CN114381482A - Preparation method of waxy wheat resistant starch - Google Patents
Preparation method of waxy wheat resistant starch Download PDFInfo
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- CN114381482A CN114381482A CN202111651682.9A CN202111651682A CN114381482A CN 114381482 A CN114381482 A CN 114381482A CN 202111651682 A CN202111651682 A CN 202111651682A CN 114381482 A CN114381482 A CN 114381482A
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/16—Preparation of compounds containing saccharide radicals produced by the action of an alpha-1, 6-glucosidase, e.g. amylose, debranched amylopectin
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Abstract
The invention belongs to the technical field of starch processing, and particularly relates to a preparation method of waxy wheat resistant starch. According to the method, waxy wheat starch and high-amylose corn starch are used as substrates, pullulanase is used for debranching, and branching enzyme is used for high-branching treatment, so that waxy wheat resistant starch is obtained. The invention adopts a complex enzyme technology of synergistic action of pullulanase and branching enzyme, obviously reduces the blood sugar value of waxy wheat starch by means of eliminating steric hindrance of waxy wheat branched chains and then linking long side chains to improve the uniformity of the chain length, and improves the application value of the waxy wheat starch. The method has the advantages of simple and convenient operation, high feasibility, no toxicity, no harm, safety, no pollution and low production cost.
Description
Technical Field
The invention belongs to the technical field of starch processing, and particularly relates to a preparation method of waxy wheat resistant starch.
Background
Wheat is a traditional staple food of Chinese people and plays an important role in diet, wherein starch is a main energy substance of human beings, and the hydrolysis rate of the starch determines the formation rate of absorbable sugar, so that the postprandial blood sugar level of the body is directly influenced. According to the release rate of glucose and its absorption in the gastrointestinal tract, Englyst et al have classified starch into fast-digestible, slow-digestible and resistant starches.
The waxy wheat can improve the quality and the taste of the noodles, prolong the retention time of the bread on a shelf, improve the taste and have great market potential. However, waxy wheat starch has poor enzymolysis resistance due to its low amylose content (less than 1%), and has a rapid increase in blood sugar after eating, which is not good for human health. With the increasing incidence of obesity and diet-related diseases, starch digestibility has become a nutritional concern. The modification technology of the biological enzyme method is one of starch modification means, and has the advantages of high reaction efficiency, environmental protection and the like, so that the modification technology becomes a hot point in recent research. How to prepare waxy wheat resistant starch by using a biological enzyme method is also provided.
Disclosure of Invention
The invention aims to provide a preparation method of waxy wheat resistant starch, and the prepared modified starch has higher enzymolysis resistance and can obviously reduce the blood sugar value of the waxy wheat starch.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a waxy wheat resistant starch is prepared from waxy wheat starch and high-amylose corn starch through debranching with pullulanase, and high-branching with branching enzyme.
Further, adding water into a substrate to prepare starch milk, and then pre-gelatinizing; adding pullulanase for reaction, and inactivating enzyme to obtain enzymatic hydrolysate A; then adding branching enzyme for reaction and inactivating enzyme to obtain enzymolysis liquid B; and washing and separating the enzymolysis liquid B to obtain the waxy wheat resistant starch.
Preferably, the mass ratio of the waxy wheat starch to the high amylose corn starch in the substrate is 1:0.5-1: 0.3.
The amylose content of the high amylose corn starch is 70%.
The mass concentration of the starch milk is 4-8%, the pH of the starch milk is adjusted to 4.4-4.6, and then the starch milk is heated in a boiling water bath and stirred for 30-45 minutes for pre-gelatinization to obtain starch gelatinization liquid.
The adding amount of the pullulanase is 4-6% of the mass of the substrate, and the reaction is carried out for 4-6 hours at 50-55 ℃.
Cooling the enzymolysis liquid A to room temperature, then adjusting the pH value to 6.3-6.6, and then adding the branching enzyme to react at 58-62 ℃ for 24-30 hours.
The amount of branching enzyme added is 2-4ml branching enzyme per 100g substrate.
The step of washing and separating the enzymolysis liquid B to obtain waxy wheat resistant starch can be carried out as follows but is not limited to the following steps: and centrifuging the enzymatic hydrolysate B for 10min under the condition of 4000r/min to obtain a precipitate, adding 3 times of ethanol with the volume concentration of 50% into the precipitate, centrifuging for 10min under the condition of 4000r/min to obtain the precipitate, recording as one-time alcohol washing, performing alcohol washing for 2 times in total, then adding 3 times of deionized water into the precipitate, centrifuging for 10min under the condition of 4000r/min to obtain the precipitate, recording as one-time water washing, performing water washing for 2 times in total, performing sample purification, obtaining a reactant, freeze-drying, crushing, and sieving with a 100-mesh sieve to obtain the modified starch.
Preferably, the preparation method of the waxy wheat resistant starch comprises the following steps:
(1) mixing waxy wheat starch and high amylose corn starch according to the proportion of 1:0.5 to be used as a substrate;
(2) adding pure water to prepare starch milk with the concentration of 4% (w/w), adjusting the pH of the starch milk to 4.5, and heating and stirring in a boiling water bath for 30 minutes for pre-gelatinization;
(3) cooling to room temperature, adding pullulanase into the starch pasting liquid, reacting for 4 hours, heating in a boiling water bath to inactivate enzyme to obtain enzymatic hydrolysate A;
(4) cooling the enzymolysis solution A to room temperature, adjusting the pH to 6.5, adding branching enzyme, reacting for 24 hours, heating in boiling water to inactivate enzyme to obtain enzymolysis solution B;
(5) cooling the enzymolysis liquid B to room temperature, and sequentially carrying out alcohol washing (or alcohol precipitation) twice and water washing twice on the enzymolysis liquid; freeze drying the precipitate, crushing and sieving to obtain the modified starch.
The invention provides a preparation method of waxy wheat resistant starch, which takes waxy wheat starch and high amylose corn starch as substrates and utilizes the synergistic effect of pullulanase and branching enzyme to improve the chain length uniformity of the starch. The high amylose corn starch serves as a donor, long side chains are provided for the waxy wheat starch, and short side chains A-chains (DP 6-12) and B1-chains (DP 13-24) are hydrolyzed through debranching action of pullulanase, so that steric hindrance between waxy wheat starch branches is eliminated, and long chain generation is facilitated. Branching enzymes act mainly on long chains of high-amylose corn starch to transfer glycosyl residues to the same or different linear or branched molecules, thereby increasing long-side chains B2chains (DP 25-36) and B3 chains (DP >36), improving the uniformity of the chain length of the side chains, enhancing the enzymolysis resistance of the starch, and predicting that the blood glucose level (pGI) is reduced.
Pullulanase (Pullulanase) is a class of starch debranching enzymes that specifically cleaves the alpha-1, 6-glycosidic bond of an amylose branch point and acts on the smallest unit of branching, for example, by hydrolysing a side branch consisting of 2-3 glucose residues. Starch Branching enzymes (Branching enzymes) catalyze the cleavage of alpha-1, 4-glucosidic bonds in starch molecules and link the resulting glucose units to the receptor chains via alpha-1, 6-glucosidic bonds, which are more resistant to hydrolysis by digestive enzymes than alpha-1, 4-glucosidic bonds, thus increasing the content of slowly digestible starch and further improving the resistance to enzymatic hydrolysis. Therefore, the two enzymes act on the starch synergistically, pullulanase hydrolyzes shorter side chains, and the rear branching enzyme transfers the middle and long side chains of the donor to the starch chain of the acceptor, so that the number of alpha-1, 6-glucosidic bonds of the starch is increased, the enzymolysis resistance of the starch is enhanced, the average chain length can be prolonged, the uniformity of the chain length of side chains is improved, and the modified starch has nutrition and application functions.
Compared with the prior art, the invention has the following advantages:
the invention adopts a complex enzyme technology of synergistic action of pullulanase and branching enzyme, obviously reduces the blood sugar value of waxy wheat starch by means of eliminating steric hindrance of waxy wheat branched chains and then linking long side chains to improve the uniformity of the chain length, and improves the application value of the waxy wheat starch. The waxy wheat can improve the quality and the taste of the noodles, prolong the retention time of the bread on a shelf, improve the taste, have great market potential, and simultaneously use the high amylose corn starch as a donor, thereby further widening the application fields of the waxy wheat and the high amylose corn. The method has the advantages of simple and convenient operation, high feasibility, no toxicity, no harm, safety, no pollution and low production cost.
Drawings
FIG. 1 is a graph showing the change in glucose content during the enzymatic hydrolysis of the examples and untreated samples;
FIG. 2 is a graph showing the change in glucose content during the enzymatic hydrolysis in examples and comparative example 1;
FIG. 3 is a graph showing the change in glucose content during the enzymatic hydrolysis in examples and comparative example 2;
FIG. 4 is a graph showing the change in glucose content during the enzymatic hydrolysis in examples and comparative example 3;
FIG. 5 is a graph showing the change in glucose content during enzymatic hydrolysis in examples and comparative example 4.
Detailed Description
The technical solution of the present invention is illustrated by the following specific examples, but the scope of the present invention is not limited thereto:
the pullulanase adopted in the following examples has the enzyme activity of 1000U/g, and is a product of Shanghai Michelin Biochemical technology Co., Ltd; the enzyme activity of the adopted branching enzyme is 660U/mL, and the product is a product of Denmark Novoxil. The enzyme activity of the alpha-amylase adopted in the comparative example is 40000U/mL, the enzyme activity of the beta-amylase is 102000U/g, and the two are both products of Beijing Soilebao company.
Example 1
A preparation method of waxy wheat resistant starch comprises the following steps:
weighing a mixture (mass ratio is 1:0.5) of waxy wheat starch and high amylose corn starch as a substrate, adding distilled water to prepare 4% (mass concentration) starch milk, adjusting the pH of the starch milk to about 4.5 by using 2mol/L sodium hydroxide and hydrochloric acid, heating in a boiling water bath, stirring for 30min, pre-gelatinizing, and then rapidly cooling. Cooling to room temperature, adding pullulanase (1000U/g) with the mass of 6% of that of the substrate into the starch pasting liquid, reacting for 4 hours at 53 ℃, heating in a boiling water bath for 10 minutes to inactivate enzyme, and obtaining enzymolysis liquid A. Cooling the enzymolysis solution A to room temperature, adjusting pH to 6.5, adding 2% (v/w) (adding 2ml of substrate 100 g) of branching enzyme into the enzymolysis solution A, reacting for 24 hours at 60 ℃, heating in boiling water bath for 10 minutes to inactivate enzyme, and obtaining enzymolysis solution B.
And (3) washing and separating the enzymolysis liquid B: centrifuging for 10min under the condition of 4000r/min to obtain a precipitate, adding 50% ethanol (v/v) with the volume being 3 times of that of the precipitate into the precipitate, centrifuging for 10min under the condition of 4000r/min to obtain the precipitate, recording as one-time alcohol washing, carrying out 2-time alcohol washing, then adding deionized water with the volume being 3 times of that of the precipitate, centrifuging for 10min under the condition of 4000r/min to obtain the precipitate, recording as one-time water washing, carrying out 2-time water washing, carrying out sample purification, carrying out freeze drying on the obtained reactant, crushing and sieving by a 100-mesh sieve to obtain the waxy wheat resistant starch.
To fully compare the resistance to enzymatic hydrolysis of the examples with that of the untreated sample, untreated waxy wheat starch was used as untreated sample 1, and a mixture of untreated waxy wheat starch and high amylose corn starch (mass ratio 1:0.5) was used as untreated sample 2.
In vitro digestion simulation experiments were carried out on the waxy wheat resistant starch obtained in the examples and the untreated sample 1 and the untreated sample 2, the change of the glucose content during digestion is shown in fig. 1, the predicted blood glucose value (pGI) of the samples in the examples is 34.01 ± 0.86, and pGI of the untreated sample 1 and the untreated sample 2 is 72.99 ± 2.48 and 61.22 ± 2.54, respectively.
Comparative example 1: substrate treated with pullulanase only
Weighing a mixture (mass ratio is 1:0.5) of waxy wheat starch and high amylose corn starch as a substrate, adding distilled water to prepare 4% starch milk, adjusting the pH of the starch milk to about 4.5 by using 2mol/L sodium hydroxide and hydrochloric acid, heating in a boiling water bath for 30min, stirring, pre-gelatinizing, and rapidly cooling to room temperature. 6% pullulanase (1000U/g) was added based on the mass of the substrate and stirred in a water bath at 53 ℃ for 4 h. After the reaction is finished, enzyme is inactivated in boiling water bath for 10 min. Obtaining enzymolysis liquid, and carrying out washing separation and other treatments on the enzymolysis liquid B in the embodiment to obtain the pullulanase modified starch.
In vitro digestion simulation measurements were carried out under the same conditions as in the samples of examples, and the change in glucose content during digestion is shown in FIG. 2, and the predicted blood glucose level (pGI) of comparative example 1 was 39.05. + -. 1.72.
Comparative example 2: substrate branching enzyme only treatment
Weighing a physical mixture (mass ratio is 1:0.5) of waxy wheat starch and high amylose corn starch as a substrate, adding distilled water to prepare 4% starch milk, adjusting the pH of the starch milk to about 6.5 by using 2mol/L sodium hydroxide and hydrochloric acid, heating in a boiling water bath, stirring for 30min, pre-gelatinizing, and rapidly cooling. Adding 2% (v/w) (100g of substrate is added with 2ml) of branching enzyme into the starch pasting liquid based on the mass of the substrate, reacting for 24 hours at 60 ℃, inactivating the enzyme for 10min in boiling water bath after the reaction is finished to obtain an enzymolysis liquid, and carrying out washing separation and other treatments on the enzymolysis liquid B in the embodiment on the enzymolysis liquid to obtain the branching enzyme modified starch.
In vitro digestion simulation measurements were performed under the same conditions as the samples of the examples, the change in glucose content during digestion is shown in FIG. 3, and the predicted blood glucose values (pGI) of comparative example 2 are: 44.52 + -0.24.
Comparative example 3: treating the substrate with alpha-amylase and branching enzyme
Weighing a mixture of waxy wheat starch and high amylose corn starch (mass ratio is 1:0.5) as a substrate, adding distilled water to prepare 4% starch milk, adjusting the pH of the starch milk to 6.9, and heating and stirring in a boiling water bath for 30 minutes to perform pre-gelatinization. Cooling to room temperature, adding alpha-amylase (40000U/mL) with 0.13% substrate (0.13 mL per 100g substrate) into the starch gelatinized liquid, reacting at 53 deg.C for 4 hr, heating in boiling water bath for 10min to inactivate enzyme, and obtaining enzymolysis liquid A. Cooling the enzymolysis solution A to room temperature, adjusting pH to 6.5, adding 2% (v/w) (adding 2ml of substrate 100 g) of branching enzyme into the enzymolysis solution A, reacting for 24 hours at 60 ℃, heating in boiling water bath for 10 minutes to inactivate enzyme, and obtaining the enzymolysis solution. And (3) washing and separating the enzymolysis liquid B in the embodiment and the like to obtain the alpha-amylase and branching enzyme enzymolysis products.
In vitro digestion simulation measurements were performed under the same conditions as the samples of the examples, the change in glucose content during digestion is shown in FIG. 4, and the predicted blood glucose values (pGI) of comparative example 3 are: 43.10 + -0.16.
Comparative example 4: treating the substrate with beta-amylase and branching enzyme
Weighing a mixture of waxy wheat starch and high amylose corn starch (mass ratio is 1:0.5) as a substrate, adding distilled water to prepare 4% starch milk, adjusting the pH of the starch milk to 5.2, and heating and stirring in a boiling water bath for 30 minutes to perform pre-gelatinization. Cooling to room temperature, adding beta-amylase (102000U/g) with the mass of 0.08 percent of that of the substrate into the starch pasting liquid, reacting for 4 hours at 53 ℃, heating in boiling water bath for 10 minutes to inactivate the enzyme, and obtaining enzymatic hydrolysate A. Cooling the enzymolysis solution A to room temperature, adjusting pH to 6.5, adding 2% (v/w) (adding 2ml of substrate 100 g) of branching enzyme into the enzymolysis solution A, reacting for 24 hours at 60 ℃, heating in boiling water bath for 10 minutes to inactivate enzyme, and obtaining the enzymolysis solution. And (3) washing and separating the enzymolysis liquid B in the embodiment and the like to obtain a beta-amylase and branching enzyme enzymolysis product.
In vitro digestion simulation measurements were performed under the same conditions as the samples of the examples, and the change in glucose content during digestion is shown in FIG. 5, and the predicted blood glucose values (pGI) of comparative example 4 are: 48.61 + -0.45.
pGI and the chain length distribution are determined by the following method:
(1) in vitro digestion simulation test determination pGI concrete procedures:
the assay of sample pGI was performed using GI20 in vitro simulated digestion system (NutraScan, australian NI limited). Weighing 50mg starch sample in a digestion tube, sequentially adding 2mL saliva alpha-amylase, reacting for 5min, adding 5mL pepsin, reacting for 30min, and finally adding 5mL pancreatin mixed solution. Wherein, the saliva alpha-amylase (250U/mL) is prepared by Sigma porcine pancreatic alpha-amylase A-3176 and 500KU type VIB in pH7 carbonic acid buffer solution; pepsin, prepared by dissolving 65mg of pepsin (Sigma P-6887, made from porcine pepsin) in 65mL of 0.02M hydrochloric acid solution; a pancreatic enzyme mixture (pH6.0) was prepared by dissolving 130mg of pancreatic enzyme (Sigma P1750 made from porcine pancreatic enzyme) and 58.8mg of Amyloglucosidase (Sigma A7420 made from 31.2U/mg of Aspergillus niger) in 120mL of 0.2M sodium acetate buffer pH 6.0.
The GI of the sample was predicted by measuring the glucose content using a glucose analyzer. pGI is calculated as follows:
wherein C is the glucose content (mg/mL) analyzed by a glucose analyzer; v is (total volume-consumed volume) (mL); a is glucose equivalent (mg) (total available carbohydrate/0.9).
(2) Chain length distribution specific operation:
weighing 5mg of starch, suspending in 5mL of water, carrying out boiling water bath for 60min, and intermittently and uniformly mixing by vortex; 50uL of sodium acetate (0.6M, pH 4.4), 10uL of NaN were added3(2% w/v) and 10uL isoamylase (1400U), incubated at 37 ℃ for 24 h. Adding 0.5% (w/v) sodium borohydride solution, mixing uniformly by vortex, and standing for 20 h. 600 μ L of the suspension was placed in a centrifuge tube and dried by nitrogen blowing at room temperature. Dissolve in 30. mu.L 1M NaOH for 60min, then add 570uL water to dilute, centrifuge at 12000rpm for 5min, and take the supernatant for sample. The assay was performed using a Thermo ICS5000 ion chromatography system (ICS500+, Thermo Fisher Scientific, USA) using DionexTM CarboPacTMPA100(250 x 4.0mm,10um) liquid chromatography column. The proportion of different starch chain lengths is calculated according to the following formula:
relative ratio of different starch chain lengths peak area/total peak area 100%.
Table 1 below specifically illustrates pGI and the chain length distribution of the samples obtained in the examples, untreated samples, and comparative examples after in vitro digestion simulation.
TABLE 1 pGI and chain Length distribution for examples and comparative examples 1-4
Claims (8)
1. A preparation method of waxy wheat resistant starch is characterized in that waxy wheat starch and high amylose corn starch are used as substrates, pullulanase is adopted for debranching, and branching enzyme is used for high branching treatment, so that the waxy wheat resistant starch is obtained.
2. The method for preparing waxy wheat resistant starch according to claim 1, wherein the substrate is added with water to prepare starch milk, followed by pregelatinization; adding pullulanase for reaction, and inactivating enzyme to obtain enzymatic hydrolysate A; then adding branching enzyme for reaction and inactivating enzyme to obtain enzymolysis liquid B; and washing and separating the enzymolysis liquid B to obtain the waxy wheat resistant starch.
3. The method of preparing waxy wheat resistant starch as claimed in claim 2 wherein the mass ratio of waxy wheat starch to high amylose corn starch in the substrate is 1:0.5 to 1: 0.3.
4. The method for preparing waxy wheat resistant starch as claimed in claim 2, wherein the mass concentration of the starch milk is 4% -8%, the pH of the starch milk is adjusted to 4.4-4.6, and then the starch milk is heated and stirred in boiling water bath for 30-45 minutes to carry out pre-gelatinization to obtain starch gelatinization liquid.
5. The method for preparing waxy wheat resistant starch according to claim 2, wherein pullulanase is added in an amount of 4% to 6% by mass of the substrate, and reacted at 50 to 55 ℃ for 4 to 6 hours.
6. The method for preparing waxy wheat resistant starch according to claim 2, wherein the enzymolysis solution a is cooled to room temperature, then the pH is adjusted to 6.3-6.6, and then the branching enzyme is added to react at 58-62 ℃ for 24-30 hours.
7. The method of preparing waxy wheat resistant starch according to claim 6 wherein the branching enzyme is added in an amount of 2-4ml per 100g of substrate.
8. The method for preparing waxy wheat resistant starch according to any one of claims 1 to 7 wherein the high amylose corn starch has an amylose content of 70%.
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