CN110819668A - Method for extracting oligosaccharide in starch - Google Patents
Method for extracting oligosaccharide in starch Download PDFInfo
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- CN110819668A CN110819668A CN201911095402.3A CN201911095402A CN110819668A CN 110819668 A CN110819668 A CN 110819668A CN 201911095402 A CN201911095402 A CN 201911095402A CN 110819668 A CN110819668 A CN 110819668A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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Abstract
The invention discloses a method for extracting oligosaccharide in starch, which comprises the following steps: 1) pretreating starch with ethanol and petroleum ether, and drying; 2) adding water into the dried starch, stirring, adjusting the pH value to 8-9 with sodium hydroxide, and filtering to obtain filtrate and precipitate; 3) adding water into the precipitate, extracting with amylase, adjusting pH to 8-9 with sodium hydroxide, and vacuum filtering to obtain filtrate and precipitate; 4) adjusting the pH value of the filtrate obtained in the steps 2) and 3) to 5-6, centrifuging, collecting supernatant, and concentrating to obtain concentrated solution; 5) filtering the concentrated solution to obtain clear liquid, performing ultrafiltration, and collecting trapped fluid; 6) concentrating the trapped fluid again, performing enzymolysis by trypsin, centrifuging, collecting supernatant, and performing alcohol precipitation by ethanol; 7) discarding the supernatant, redissolving the precipitate with ammonium sulfate, centrifuging, and collecting the supernatant; 8) dialyzing the supernatant, purifying, and freeze-drying to obtain the final product. The invention mainly uses enzymolysis preparation, saves energy, protects environment, has good safety and lays a foundation for the development and utilization of oligosaccharide.
Description
Technical Field
The present invention relates to the field of biological extraction. More particularly, relates to a method for extracting oligosaccharide in starch.
Background
The oligosaccharide is also called oligosaccharide, is a novel starch sugar generated by the catalytic action of enzyme on starch, and has the main functions of: (1) improving micro-ecological environment in human body, facilitating proliferation of Bacillus bifidus and other beneficial bacteria, producing organic acid through metabolism to reduce intestinal pH, inhibiting growth of Salmonella and putrefying bacteria in intestine, regulating gastrointestinal function, inhibiting intestinal putrefying substance, changing stool character, preventing and treating constipation, increasing vitamin synthesis, and improving immunity; (2) the oligosaccharide is similar to water-soluble plant fiber, and can improve blood lipid metabolism, and reduce cholesterol and triglyceride content in blood; (3) the oligosaccharide is not dependent on insulin, does not increase blood sugar, and is suitable for hyperglycemia people and diabetes patients; (4) because the fat is difficult to be hydrolyzed by salivary enzyme and small intestine digestive enzyme, the calorific value is very low, and the fat is rarely converted into fat; (5) it is not formed into matrix by dental caries bacteria, and has no bacteria coagulation effect, and can be used for preventing dental caries.
As a food ingredient, the oligosaccharide is widely applied to dairy products, lactobacillus beverages, bifidobacterium yogurts, starch foods and health-care foods, particularly foods for infants and old people. In the health food series, there are also oral liquids prepared from oligosaccharide as raw material, which are directly used for regulating intestinal flora, loosening bowel to relieve constipation, regulating blood lipid, regulating immunity, etc. It integrates nutrition, health care and food therapy into a whole, is a novel functional sugar source for replacing cane sugar, is a new generation functional food for the 'future' of twenty-first century, is a new product with wide application range and promising prospect, and is quite popular internationally in recent years.
Therefore, it is desirable to provide a method for efficiently extracting oligosaccharides.
Disclosure of Invention
The invention aims to provide an extraction method of oligosaccharide in starch, which improves the utilization rate of starch, saves energy, protects environment and has high safety.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for extracting oligosaccharide from starch comprises the following steps:
1) pretreatment: refluxing starch ethanol and petroleum ether at 60-80 deg.C for 2-5 hr, filtering, and drying at 50-60 deg.C to constant mass;
2) adding water into the dried starch at 40-60 ℃, fully stirring, adding anhydrous sodium hydroxide into the solution, adjusting the pH value to 8-9, and filtering to obtain filtrate and precipitate;
3) adding 3-5 times of water into the precipitate obtained in the step 2) for secondary extraction, adding amylase into the solution for enzymolysis, adjusting the pH value to 8-9 by using sodium hydroxide, and performing suction filtration to obtain filtrate and precipitate;
4) adjusting the pH value of the filtrate obtained in the steps 2) and 3) to 5-6, centrifuging again, collecting supernatant, and concentrating the supernatant to obtain concentrated solution;
5) filtering the concentrated solution obtained in the step 4) to obtain clear liquid, performing ultrafiltration, and collecting trapped fluid;
6) concentrating the collected trapped fluid again, adding trypsin, performing enzymolysis, centrifuging for 15min, collecting supernatant, and adding 65-75% ethanol for alcohol precipitation;
7) standing at 0-4 deg.C for 24h, discarding supernatant, redissolving the precipitate with 20% ammonium sulfate, centrifuging after completely dissolving, and collecting supernatant;
8) putting the supernatant into a dialysis bag for dialysis for 1d, purifying, and freeze drying to obtain starch oligosaccharide.
Further, the volume ratio of the ethanol to the petroleum ether is 3-5: 1;
further, the concentration of the amylase in the step 3) is 40u/100 mL; the enzymolysis is carried out for 2 to 4 hours at the temperature of between 40 and 50 ℃.
Further, the ultrafiltration in the step 5) is carried out by adopting an ultrafiltration membrane with the cut-off molecule of 10000Da-50000 Da.
Further, the concentration of the trypsin in the step 6) is 40-60u/100ml, and the enzymolysis is carried out for 10-30min at 70 ℃.
The invention has the following beneficial effects:
the method mainly takes enzymolysis preparation as a main part, is energy-saving and environment-friendly, has good safety, and lays a foundation for the development and utilization of the oligosaccharide in the starch. In addition, the oligosaccharide obtained by the method has high purity which can reach more than 90 percent.
Detailed Description
In order to more clearly illustrate the invention, the invention is further described below in connection with preferred embodiments. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.
Example 1 extraction of oligosaccharide from starch
A method for extracting oligosaccharide from starch comprises the following steps:
1) pretreatment: refluxing starch with ethanol and petroleum ether at a volume ratio of 3:1 at 60 deg.C for 2 hr, filtering, and drying at 50 deg.C to constant mass;
2) adding water into the dried starch at 40 ℃, fully stirring, adding anhydrous sodium hydroxide into the solution, adjusting the pH value to 8, and filtering to obtain filtrate and precipitate;
3) adding 3 times volume of water into the precipitate in the step 2) for secondary extraction, adding 40u/100mL amylase into the solution, carrying out enzymolysis for 2 hours at 40 ℃, adjusting the pH value to 8 by using sodium hydroxide, and carrying out suction filtration to obtain filtrate and precipitate.
4) Adjusting the pH value of the filtrate obtained in the steps 2) and 3) to 5, centrifuging again, collecting supernatant, and concentrating the supernatant to obtain concentrated solution;
5) filtering the concentrated solution obtained in the step 4) to obtain clear liquid; filtering with an ultrafiltration membrane with a molecular interception direction of 10000Da, and collecting the trapped fluid;
6) concentrating the collected trapped fluid again, adding 40u/100ml trypsin, reacting at 70 ℃ for 10min, centrifuging for 15min, collecting supernatant, and adding 65% ethanol for alcohol precipitation;
7) standing at 0 deg.C for 24h, discarding supernatant, redissolving the precipitate with 20% ammonium sulfate, centrifuging after completely dissolving, and collecting supernatant;
8) putting the supernatant into a dialysis bag for dialysis for 1d, purifying, and freeze-drying to obtain starch oligosaccharide;
the determination shows that the purity of oligosaccharide is high and reaches 90 percent.
Example 2 extraction of oligosaccharide from starch
A method for extracting oligosaccharide from starch comprises the following steps:
1) pretreatment: refluxing starch with ethanol and petroleum ether at a volume ratio of 5:1 at 80 deg.C for 5 hr, filtering, and drying at 60 deg.C to constant mass;
2) adding water into the dried starch at 60 ℃, fully stirring, adding anhydrous sodium hydroxide into the solution, adjusting the pH value to 9, and filtering to obtain filtrate and precipitate;
3) adding 5 times volume of water into the precipitate obtained in the step 2) for secondary extraction, adding 40u/100mL amylase into the solution, carrying out enzymolysis for 4 hours at 50 ℃, adjusting the pH value to 9 by using sodium hydroxide, and carrying out suction filtration to obtain filtrate and precipitate.
4) Adjusting the pH value of the filtrate obtained in the steps 2) and 3) to 6, centrifuging again, collecting supernatant, and concentrating the supernatant to obtain concentrated solution;
5) filtering the concentrated solution obtained in the step 4) to obtain clear liquid; filtering with 50000Da ultrafiltration membrane by using cut-off molecules, and collecting the cut-off solution;
6) concentrating the collected trapped fluid again, adding 60u/100ml trypsin, reacting at 70 deg.C for 30min, centrifuging for 15min, collecting supernatant, and adding 75% ethanol for alcohol precipitation;
7) standing at 4 deg.C for 24h, discarding supernatant, redissolving the precipitate with 20% ammonium sulfate, centrifuging after completely dissolving, and collecting supernatant;
8) putting the supernatant into a dialysis bag for dialysis for 1d, purifying, and freeze-drying to obtain starch oligosaccharide;
the determination shows that the purity of oligosaccharide is higher and reaches 95 percent.
Example 3 extraction of oligosaccharide from starch
A method for extracting oligosaccharide from starch comprises the following steps:
1) pretreatment: refluxing starch with ethanol and petroleum ether at a volume ratio of 4:1 at 70 deg.C for 4 hr, filtering, and drying at 55 deg.C to constant mass;
2) adding water into the dried starch at 50 ℃, fully stirring, adding anhydrous sodium hydroxide into the solution, adjusting the pH value to 8, and filtering to obtain filtrate and precipitate;
3) adding 4 times volume of water into the precipitate obtained in the step 2) for secondary extraction, adding 40u/100mL amylase into the solution, carrying out enzymolysis for 3 hours at 45 ℃, adjusting the pH value to 9 by using sodium hydroxide, and carrying out suction filtration to obtain filtrate and precipitate.
4) Adjusting the pH value of the filtrate obtained in the steps 2) and 3) to 5, centrifuging again, collecting supernatant, and concentrating the supernatant to obtain concentrated solution;
5) filtering the concentrated solution obtained in the step 4) to obtain clear liquid; filtering with an ultrafiltration membrane with retention molecule of 30000Da, and collecting the retention solution;
6) concentrating the collected trapped fluid again, adding 50u/100ml trypsin, reacting at 70 deg.C for 20min, centrifuging for 15min, collecting supernatant, and adding 70% ethanol for alcohol precipitation;
7) standing at 2 deg.C for 24h, discarding supernatant, redissolving the precipitate with 20% ammonium sulfate, centrifuging after completely dissolving, and collecting supernatant;
8) putting the supernatant into a dialysis bag for dialysis for 1d, purifying, and freeze-drying to obtain starch oligosaccharide;
the oligosaccharide purity is high and reaches 92 percent through determination.
Comparative example 1 extraction method of oligosaccharide in starch
Adding 100L of starch into water, centrifuging with a three-leg centrifuge at 4000g for 15min, and vacuum concentrating the supernatant at 50.0 deg.C to obtain concentrated solution with volume about 1/5 of the original solution; adding absolute ethanol into the concentrated solution, controlling the ethanol precipitation concentration to be 65%, the precipitation temperature to be 15.0 ℃, and the precipitation time to be 18 h; the mixture was centrifuged (3000rpm, 30min) and the alcohol precipitate was collected. Freezing and drying to obtain the oligosaccharide, wherein the purity of the oligosaccharide is only 40%.
It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention, and it will be obvious to those skilled in the art that other variations or modifications may be made on the basis of the above description, and all embodiments may not be exhaustive, and all obvious variations or modifications may be included within the scope of the present invention.
Claims (5)
1. The method for extracting the oligosaccharide in the starch is characterized by comprising the following steps of:
1) pretreatment: refluxing starch ethanol and petroleum ether at 60-80 deg.C for 2-5 hr, filtering, and drying at 50-60 deg.C to constant mass;
2) adding water into the dried starch at 40-60 ℃, fully stirring, adding anhydrous sodium hydroxide into the solution, adjusting the pH value to 8-9, and filtering to obtain filtrate and precipitate;
3) adding 3-5 times of water into the precipitate obtained in the step 2) for secondary extraction, adding amylase into the solution for enzymolysis, adjusting the pH value to 8-9 by using sodium hydroxide, and performing suction filtration to obtain filtrate and precipitate;
4) adjusting the pH value of the filtrate obtained in the steps 2) and 3) to 5-6, centrifuging again, collecting supernatant, and concentrating the supernatant to obtain concentrated solution;
5) filtering the concentrated solution obtained in the step 4) to obtain clear liquid, performing ultrafiltration, and collecting trapped fluid;
6) concentrating the collected trapped fluid again, adding trypsin, performing enzymolysis, centrifuging for 15min, collecting supernatant, and adding 65-75% ethanol for alcohol precipitation;
7) standing at 0-4 deg.C for 24h, discarding supernatant, redissolving the precipitate with 20% ammonium sulfate, centrifuging after completely dissolving, and collecting supernatant;
8) putting the supernatant into a dialysis bag for dialysis for 1d, purifying, and freeze drying to obtain starch oligosaccharide.
2. The extraction method according to claim 1, wherein the volume ratio of ethanol to petroleum ether is 3-5: 1.
3. The extraction method according to claim 1, wherein the amylase of step 3) is at a concentration of 40u/100 mL; the enzymolysis is carried out for 2 to 4 hours at the temperature of between 40 and 50 ℃.
4. The extraction method according to claim 1, wherein the ultrafiltration in step 5) is performed by ultrafiltration using an ultrafiltration membrane with a cut-off of 10000Da to 50000 Da.
5. The extraction method according to claim 1, wherein the concentration of the trypsin in step 6) is 40-60u/100ml, and the enzymolysis is carried out at 70 ℃ for 10-30 min.
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