CN109022519B - Method for preparing high-purity resistant starch under low-temperature condition - Google Patents
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Abstract
The invention discloses a method for preparing high-purity resistant starch under a low-temperature condition. After starch or resistance-increasing starch is gelatinized, pepsin, low-temperature alpha-amylase and saccharifying enzyme are sequentially subjected to enzymolysis, and micromolecular sugar is washed off, so that high-purity resistant starch is finally obtained. The method can prepare the resistant starch product with the resistant starch purity of more than 70 percent (up to 88.05 +/-0.45 percent) under the condition of low energy consumption.
Description
Technical Field
The invention belongs to the technical field of food processing, and relates to a method for preparing high-purity resistant starch.
Background
The resistant starch is not digested and degraded by enzyme liquid in gastrointestinal tract, and has similar physiological function as dietary fiber. The existing research results show that the resistant starch can be fermented and degraded by intestinal microorganisms in rectum, has a regulating effect on the intestinal microbial flora structure, further influences the intestinal hormone level and the physiological functions of pituitary, gonad and pancreas, and plays roles in reducing blood sugar, blood fat and the like. In recent years, the number of people with hypertension, hyperglycemia and hyperlipidemia in China is increasing continuously, and the number of diabetes in China is nearly 2 hundred million by the statistical data at the end of 2016. To control the spreading trend of diabetes in China and prevent and treat diabetes, the daily food needs to be started. The structure of the starch is adjusted by advanced process technology means, and the digestion characteristic of the starch is further controlled, so that the starch is an important direction for the development of starch products and meets the requirements of the ' healthy Chinese 2030 ' programming compendium '.
At present, the research on the preparation method of the resistant starch at home and abroad mainly focuses on adopting physical and (or) chemical related methods to increase the digestibility (resistance increase) of the resistant starch, and the purity of the obtained resistant starch product is lower. Although some methods for purifying resistant starch are reported, the methods have the problems of complex operation process, high energy consumption and the like, are not suitable for industrial production, and are only applied to laboratory research. Such as: in order to facilitate the physicochemical analysis and structural characterization of the resistant starch sample, researchers use an organic solvent for desugaring treatment, thereby improving the purity. For example, in order to facilitate the crystallinity and DSC analysis of the resistant starch sample, the obtained resistant starch sample is purified by populus forest et al, and the monosaccharide and oligosaccharide in the starch are dissolved mainly by 95% ethanol and removed by centrifugation, and the purity of the obtained resistant starch is about 30%. In order to accurately determine the resistant starch content, researchers also refer to the method for measuring the resistant starch content to carry out enzymolysis purification on the resistant starch content. For example, Gontilide and the like carry out enzymolysis on resistant starch by using various enzymes, but the method has large acid-base and enzyme consumption, needs high treatment conditions (90-95 ℃), and has long time consumption, such as: 4mol/LNaOH is needed to be dissolved and then adjusted to be neutral. Therefore, energy conservation, environmental protection and easy industrialization are the development directions of resistant starch purification technology.
Disclosure of Invention
The invention aims to provide a method for preparing high-purity resistant starch, aiming at the defects of the prior art, and the high-purity resistant starch product (the product purity can reach 88.05 +/-0.45)%) can be obtained after the starch or the resistance-increasing starch is purified at low temperature by the method.
The technical scheme of the invention is as follows:
(1) gelatinizing starch: taking a proper amount of starch or resistance-increasing starch to prepare a starch suspension with the starch mass content of 5-15%, gelatinizing at 85-95 ℃ for 15-60 min, and stirring in the process of gelatinizing until the starch is completely gelatinized.
(2) Low-temperature purification: cooling the starch paste in the step (1) to 30-45 ℃, and sequentially adding pepsin (the reaction condition is that the pH is 2-2.5, the temperature is 30-45 ℃, the time is 1-2 h, and the enzyme dosage is 5-15U/g(Dry-based starch)) And low-temperature alpha-amylase (the reaction conditions are pH6.0-7.0, the temperature is 35-45 ℃, the time is 1-3 h, and the enzyme dosage is 5-10U/g(Dry-based starch)) Saccharifying enzyme (the reaction conditions are pH4.0-4.5, the temperature is 40-60 ℃, the time is 1-3 h, and the enzyme dosage is 50-100U/g(Dry-based starch)) Carrying out enzymolysis; after the enzymolysis is finished, inactivating the enzyme at high temperature; cooling to room temperature, taking precipitate by adopting a centrifugation or filtration method, mixing the precipitate with distilled water with the volume of 4 times or ethanol with the volume of more than 85% (volume fraction), stirring for 0.5-1 h at the temperature of 20-30 ℃, and standing for 1-3 h; and (3) taking the precipitate by a centrifugation or filtration method, and washing the precipitate for 2-4 times by using distilled water or ethanol with the concentration of more than 85%.
(3) And (3) drying: and (3) taking the precipitate obtained in the step (2) by adopting a centrifugation or filtration method, drying the precipitate by adopting a freeze drying or blast drying method, crushing the dried precipitate, and grinding the crushed precipitate through a 80-120-mesh sieve to obtain the high-purity resistant starch (the purity is more than 70%).
Wherein, the starch used as the raw material in the process is extracted and separated from plants rich in starch, and the starch is used as popular consumption food, the purity is more than or equal to 80 percent, and the 80-mesh sieve passing rate is 100 percent. The resistance-increasing starch is prepared from raw starch by resistance-increasing process (such as autoclave method, enzyme-autoclave compounding method, etc.).
Wherein, the pH adjustment in the process preferably adopts 0.2mol/L citric acid and sodium citrate solution as a regulator; inorganic acid (such as hydrochloric acid), other organic acid (such as tartaric acid), sodium hydroxide and the like can also be used as regulators, but certain negative effects are caused on the color and luster and the yield of the product.
Wherein the pepsin enzyme activity is 8000-10000U/g, the low-temperature alpha-amylase enzyme activity is 1500-2000U/g, the carbohydrase enzyme activity is 100000U/mL, and the commercial enzyme preparation is diluted by a citric acid-sodium citrate buffer solution before use.
Wherein, a mode of taking sediment by adopting a centrifugal or filtering method is characterized in that the centrifugal condition is 3000-4000 r/min, and the centrifugation is 5-15 min.
The process for preparing and purifying the resistant starch provided by the invention has the following advantages:
the method can prepare the resistant starch product with the resistant starch purity of more than 70 percent (up to 88.05 +/-0.45 percent) under the condition of low energy consumption.
Compared with the existing preparation of resistant starch (see table 1), the purification process is carried out at low temperature, the operation is easy, the energy consumption is low, the efficiency is high, and the industrial popularization prospect is better. Compared with the preparation method of resistant starch (2014), the method has the advantages that the RS purity of the prepared resistant starch is slightly lower than that of the method, but the enzymolysis time is reduced by half; the method adds the steps of alcohol washing and/or water washing, can remove a large amount of micromolecule sugar generated in the enzymolysis process and soluble salt substances generated in the pH adjusting process, and effectively reduces the sugar production effect of the product.
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FIG. 1 is a process flow diagram of the present invention.
Detailed Description
The invention will be described in detail below with reference to the figures and specific examples.
Example 1 preparation of Gorgon fruit resistant starch
(1) Preparing gordon euryale seed starch: commercial dry gordon euryale seed is ground into powder and sieved by a sieve of 80 meshes, 100g of gordon euryale seed powder is taken, and the weight ratio is determined according to 1: preparing a gordon euryale seed powder suspension by using a material-water ratio of 8 (g: g); adjusting the pH value of the gordon euryale seed powder suspension to 8.5 by using 0.5mol/L NaOH; stirring for 1h at room temperature, centrifuging and taking precipitate; mixing the precipitate with NaOH solution (pH 8.5, the volume of the added NaOH solution is the same as the volume of the supernatant obtained in the previous step), stirring for 1h, centrifuging, and repeating the process for 2 times; centrifuging to obtain precipitate, and washing the precipitate with distilled water until the pH is neutral; centrifuging to obtain precipitate, oven drying at 50 deg.C to constant weight, pulverizing, and sieving with 100 mesh sieve to obtain semen euryales starch.
(2) Pasting the gordon euryale seed starch: taking a proper amount of gordon euryale seed starch, preparing 10% starch milk, gelatinizing at 90 ℃ for 30min, and continuously stirring the starch in the gelatinizing process to ensure complete gelatinization.
(3) Low-temperature purification: cooling to 40 deg.C, sequentially adding pepsin (reaction condition is pH2.0, temperature is 40 deg.C, time is 1h, enzyme dosage is 15U/g(Dry-based starch)) Low temperature alpha-amylase (reaction condition is pH6.0, temperature is 45 deg.C, time is 2h, enzyme dosage is 10U/g(Dry-based starch)) Saccharifying enzyme (reaction condition is pH4.6, temperature is 60 ℃, time is 2h, enzyme dosage is 50U/g(Dry-based starch)) Carrying out enzymolysis; inactivating enzyme in boiling water bath for 15min after enzyme action; cooling to room temperature, centrifuging to obtain precipitate, mixing the precipitate with 4 times volume of 95% (volume fraction) ethanol, stirring for 0.75h, standing at 22 deg.C for 2 h; centrifuging to obtain precipitate, washing with 95% ethanol for 3 times, and centrifuging.
(4) And (3) drying: and (4) taking the precipitate obtained by centrifugation in the step (3), drying to constant weight by adopting a forced air drying method, crushing, grinding and sieving by using a 100-mesh sieve to obtain the gordon euryale seed resistant starch.
Wherein, the centrifugation conditions in the process are all 4000r/min for 5 min.
Wherein, the pH adjustment in the process is completed by 0.2mol/L of citric acid and sodium citrate solution.
Wherein, the pepsin enzyme activity is 10000U/g, the low-temperature alpha-amylase enzyme activity is 2000U/g, the saccharifying enzyme activity is 100000U/mL, and the commercial enzyme preparation is diluted by a citric acid-sodium citrate buffer solution before use.
The purity of the gordon euryale seed resistant starch prepared by the process is (75.04 +/-0.11)%.
The method for determining the purity of the resistant starch is suitably modified by the enzyme-gravity method recommended by the American society for analytical chemistry (AOAC). The method mainly comprises the following steps: taking 0.500g of sample, uniformly dispersing the sample in 5mL of citric acid-sodium citrate buffer solution (pH6.0), and placing the solution in a water bath at 90 ℃ for 30 min; adding 500U/g(Dry-based starch)High temperature resistant alpha-amylase, heating in water bath at 90 deg.C for 30min, and stirring continuously; inactivating enzyme at 100 deg.C for 15min, and cooling to 60 deg.C; adjusting the pH value to about 4.6 by using 0.2mol/L citric acid; adding 5000U/g(Dry-based starch)Saccharifying enzyme, performing water bath reaction at 60 deg.C for 30min, inactivating enzyme at 100 deg.C for 15min, and cooling to room temperature; filtering the starch hydrolysate with Buchner funnel, and washing the precipitate with distilled water for 3 times; drying the precipitate at 80 deg.C, keeping constant weight at 105 deg.C, cooling, weighing, deducting protein and ash content, and calculating to obtain resistant starch content.
Example 2 preparation of resistant starch from Gorgon fruit
(1) Preparing the gorgon fruit anti-starch: mixing 100g of semen euryales starch with distilled water to obtain 20% (g: g) starch suspension, gelatinizing in 90 deg.C water bath for 30min, cooling to 70 deg.C, adjusting pH to 6.0 with citric acid, and adding 4U/g(Dry-based starch)Allowing medium temperature alpha-amylase to act for 25min, inactivating enzyme at high temperature, and cooling starch paste to 60 deg.C; adjusting pH of the starch paste to 4.6, adding 4U/g(Dry-based starch)The pullulanase is acted for 1.5h, and the enzyme is inactivated at high temperature. And cooling the treated starch paste at 25 ℃ for 1h, and refrigerating the cooled starch paste in a refrigerator at 4 ℃ for regeneration for 24 h. Centrifuging at 4000r/min for 5min, collecting precipitate, drying at 50 deg.C, grinding, and sieving with 100 mesh sieve to obtain semen euryales anti-starch.
(2) Pasting the starch resistance-increasing gordon euryale seed: taking a proper amount of gordon euryale seed anti-starch to prepare 10% starch milk, gelatinizing at 90 ℃ for 30min, and continuously stirring the starch in the gelatinizing process to ensure complete gelatinizing.
(3) Low-temperature purification: cooling to 30 deg.C, sequentially adding pepsin (reaction condition is pH2.0, temperature is 40 deg.C, time is 1h, enzyme dosage is 15U/g(Dry-based starch)) Low temperature alpha-amylase (reaction condition is pH6.0, temperature is 45 deg.C, time is 2h, enzyme dosage is 10U/g(Dry-based starch)) Saccharifying enzyme (reaction condition is pH4.6, temperature is 60 ℃, time is 2h, enzyme dosage is 100U/g(Dry-based starch)) Carrying out enzymolysis; inactivating enzyme in boiling water bath for 15min after enzyme action is completed; cooling to room temperature, centrifuging at 4000r/min for 10min, adding distilled water with volume 4 times of that of the precipitate, stirring for 1h, and standing at 25 ℃ for 1 h; the precipitate was taken by centrifugation at 4000r/min for 10min and washed 3 times with distilled water.
(4) And (3) drying: centrifuging at 4000r/min for 5min, centrifuging to obtain the precipitate in step (3), freeze-drying until the water content is 4%, pulverizing, and grinding with 100 mesh sieve to obtain semen euryales resistant starch.
Wherein, the pH adjustment in the process is completed by 0.2mol/L of citric acid and sodium citrate solution.
Wherein, the pepsin enzyme activity is 10000U/g, the low-temperature alpha-amylase enzyme activity is 2000U/g, the saccharifying enzyme activity is 100000U/mL, and the commercial enzyme preparation is diluted by a citric acid-sodium citrate buffer solution before use.
The purity of the gordon euryale seed resistant starch prepared by the process is (88.05 +/-0.45)%. The resistant starch purity determination method employed the enzyme-gravity method recommended by the american society for analytical chemistry (AOAC) (as shown in example 1).
TABLE 1 patent (part) of the preparation of resistant starch already disclosed
(Note: the RS purity shown in the table is expressed in terms of the resistant starch content in the product, and there is no national unified standard for determination of the resistant starch content at present, there is no method for determining the resistant starch content shown in the above patent or the determination method used is different from that in the present invention, drying, centrifugation, filtration are not considered to be time-consuming)
The above embodiments are not intended to limit the present invention, and the present invention is not limited to the above embodiments, and all embodiments are within the scope of the present invention as long as the requirements of the present invention are met.
Claims (6)
1. A method for preparing high-purity resistant starch under low-temperature conditions is characterized by comprising the following steps:
(1) gelatinizing starch: taking a proper amount of starch, preparing a starch suspension with the starch mass content of 5% -15%, gelatinizing at 85-95 ℃ for 15-60 min, and stirring in the gelatinizing process until the starch is completely gelatinized;
(2) low-temperature purification: cooling the starch paste in the step (1) to 30-45 ℃, and sequentially adding pepsin, low-temperature alpha-amylase and glucoamylase for enzymolysis; after the enzymolysis is finished, inactivating the enzyme at high temperature; cooling to room temperature, taking the precipitate, mixing the precipitate with distilled water with the volume of 4 times that of the precipitate or ethanol with the volume fraction of more than 85%, stirring at 20-30 ℃ for 0.5-1 h, and standing for 1-3 h; taking the precipitate, and washing the precipitate for 2-4 times by using distilled water or ethanol with the volume fraction of more than 85%;
the reaction conditions of the pepsin are that the pH is 2-2.5, the temperature is 30-45 ℃, the time is 1-2 hours, and the enzyme dosage is 5-15U/g;
the reaction conditions of the low-temperature alpha-amylase are that the pH value is 6.0-7.0, the temperature is 35-45 ℃, the time is 1-3 hours, and the enzyme dosage is 5-10U/g;
the reaction conditions of the saccharifying enzyme are that the pH value is 4.0-4.5, the temperature is 40-60 ℃, the time is 1-3 h, and the enzyme dosage is 50-100U/g;
(3) and (3) drying: and (3) taking the precipitate obtained in the step (2) by adopting a centrifugation or filtration method, drying, crushing, grinding and sieving by using a 80-120-mesh sieve to obtain the high-purity resistant starch.
2. The method for preparing resistant starch by purification under low temperature conditions as claimed in claim 1, wherein a solution of citric acid and sodium citrate with pH adjustment preferably of 0.2mol/L is used as the adjusting agent.
3. The method for preparing resistant starch through purification at low temperature as claimed in claim 1, wherein the pepsin enzyme activity is 8000-10000U/g, the low temperature alpha-amylase enzyme activity is 1500-2000U/g, and the saccharifying enzyme activity is 100000U/mL.
4. The method for purifying and preparing resistant starch at low temperature according to claim 1, wherein the precipitate is obtained by centrifugation or filtration, wherein the centrifugation is carried out at 3000-4000 r/min for 5-15 min.
5. The method for purifying and preparing resistant starch under low temperature conditions as claimed in claim 1, wherein the drying method is freeze drying or air drying.
6. The method for purifying and producing resistant starch under low temperature conditions of claim 1 wherein said starch is a fortified starch.
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| CN102533908A (en) * | 2012-01-04 | 2012-07-04 | 江南大学 | Method for preparing porous starch with high oil absorption rate through preprocessing starch |
| CN103992489A (en) * | 2014-03-14 | 2014-08-20 | 周忠凯 | Novel green processing method for increasing resistant starch content through crosslinking of starch and chitosan |
| CN107299125A (en) * | 2017-08-16 | 2017-10-27 | 山东百龙创园生物科技股份有限公司 | A kind of preparation method of colourless resistant starch |
| CN108018322A (en) * | 2018-01-24 | 2018-05-11 | 浙江树人学院 | A kind of preparation method of corn porous starch |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102533908A (en) * | 2012-01-04 | 2012-07-04 | 江南大学 | Method for preparing porous starch with high oil absorption rate through preprocessing starch |
| CN103992489A (en) * | 2014-03-14 | 2014-08-20 | 周忠凯 | Novel green processing method for increasing resistant starch content through crosslinking of starch and chitosan |
| CN107299125A (en) * | 2017-08-16 | 2017-10-27 | 山东百龙创园生物科技股份有限公司 | A kind of preparation method of colourless resistant starch |
| CN108018322A (en) * | 2018-01-24 | 2018-05-11 | 浙江树人学院 | A kind of preparation method of corn porous starch |
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