Lys Gly Glu Thr Pro Ile Thr Val Glu Lys Gln Ser Ala Leu
675 680 685
Val Glu Ser Asn Trp Arg Asp Arg Met Lys Gln Ser Leu Ser Lys Asn
690 695 700
Ala Thr Leu Asp Lys Leu Asp Pro Asp Pro Ala Ile Asn Ser Leu Glu
705 710 715 720
Lys Leu Val Ala Asp Asp Leu Val Asn Arg Ser Gln Ser Ser Asp Lys
725 730 735
Asp Ser Ser Thr Ile Pro Asn Tyr Ser Ile Val His Ala His Asp Lys
740 745 750
Asp Ile Gln Asp Thr Val Ile His Ile Met Lys Ile Val Asn Asn Asn
755 760 765
Pro Asn Ile Ser Met Ser Asp Phe Thr Met Gln Gln Leu Gln Asn Gly
770 775 780
Leu Lys Ala Phe Tyr Glu Asp Gln His Gln Ser Val Lys Lys Tyr Asn
785 790 795 800
Gln Tyr Asn Ile Pro Ser Ala Tyr Ala Leu Leu Leu Thr Asn Lys Asp
805 810 815
Thr Val Pro Arg Val Phe Tyr Gly Asp Met Tyr Gln Asp Tyr Gly Asp
820 825 830
Asp Leu Asp Gly Gly Gln Tyr Met Ala Thr Lys Ser Ile Tyr Tyr Asn
835 840 845
Ala Ile Glu Gln Met Met Lys Ala Arg Leu Lys Tyr Val Ala Gly Gly
850 855 860
Gln Ile Met Ala Val Thr Lys Ile Lys Asn Asp Gly Ile Asn Lys Asp
865 870 875 880
Gly Thr Asn Lys Ser Gly Glu Val Leu Thr Ser Val Arg Phe Gly Lys
885 890 895
Asp Ile Met Asp Ala Gln Gly Gln Gly Thr Ala Glu Ser Arg Asn Gln
900 905 910
Gly Ile Gly Val Ile Val Ser Asn Ser Ser Gly Leu Glu Leu Lys Asn
915 920 925
Ser Asp Ser Ile Thr Leu His Met Gly Ile Ala His Lys Asn Gln Ala
930 935 940
Tyr Arg Ala Leu Met Leu Thr Asn Asp Lys Gly Ile Val Asn Tyr Asp
945 950 955 960
Gln Asp Asn Asn Ala Pro Ile Ala Trp Thr Asn Asp His Gly Asp Leu
965 970 975
Ile Phe Thr Asn Gln Met Ile Asn Gly Gln Ser Asp Thr Ala Val Lys
980 985 990
Gly Tyr Leu Asn Pro Glu Val Ala Gly Tyr Leu Ala Val Trp Val Pro
995 1000 1005
Val Gly Ala Asn Asp Asn Gln Asp Ala Arg Thr Val Thr Thr Asn
1010 1015 1020
Gln Lys Asn Thr Asp Gly Lys Val Leu His Thr Asn Ala Ala Leu
1025 1030 1035
Asp Ser Lys Leu Met Tyr Glu Gly Phe Ser Asn Phe Gln Lys Met
1040 1045 1050
Pro Thr Arg Gly Asn Gln Tyr Ala Asn Val Val Ile Thr Lys Asn
1055 1060 1065
Ile Asp Leu Phe Lys Ser Trp Gly Ile Thr Asp Phe Glu Leu Ala
1070 1075 1080
Pro Gln Tyr Arg Ser Ser Asp Gly Lys Asp Ile Thr Asp Arg Phe
1085 1090 1095
Leu Asp Ser Ile Val Gln Asn Gly Tyr Gly Leu Ser Asp Arg Tyr
1100 1105 1110
Asp Leu Gly Phe Lys Thr Pro Thr Lys Tyr Gly Thr Asp Gln Asp
1115 1120 1125
Leu Arg Lys Ala Ile Glu Arg Leu His Gln Ala Gly Met Ser Val
1130 1135 1140
Met Ala Asp Phe Val Ala Asn Gln Ile Tyr Gly Leu His Ala Asp
1145 1150 1155
Lys Glu Val Val Ser Ala Gln His Val Asn Ile Asn Gly Asp Thr
1160 1165 1170
Lys Leu Val Val Asp Pro Arg Tyr Gly Thr Gln Met Thr Val Val
1175 1180 1185
Asn Ser Val Gly Gly Gly Asp Tyr Gln Ala Lys Tyr Gly Gly Glu
1190 1195 1200
Tyr Leu Asp Thr Ile Ser Lys Leu Tyr Pro Gly Leu Leu Leu Asp
1205 1210 1215
Ser Asn Gly Gln Lys Ile Asp Leu Ser Thr Lys Ile Lys Glu Trp
1220 1225 1230
Ser Ala Lys Tyr Leu Asn Gly Ser Asn Ile Pro Gln Val Gly Met
1235 1240 1245
Gly Tyr Val Leu Lys Asp Trp Asn Asn Gly Gln Tyr Phe His Ile
1250 1255 1260
Leu Asp Lys Glu Gly Gln Tyr Ser Leu Pro Thr Gln Leu His His
1265 1270 1275
His His His His
1280
<210> 2
<211> 3858
<212> DNA
<213> Artificial sequence
<400> 2
catatggctg atacacagac gcccgtaggg acaactcagt cccaacagga ccttactggg 60
cagacaggtc aggataaacc aactactaaa gaggtcatcg acaagaagga gcctgtacct 120
caggtatctg cacagaacgt gggcgatctg tcggccgatg ccaagacgcc caaggctgac 180
gacaagcagg atacccagcc cactaacgcg caattgccgg atcaaggtaa caagcaaaca 240
aactcgaact cagataaagg cgttaaggaa tctactacgg cccccgtgaa gacgactgat 300
gttccatcca agagtgtcgc cccggagacg aacacgtcca ttaatggtgg tcaatatgtc 360
gagaaagacg ggcagttcgt gtacattgat caatctggca agcaagtttc aggacttcag 420
aacattgagg gtcacacgca atatttcgat cccaaaacgg ggtatcagac aaagggagag 480
ttaaagaata ttgacgacaa cgcgtactat tttgacaaga atagtgggaa tggacgcaca 540
tttacaaaaa ttagtaacgg ttcgtactca gagaaagacg gcatgtggca gtatgttgat 600
tcgcacgaca aacagcccgt taagggttta tatgatgtag agggaaattt acaatacttc 660
gatcttagta ccggaaatca agcaaagcat caaattcgct ctgtcgatgg cgtcacttac 720
tacttcgacg ccgattcagg taacgcaacg gctttcaaag ctgtgacgaa cggacgttac 780
gctgagcaaa ccactaagga taaagatggt aacgagacct cgtactgggc gtatcttgat 840
aaccaaggga acgcgatcaa gggcttgaac gatgttaatg gggagatcca atacttcgat 900
gaacatacag gtgaacagtt gaaaggccac accgctacag tagatggcac cacttattac 960
tttgagggaa ataaagggaa cctggtctct gttgtcaata ctgctccgac aggacagtat 1020
aaaatcaatg gggataacgt ctactactta gacaacaaca atgaagccat caagggtctg 1080
tacggaatca acggtaatct gaactacttt gatttggcaa ccggtattca attgaaggga 1140
caggccaaaa acatcgatgg catcggatac tacttcgatc aaaacaatgg gaatggagag 1200
tatcgttact cgctgaccgg acctgtcgta aaagacgtgt acagccaaca taatgccgtg 1260
aacaacttat cggcaaacaa cttcaagaat ttagttgacg gctttttgac ggcggagaca 1320
tggtaccgcc ccgcgcaaat cttatctcat ggaacggatt gggtggcttc gactgacaag 1380
gacttccgcc cgttaattac cgtgtggtgg cctaataagg acatccaagt taattatctg 1440
aagctgatgc aacaaatcgg tattcttgat aacagtgttg tattcgacac caataacgat 1500
caattggtat taaacaaggg agccgagtca gcacaaatcg gaattgagaa gaaggtttca 1560
gagactggca acaccgactg gcttaatgaa ttgttatttg ctccaaatgg caatcaaccg 1620
tcattcatca aacagcaata tctttggaac gtcgatagtg agtatcccgg aggttggttt 1680
caggggggat accttgccta tcaaaattca gacctgacgc cgtatgctaa tactaatcca 1740
gactaccgca cgcacaacgg gctggagttc ttattggcaa acgacgttga taactcaaac 1800
ccggtcgtgc aggcagaaca gttaaattgg ctgtactact tgatgaattt cggccaaatc 1860
acggccaatg attcgaatgc aaactttgac tctatgcgca tcgatgctat ttccttcgtt 1920
gatccacaga tcgcaaaaaa agcgtacgat cttcttgata agatgtacgg acttaccgac 1980
aatgaggcgg ttgctaacca acacatttct attgtagaag ctccaaaggg ggagactcca 2040
atcacggtcg aaaagcaatc ggcactggtt gaatctaatt ggcgcgatcg tatgaaacag 2100
tccttgagca agaacgctac tttggataag ctggacccgg acccggccat taactcactg 2160
gaaaaattag tcgccgatga cttagttaac cgctctcaat catccgataa agactcctcc 2220
accatcccta attattctat tgtccacgct cacgataagg acatccaaga tacggttatc 2280
catattatga agattgtgaa taataacccg aacatcagca tgagcgactt cacaatgcaa 2340
cagctgcaga atggacttaa agcgttttat gaggatcaac atcaatctgt caagaaatat 2400
aatcaatata atatcccgtc ggcgtacgcc ctgttactga ctaataagga cacggtgcct 2460
cgcgtctttt atggtgacat gtaccaagat tacggggacg atcttgatgg cgggcaatat 2520
atggctacga agtccatcta ttataatgct atcgaacaga tgatgaaagc ccgtttaaag 2580
tatgttgccg gtggacagat catggcagtg acaaaaatta agaatgatgg cattaataag 2640
gacggcacca ataagtctgg agaggtatta acctcggttc gctttggcaa ggatattatg 2700
gacgcgcagg gacagggaac agcagaaagc cgcaatcagg gaatcggcgt tatcgtatcc 2760
aattccagcg gcctggaact taaaaatagt gactcgatca cacttcacat gggaatcgct 2820
cacaagaatc aagcctatcg cgcattaatg cttacaaacg acaaaggcat tgtgaactac 2880
gaccaagaca ataatgctcc cattgcttgg acgaatgacc acggggacct gattttcaca 2940
aaccagatga tcaatggaca atcagacact gcagtcaaag gctacttaaa cccggaagtt 3000
gccgggtacc tggctgtgtg ggttccagtt ggggctaacg acaaccaaga cgcgcgtaca 3060
gtgacgacga accaaaagaa tacagatgga aaagtgctgc atacgaacgc cgcacttgac 3120
tctaaactta tgtacgaggg tttttctaat ttccaaaaaa tgcctacacg tggtaatcag 3180
tacgcgaacg tggtaatcac taaaaacatc gatctgttta aaagctgggg aatcactgac 3240
tttgaacttg cgccgcaata ccgttcctca gacggcaaag acatcactga tcgttttctt 3300
gattcgattg tacaaaacgg ttatggtctt tcagaccgtt acgacttggg ctttaaaacg 3360
ccgacaaaat atggcacgga tcaagattta cgcaaggcca tcgagcgtct tcatcaagca 3420
ggcatgtcag taatggcgga ttttgtcgcc aatcagattt atggactgca tgcggacaaa 3480
gaagtcgtat ccgctcagca tgtgaatatc aatggggata cgaaacttgt tgttgatccg 3540
cgctacggca cacaaatgac cgttgttaat tctgtaggtg gtggtgatta ccaagcgaag 3600
tacggcggtg agtacttgga cacgatctct aaactttatc caggcttatt acttgattcc 3660
aacgggcaga aaattgactt aagcaccaag attaaggaat ggagcgcgaa atatttaaat 3720
gggtctaata ttccgcaagt agggatgggg tatgtcctga aagattggaa caacgggcaa 3780
tatttccata tcttggacaa ggagggtcag tattcgttgc ctacccagct gcaccaccat 3840
caccaccatt aagaattc 3858

Claims (10)

1. A method for preparing low calorie dextrin, characterized in that 4, 6-alpha-glucosyltransferase and branched sucrase are used to convert starch to form low calorie dextrin.
2. The method of claim 1, wherein said 4, 6-alpha-glucosyltransferase is derived from Lactobacillus fermentum (Lactobacillus fermentum); the branched sucrase is derived from Leuconostoc citreum (Leuconostoc ccitreum) NRRL B-742.
3. The method according to claim 2, wherein the branched sucrase is added in an amount of 0.5 to 5U/mL in the reaction system.
4. The method as claimed in claim 3, wherein the 4,6- α -glucosyltransferase is added to the reaction system in an amount of 2500-.
5. The method of claim 4, wherein the starch is a cereal starch, or a potato starch, a starch derivative, or a starch-containing material.
6. The method according to claim 5, wherein the starch is prepared into a suspension, and the suspension is gelatinized and liquefied to prepare a reaction solution; catalyzing reaction liquid by using pullulanase and 4, 6-alpha-glucosyltransferase; adding sucrose and branched sucrase after the catalysis is finished, and reacting for 20-30h at 35-40 ℃; after the reaction is finished, inactivating enzyme in the reaction liquid, adding alpha-amylase for reaction for 20-40min, adding amyloglucosidase for reaction for 20-40 min; digesting the prepared reaction solution, filtering and drying to obtain a finished product.
7. The method according to claim 6, wherein the final concentration of sucrose in the reaction system is 2-40 g/100 mL.
8. The method of claim 7, wherein the starch is present in a suspension of 10% to 30%.
9. A resistant dextrin prepared by the process of any one of claims 1 to 8.
10. Use of the method according to any one of claims 1 to 8, or the resistant dextrin according to claim 9 in the food or cosmetic field.
CN202011475893.7A 2020-12-15 2020-12-15 Method for preparing alpha-1, 3 glycosidic bond modified low-calorie dextrin Pending CN112553270A (en)

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CN112708649A (en) * 2021-02-06 2021-04-27 江南大学 Method for producing isomaltooligosaccharide by multi-enzyme coupling
CN115053965A (en) * 2022-07-11 2022-09-16 山东省食品发酵工业研究设计院 Method for preparing resistant dextrin with higher indigestibility by using ultrahigh pressure treatment
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Publication number Priority date Publication date Assignee Title
CN112708649A (en) * 2021-02-06 2021-04-27 江南大学 Method for producing isomaltooligosaccharide by multi-enzyme coupling
CN115067392A (en) * 2022-06-14 2022-09-20 江南大学 Enzymatic preparation method of low-calorie high-protein oat beverage
CN115053965A (en) * 2022-07-11 2022-09-16 山东省食品发酵工业研究设计院 Method for preparing resistant dextrin with higher indigestibility by using ultrahigh pressure treatment
CN115053965B (en) * 2022-07-11 2023-09-08 山东省食品发酵工业研究设计院 Method for preparing resistant dextrin with stronger indigestibility by utilizing ultrahigh pressure treatment

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