CN107827992B - Preparation method of high-purity porphyra yezoensis polysaccharide α -amylase inhibitor - Google Patents
Preparation method of high-purity porphyra yezoensis polysaccharide α -amylase inhibitor Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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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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- 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
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Abstract
The invention discloses a preparation method of high-purity porphyra yezoensis polysaccharide α -amylase inhibitor, belonging to the field of preparation of plant active ingredients.A porphyra yezoensis is firstly ground, sieved and then extracted with water, then a supernatant obtained by centrifugation is added with protease for enzymolysis, enzyme deactivation and ultrafiltration are carried out on the supernatant obtained by centrifugation, and the obtained trapped fluid is dried by spraying to obtain the porphyra yezoensis polysaccharide α -amylase inhibitor.
Description
Technical Field
The invention relates to a preparation method of a high-purity porphyra yezoensis polysaccharide α -amylase inhibitor, belonging to the field of preparation of plant active ingredients.
Background
Porphyra yezoensis (Porphyra yezoensis) has an oval shape, a long oval shape, and a purple red color or slightly green color. Laver contains abundant vitamins, minerals, proteins, fatty acids, laver polysaccharide, and other nutritional components and physiologically active substances. Laver is one of seaweeds with the most abundant polysaccharide content, and the polysaccharide content in laver varies with the species, growth time, place, etc. of algae, and is generally 20% -40%.
Diabetes is one of the major diseases seriously threatening the life and health of human in the 21 st century. The incidence of the disease is related to genetic factors, life style, dietary structure, age and other factors, and is a common metabolic disease. Epidemiological research in recent years shows that postprandial hyperglycemia is more important for the occurrence and development of chronic complications of type 2 diabetes than fasting hyperglycemia, and has close relation with the occurrence and development of diabetic macrovascular and microvascular complications; and strictly controlling the abnormal rise of postprandial blood sugar can delay the transformation of the patients with impaired glucose tolerance to type 2 diabetes. Therefore, the intervention treatment of postprandial hyperglycemia has very important significance in the aspect of preventing and treating type 2 diabetes.
α -amylase inhibitor can be protein, polypeptide or polysaccharide, since the 70 s of the 20 th century, it has been widely studied because of its medical value α -amylase inhibitor, α -AI has been isolated from seeds of plants such as beans, wheat, wild amaranth, etc., α -AI with higher activity is extracted from white kidney bean, it has stronger inhibiting effect on mammal pancreas α -amylase, it has been applied as diet health food abroad, α -AI with high activity of protein and polypeptide but incomplete removal of phytoagglutinin exists, or acid and alkali conditions are needed in the extraction process to cause serious influence on ecological environment, and there is no high temperature resistance treatment, and anaphylactic shock occurs in serious cases, α -AI of polysaccharide has certain stability on temperature, acid and alkali and gastrointestinal digestion, it is safe and nontoxic, it can effectively prevent and manage type 2 diabetes, it has good prospect for natural hypoglycemic medicine, polysaccharide has stronger inhibiting effect on temperature, acid and alkali digestion and gastrointestinal digestion, polysaccharide has strong inhibiting effect on polysaccharide 2-AI, polysaccharide has been purified by conventional methods, such as polysaccharide, polysaccharide has been purified by ethanol extraction, polysaccharide has been purified by a step-by-extraction method, polysaccharide extraction method has been used for industrial extraction, polysaccharide extraction method has strong inhibiting effect on polysaccharide extraction, polysaccharide extraction method has been used for extraction, polysaccharide extraction method for extraction.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a method for combining enzymolysis and ultrafiltration, which is an industrial preparation method of a high-purity porphyra yezoensis polysaccharide α -amylase inhibitor with high preparation efficiency, high product temperature stability, acid and alkali resistance and gastrointestinal tract digestion resistance, and is beneficial to the actual production and application of the porphyra yezoensis α -amylase inhibitor.
It is a first object of the present invention to provide a method for preparing a high-purity porphyra yezoensis polysaccharide α -amylase inhibitor, comprising the steps of:
(1) grinding: grinding Porphyra yezoensis, and sieving;
(2) water extraction: mixing the laver powder with water, stirring and extracting to obtain an extracting solution;
(3) centrifuging: centrifuging the extracting solution in the step (2) and taking supernatant;
(4) adjusting the pH value: adjusting the pH value of the supernatant in the step (3) to 3.0-8.0;
(5) enzymolysis: adding protease into the solution obtained in the step (4) under a stirring state for enzymolysis to obtain an enzymolysis solution;
(6) enzyme deactivation: inactivating the enzyme of the enzymolysis liquid in the step (5);
(7) centrifuging: centrifuging the solution of the step (6) to obtain a supernatant;
(8) and (3) ultrafiltration: carrying out ultrafiltration on the supernatant obtained in the step (7), and collecting trapped fluid;
(9) and (4) drying, namely performing spray drying on the trapped fluid obtained in the step (8) to obtain a powdery α -amylase inhibitor product.
In one embodiment of the invention, the sieving in the step (1) is performed by sieving with a 60-80 mesh sieve.
In one embodiment of the invention, the extraction temperature in the step (2) is 50-90 ℃, and the extraction time is 2-4 h.
In one embodiment of the present invention, the amount of the protease added in the step (5) is 1 to 5% by weight of the solution in the step 4.
In one embodiment of the invention, the enzymolysis temperature in the step (5) is 40-60 ℃, and the enzymolysis time is 2-4 h.
In one embodiment of the present invention, the enzyme deactivation conditions in step (6) are: adjusting the pH value to 6.5-7.5, and inactivating the enzyme in water bath at the temperature of 60-80 ℃ for 10-20 min.
In one embodiment of the present invention, the ultrafiltration membrane in step (8) has a molecular cut-off of 1-10 kDa.
In an embodiment of the present invention, the method specifically includes:
(1) grinding: grinding porphyra yezoensis into powder, and screening the powder through a 60-80-mesh sieve;
(2) water extraction: mixing the laver powder obtained in the step (1) with water according to a mass ratio of 1: 20-1: 30, and stirring for 2-4 h at 50-90 ℃;
(3) centrifuging: centrifuging the extracting solution obtained in the step (2) at 4000-8000 r/min for 10-20 min, and taking supernatant;
(4) adjusting the pH value: adjusting the pH value of the supernatant in the step (3) to 3.0-8.0;
(5) enzymolysis: adding protease accounting for 1-5% of the weight of the solution in the step (4) to carry out enzymolysis on the hybrid protein under the stirring state, and stirring for 2-4 hours at 40-60 ℃ to obtain an enzymolysis solution;
(6) enzyme deactivation: adjusting the pH value of the enzymolysis liquid in the step (5) to 6.5-7.5, and inactivating the enzyme in water bath at the temperature of 60-80 ℃ for 10-20 min;
(7) centrifuging: centrifuging the solution obtained in the step (6) at 4000-8000 r/min for 10-20 min to obtain a supernatant;
(8) and (3) ultrafiltration: enabling the supernatant obtained in the step (7) to pass through an ultrafiltration membrane with the molecular weight cutoff of 1-10 kD, and collecting the cutoff liquid;
(9) and (4) drying, namely spray drying the trapped fluid in the step (8), and carrying out superfine grinding to obtain a powdery α -amylase inhibitor product.
The second object of the present invention is to provide an α -amylase inhibitor obtained by the above method.
The third purpose of the invention is to provide the application of the α -amylase inhibitor in preparing hypoglycemic drugs and health-care foods, and the invention has the following beneficial effects:
the extraction method of the high-purity porphyra yezoensis α -amylase inhibitor is realized by combining enzymolysis with ultrafiltration, has the characteristics of simplicity, easiness in operation and control, high purity, high biological activity and good stability, the activity of the α -amylase inhibitor can reach 8000U/g, and the prepared porphyra yezoensis α -amylase inhibitor has certain strength stability on temperature, acid and alkali and gastrointestinal digestion, is safe and nontoxic, can effectively prevent and manage type 2 diabetes, and has good development prospect as a natural hypoglycemic medicament.
Drawings
FIG. 1: extracting a polysaccharide molecular weight distribution map by combining enzymolysis and ultrafiltration of porphyra yezoensis;
FIG. 2 is a graph showing the stability of Porphyra yezoensis α -amylase inhibitor against temperature;
FIG. 3 is a graph showing stability to acid and base of Porphyra yezoensis α -amylase inhibitor;
FIG. 4 is a graph of the stability of Porphyra yezoensis α -amylase inhibitor against gastrointestinal digestion;
FIG. 5: the porphyra yezoensis traditional method extracts a polysaccharide molecular weight distribution diagram.
Detailed Description
The present invention is further illustrated by the following specific examples, but it should be understood that the present invention is not limited thereto.
α -determination method of amylase inhibitor activity
Adding 0.25mL of α -amylase solution and 0.25mL of sample into 0.5mL of 0.2mol/LPBS (pH6.9), reacting in a water bath at 37 ℃ for 10min, adding 0.5mL of 1% soluble starch solution, accurately reacting for 5min, adding 1mLDNS reagent, quickly cooling in a boiling water bath for 10min, adding 5mL of deionized water, and measuring the absorbance at a wavelength of 540 nm.
The inhibition rate of α -amylase by the sample can be calculated according to formula (1):
wherein A1, A2, A3 and A4 are absorbance values at 540nm for blank tube, blank control tube, inhibition tube and inhibition control tube, respectively.
Example 1 preparation of Porphyra yezoensis polysaccharide α -amylase inhibitor
Pulverizing Porphyra yezoensis, sieving with 80 mesh sieve, and collecting Porphyra yezoensis powder;
adding 1000mL of deionized water into 50g of laver powder, mixing, and stirring and extracting at 70 ℃ for 2 h;
centrifuging the extract at 4 deg.C and 8000r/min for 20min, and collecting supernatant;
adjusting the pH of the supernatant to 3.0 by using 1mol/LHCl solution, adding 1% protease for enzymolysis of the hybrid protein, stirring at 60 ℃ for 2 hours, adjusting the pH of the enzymolysis solution to 6.5 by using 1mol/LNaOH solution, inactivating the enzyme in water bath at 80 ℃ for 20min, centrifuging at 8000r/min for 20min, and taking the supernatant;
ultrafiltering the supernatant with ultrafiltration membrane with pore diameter of 10kDa, and collecting the trapped fluid;
spray drying the trapped liquid to obtain a powdery α -amylase inhibitor product, and measuring the molecular weight distribution of polysaccharide by adopting HPLC (high performance liquid chromatography). the measurement result shows that the activity of the α -amylase inhibitor in the α -amylase inhibitor product is 8000U/g, as shown in figure 1, the polysaccharide extracted by the invention has uniform molecular weight and has stronger inhibition effect on α -amylase.
Example 2 preparation of Porphyra yezoensis polysaccharide α -amylase inhibitor
Pulverizing Porphyra yezoensis, sieving with 60 mesh sieve, and collecting Porphyra yezoensis powder;
adding 1000mL of deionized water into 50g of laver powder, mixing, and stirring and extracting at 50 ℃ for 2 h;
centrifuging the extract at 4 deg.C and 8000r/min for 20min, and collecting supernatant;
adjusting the pH of the supernatant to 8.0 by using 1mol/L NaOH solution, adding 2% protease for enzymolysis of the hybrid protein, stirring at 50 ℃ for 2h, adjusting the pH of the enzymolysis solution to 7.5 by using 1mol/L LHCl solution, inactivating the enzyme in water bath at 80 ℃ for 20min, centrifuging at 8000r/min for 20min, and taking the supernatant;
ultrafiltering the supernatant with ultrafiltration membrane with pore diameter of 10kDa, and collecting the trapped fluid;
spray drying the trapped fluid to obtain α -amylase inhibitor powder.
An appropriate amount of α -amylase inhibitor product is dissolved in deionized water, and the activity of the α -amylase inhibitor in the α -amylase inhibitor product is measured, wherein the measurement result shows that the activity of α -amylase inhibitor in the α -amylase inhibitor product is 5000U/g.
Example 3 preparation of Porphyra yezoensis polysaccharide α -amylase inhibitor
Pulverizing Porphyra yezoensis, sieving with 80 mesh sieve, and collecting Porphyra yezoensis powder;
adding 1500mL of deionized water into 50g of laver powder, mixing, and stirring and extracting at 60 ℃ for 2 h;
centrifuging the extract at 4 deg.C and 8000r/min for 20min, and collecting supernatant;
adjusting the pH of the supernatant to 5.0 by using 1mol/LHCl solution, adding 4% protease for enzymolysis of the hybrid protein, stirring at 40 ℃ for 2 hours, adjusting the pH of the enzymolysis solution to 7.0 by using 1mol/LNaOH solution, inactivating the enzyme in water bath at 60 ℃ for 20min, centrifuging at 8000r/min for 20min, and taking the supernatant;
ultrafiltering the supernatant with ultrafiltration membrane with aperture of 1kDa, and collecting the trapped fluid;
and (3) spray-drying the trapped liquid to obtain a powdered α -amylase inhibitor product, wherein the determination result shows that the activity of the α -amylase inhibitor in the α -amylase inhibitor product is 3600U/g.
Example 4 preparation of Porphyra yezoensis polysaccharide α -amylase inhibitor
Pulverizing Porphyra yezoensis, sieving with 80 mesh sieve, and collecting Porphyra yezoensis powder;
adding 1500mL of deionized water into 50g of laver powder, mixing, and stirring and extracting at 70 ℃ for 2 h;
centrifuging the extract at 4 deg.C and 8000r/min for 20min, and collecting supernatant;
adjusting the pH of the supernatant to 8.0 by using 1mol/LNaOH solution, adding 5% protease for enzymolysis of the hybrid protein, stirring at 60 ℃ for 2 hours, adjusting the pH of the enzymolysis solution to 6.5 by using 1mol/HCl solution, inactivating the enzyme in water bath at 80 ℃ for 20min, centrifuging at 8000r/min for 20min, and taking the supernatant;
ultrafiltering the supernatant with ultrafiltration membrane with aperture of 1kDa, and collecting the trapped fluid;
and (3) spray-drying the trapped liquid to obtain a powdered α -amylase inhibitor product, wherein the determination result shows that the activity of the α -amylase inhibitor in the α -amylase inhibitor product is 3100U/g.
Example 5 Porphyra yezoensis polysaccharide α -amylase inhibitor tolerance
The α -amylase inhibitor product prepared in the example 1 is dissolved in deionized water in a proper amount, 5mL of the product water solution is taken and subpackaged in test tubes, after the treatment for 20min in a constant-temperature water bath kettle at 50, 60, 70, 80 and 90 ℃, running water is rapidly cooled to room temperature, and the activity of the α -amylase inhibitor in the α -amylase inhibitor product is determined, as shown in figure 2, after the product is heated at 90 ℃ for 30min, the inhibition activity is basically not changed, and the product has good heat-resistant stability.
Dissolving a proper amount of the α -amylase inhibitor product prepared in example 1 in deionized water, taking 100mL of a product aqueous solution, adjusting the pH of the product aqueous solution to 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 respectively at room temperature by using 1mol/LHCL or 1mol/mLNaOH, continuously stirring for 30min, immediately adjusting the pH back to the pH value of the stock solution, centrifuging for 20min at 8000r/min, taking a supernatant, and measuring the inhibitory activity of the supernatant on α -amylase, wherein the product has a stable chemical structure between the pH value of 2 and 10, is not affected by the inhibitory activity and has wide acid-base tolerance as shown in figure 3.
The α -amylase inhibitor product prepared in the embodiment 1 is dissolved in deionized water in a proper amount, a product water solution is taken and put in an enzyme reactor at 37 ℃, the pH value is adjusted to 2.0 by 1mol/LHCL, 1% pepsin is added to simulate the gastric environment, 20mL of enzyme is inactivated every 30min in the treatment process and then is measured, 1mol/LNaOH is used to adjust the pH value to 8.2, 1% trypsin is added to simulate the intestinal environment, 20mL of enzyme is inactivated every 30min in the treatment process and then is measured, as shown in figure 4, the inhibition activity is always in a stable state when the gastrointestinal tract is simulated, therefore, the α -amylase inhibitor activity can play a good role in reducing the blood sugar in the gastrointestinal tract, and the physiological effect is reliable.
Comparative example 1 preparation of Porphyra yezoensis polysaccharide α -amylase inhibitor by conventional method
Pulverizing Porphyra yezoensis, sieving with 80 mesh sieve, and collecting Porphyra yezoensis powder;
adding 1000mL of deionized water into 50g of laver powder, mixing, and stirring and extracting at 70 ℃ for 2 h;
centrifuging the extract at 4 deg.C and 8000r/min for 20min, and collecting supernatant;
repeatedly precipitating with ethanol or acetone, centrifuging at 8000r/min for 20min, collecting supernatant, dissolving precipitate with deionized water, and determining polysaccharide molecular weight distribution by HPLC, wherein the determination result shows that α -amylase inhibitor activity in α -amylase inhibitor product is 1000U/g, as shown in FIG. 5, the polysaccharide extracted by conventional method has nonuniform molecular weight, and high purity polysaccharide can be obtained by further separation and purification, and the activity of α -amylase inhibitor is not high.
Control example 2 preparation of Porphyra yezoensis polysaccharide α -amylase inhibitor
In the case of protease hydrolysis, the supernatant pH was adjusted to 10.0 using 1mol/L NaOH solution, and the other steps were identical to those of example 1, and as a result, it was found that the activity of the α -amylase inhibitor in the α -amylase inhibitor product was only 1100U/g, whereas in the case of protease hydrolysis, the supernatant pH was adjusted to 2.0 using 1mol/LHCl solution, and the other steps were identical to those of example 1, and as a result, it was found that the activity of the α -amylase inhibitor in the α -amylase inhibitor product was only 2000U/g.
Control example 3 preparation of Porphyra yezoensis polysaccharide α -amylase inhibitor
And filtering the supernatant obtained after the extract is centrifuged by an ultrafiltration membrane with the molecular weight cutoff of 10kD, and then treating the supernatant by the steps of enzymolysis and enzyme deactivation of the example 1, wherein the conditions are consistent with the example 1, and the activity of the α -amylase inhibitor in the α -amylase inhibitor product is only 600U/g.
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.
Claims (3)
1. A method of preparing an α -amylase inhibitor, the method comprising any one of the following:
the first scheme is as follows:
(1) pulverizing Porphyra yezoensis, sieving with 80 mesh sieve, and collecting Porphyra yezoensis powder;
(2) mixing 50g of laver powder with 1000mL of deionized water, and stirring and extracting at 70 ℃ for 2 h;
(3) centrifuging the extract at 4 deg.C and 8000r/min for 20min, and collecting supernatant;
(4) adjusting the pH of the supernatant to 3.0 by using 1mol/LHCl solution, adding 1% protease for enzymolysis of the hybrid protein, stirring at 60 ℃ for 2 hours, adjusting the pH of the enzymolysis solution to 6.5 by using 1mol/LNaOH solution, inactivating the enzyme in water bath at 80 ℃ for 20min, centrifuging at 8000r/min for 20min, and taking the supernatant;
(5) ultrafiltering the supernatant with ultrafiltration membrane with pore diameter of 10kDa, and collecting the trapped fluid;
(6) spray drying the trapped fluid to obtain α -amylase inhibitor powder;
scheme II:
(1) pulverizing Porphyra yezoensis, sieving with 60 mesh sieve, and collecting Porphyra yezoensis powder;
(2) mixing 50g of laver powder with 1000mL of deionized water, and stirring and extracting at 50 ℃ for 2 h;
(3) centrifuging the extract at 4 deg.C and 8000r/min for 20min, and collecting supernatant;
(4) adjusting the pH of the supernatant to 8.0 by using 1mol/L NaOH solution, adding 2% protease for enzymolysis of the hybrid protein, stirring at 50 ℃ for 2h, adjusting the pH of the enzymolysis solution to 7.5 by using 1mol/L LHCl solution, inactivating the enzyme in water bath at 80 ℃ for 20min, centrifuging at 8000r/min for 20min, and taking the supernatant;
(5) ultrafiltering the supernatant with ultrafiltration membrane with pore diameter of 10kDa, and collecting the trapped fluid;
(6) spray drying the trapped fluid to obtain α -amylase inhibitor powder.
2.α -amylase inhibitor obtainable by the process of claim 1.
3. Use of the α -amylase inhibitor of claim 2 in the preparation of hypoglycemic drugs, health foods.
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| CN1687444A (en) * | 2005-03-23 | 2005-10-26 | 江苏大学 | Method for preparing peptide of decrease blood pressure in laver by using enzyme-membrane coupling technique and application thereof |
| CN102268101A (en) * | 2011-08-31 | 2011-12-07 | 南京财经大学 | Enzyme-assisted extraction method of high-purity porphyra polysaccharide |
| CN104293871A (en) * | 2014-10-11 | 2015-01-21 | 江南大学 | Method for preparing porphyra anti-oxidation peptide and comprehensively utilizing byproducts |
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| CN1687444A (en) * | 2005-03-23 | 2005-10-26 | 江苏大学 | Method for preparing peptide of decrease blood pressure in laver by using enzyme-membrane coupling technique and application thereof |
| CN102268101A (en) * | 2011-08-31 | 2011-12-07 | 南京财经大学 | Enzyme-assisted extraction method of high-purity porphyra polysaccharide |
| CN104293871A (en) * | 2014-10-11 | 2015-01-21 | 江南大学 | Method for preparing porphyra anti-oxidation peptide and comprehensively utilizing byproducts |
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