CN110283860B - Gracilaria tenuistipitata polysaccharide extracted by ultrasonic-assisted composite enzymolysis and extraction method thereof - Google Patents
Gracilaria tenuistipitata polysaccharide extracted by ultrasonic-assisted composite enzymolysis and extraction method thereof Download PDFInfo
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- C12P19/00—Preparation of compounds containing saccharide radicals
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Abstract
The invention relates to a method for extracting gracilaria tenuistipitata polysaccharide by ultrasonic-assisted composite enzymolysis; the method comprises the steps of adding a buffer solution into decolorized fine gracilaria powder, uniformly stirring, and carrying out ultrasonic treatment; adding compound enzyme for enzymolysis; the compound enzyme is bromelain and a second component enzyme, and the second component enzyme is cellulase or pectinase; after enzymolysis, inactivating enzyme by adopting boiling water bath, separating an extracting solution by centrifugation and suction filtration, and collecting supernatant; and carrying out alcohol precipitation, centrifugation, washing and drying on the extracting solution to obtain the gracilaria tenuistipitata polysaccharide extract. The extraction method has the advantages of mild conditions, one-step enzymolysis, simple operation, short extraction time, high polysaccharide yield and the like, and the obtained fine gracilaria polysaccharide has strong oxidation resistance and high nutritional and health-care values.
Description
Technical Field
The invention relates to gracilaria tenuistipitata polysaccharide, in particular to gracilaria tenuistipitata polysaccharide extracted by ultrasonic-assisted composite enzymolysis and an extraction method thereof, and the gracilaria tenuistipitata polysaccharide obtained by the method has good antioxidant activity; belongs to the technical field of functional food engineering.
Background
With the rapid increase of the number of human beings and animals on the earth, terrestrial resources are gradually reduced; to achieve sustainability of the human society, it is necessary to develop and research marine resources. Since 1970, more than 3000 natural products have been isolated from marine algae, sponges, coelenterates, mollusks, echinoderms and microorganisms, and many of them have pharmacological activities such as antifungal, antiviral, anticoagulant, antitumor, etc., and the research on marine natural products has become a popular topic at present.
In recent years, seaweeds have been favored as functional foods in developed countries such as western europe, north america, and northern europe because of their unique flavor and nutritional value. Research has shown that seaweed has the effects of preventing metabolic diseases such as obesity, gallstone, constipation, gastrointestinal diseases, and the like, preventing and resisting cancer, reducing blood pressure, preventing arteriosclerosis and thrombosis, reducing blood sugar, and the like. The hedgerow is seaweed with high economic value, is widely distributed in tropical, subtropical and temperate sea areas all over the world, and is named about 100 at present. China has abundant hedgerow resources, has more than 30 clear identifications, is one of the coastal algae cultivation varieties of south China, Guangdong, Zhejiang and Taiwan, and is mainly used as fishery cultivation feed and raw materials for producing food gum. Different species of hedgerows have evolutionary differences, so that different influences are brought to the properties of the agar, such as the coagulation capacity, the coagulation temperature and the like. Agar or agarose is commonly used in techniques such as electrophoresis, chromatography and the like in biochemical analysis and clinical tests, and the agar prevents constipation by promoting gastrointestinal motility and prevents cardiovascular diseases and colon cancer by preventing absorption of fat, cholesterol and chemical carcinogens.
The Gracilaria Tenuistipitata (Gracilaria Tenuistipitata) is the most cultivated hedgerow in China, can germinate and propagate, is easy to solve seedlings, is simpler in cultivation method, and is the main cultivation type in China at present. The research on the biological activity of the gracilaria tenuistipitata has also progressed to a certain extent, and the research on the anticancer effect of the methanol extract of the gracilaria tenuistipitata by Yeh et al shows that the methanol extract has cytotoxicity to Ca9-22 oral cancer cells and can kill the cancer cells by inducing apoptosis, causing DNA damage and oxidative stress (BMC complete. Yangtong et al have studied the antioxidant activity of boiling water extracts of four seaweeds, and the studies show that the boiling water extract of Gracilaria tenuistipitata has certain antioxidant capacity due to the presence of polyphenol compounds (Plant Foods hum. Nutr.,2009,64(3), 218-); chen et al have studied the recovery of the water extract of Gracilaria tenuistipitata on the immunocompetence of white prawn under the influence of ammonia, and the study shows that the water extract of Gracilaria tenuistipitata has an auxiliary effect on the improvement of the immunological index of white prawn and can help white prawn recover the immunocompetence earlier (Mar. drugs,2015,13(6), 3606-.
The chemical composition of the fine-base hedgerow extract strongly depends on the extraction system, and different extraction systems and conditions endow the extract with different functions. The resource development and utilization degree of the fine-base hedgehog is lower, and the extraction process of the fine-base hedgehog polysaccharide is disclosed as an ultrasonic water extraction process of a Zhao Liming subject group at present: 100W ultrasonic extracting for 30min at a solid-to-liquid ratio of 1:30 and 70 deg.C for 10h with a maximum extraction rate of 6.25% (optimum process parameters), and the obtained Gracilaria tenuistipitata polysaccharide has an optimum antioxidant index of 1.25 mg/mL-1And hydroxyl radical clearance of 51.54% (0.1 wt% hydrogen peroxide) (food industry science and technology, 2005,2(3), 67-69). Other currently disclosed hedgehog polysaccharide extraction technologies include the ultrasonic acid hedgehog polysaccharide sulfate extraction method disclosed in the Chinese patent invention 200810029075.7A, wherein the solid-liquid ratio is 1: 40-1: 60, the concentration of hydrochloric acid is 0.1-0.2%, the 500W ultrasonic extraction is performed for 15-25 min (55-65 ℃), and the highest extraction rate of polysaccharide is 10.93%. The Chinese patent application 200910001265.2A discloses a method for preparing hedgerow polysaccharide by multi-step enzymolysis reaction of hedgerow water extract lyase and protease, wherein the solid-liquid ratio is 1: 5-1: 10, the extraction is carried out for 8.5-12.5 h (30-60 ℃), and the highest extraction rate of polysaccharide is 13.42%. The extraction method of the hedgerow polysaccharide containing the protein and the hedgerow polysaccharide multi-component enzymolysis liquid based on the cellulase and compound protease stepwise enzymolysis process disclosed by the invention patent 201310694077.9A in China has the solid-liquid ratio of 1: 8-1: 14, the enzymolysis extraction is carried out for 6-12 h (40-65 ℃), and the extraction rate of the hedgerow polysaccharide is 8-10%.
Generally speaking, the yield of the hedgehog hydnum polysaccharide in the prior art is low, and the strong acid extraction has high requirements on equipment and has the problems of environmental pollution, long extraction time, high energy consumption, low yield, low efficiency and the like.
Disclosure of Invention
In order to overcome the defects of long extraction time, high energy consumption, low yield, low efficiency and low antioxidant activity of the extracted polysaccharide in the prior art, the invention provides the gracilaria tenuistipitata polysaccharide extracted by ultrasonic-assisted composite enzymolysis and the extraction method thereof, wherein the extraction rate of the gracilaria tenuistipitata polysaccharide measured by a phenol-sulfuric acid method reaches 25.01-38.60 wt%; the gracilaria tenuistipitata polysaccharide has hydroxyl radical scavenging rate of 50-66%.
The invention utilizes ultrasonic and multienzyme composite single-step enzymolysis to multiply disperse fine gracilaria powder and multiply break hedge cells, not only shortens extraction time and improves the extraction rate of fine gracilaria polysaccharide, but also has mild process conditions and simple process, the extraction rate of the fine gracilaria polysaccharide is obviously higher than that of the prior art, the clearance rate of hydroxyl radical of the obtained fine gracilaria polysaccharide reaches 50-66%, the structure of the polysaccharide is improved compared with the product of the ultrasonic water extraction process, and the in vitro antioxidant activity is obviously enhanced.
The purpose of the invention is realized by the following technical scheme:
a method for extracting gracilaria tenuistipitata polysaccharide by ultrasonic-assisted composite enzymolysis comprises the following steps:
1) adding the decolorized fine gracilaria powder into a buffer solution, uniformly stirring, and performing ultrasonic treatment;
2) after the ultrasonic treatment is finished, adding a complex enzyme for enzymolysis; the compound enzyme is bromelain and a second component enzyme, the second component enzyme is cellulase or pectinase, and the mass ratio of the bromelain to the second component enzyme is 1: 1-1: 2; the using amount of the complex enzyme is 1-5 wt% of the fine gracilaria powder;
3) after enzymolysis, inactivating enzyme by adopting boiling water bath, separating an extracting solution by centrifugation and suction filtration, and collecting supernatant;
4) and carrying out alcohol precipitation, centrifugation, washing and drying on the extracting solution to obtain the gracilaria tenuistipitata polysaccharide extract.
In order to further achieve the aim of the invention, preferably, the buffer solution is citric acid-disodium hydrogen phosphate buffer solution, the pH is 4.5-6.5, and the concentration of the disodium hydrogen phosphate is 0.05 +/-0.005 mol.L-1The mass ratio of the volume of the buffer solution to the powder is 40: 1-100: 1.
Preferably, the water bath temperature during the ultrasonic treatment is 45-65 ℃, the ultrasonic power is 90-150W, and the ultrasonic time is 10-40 min.
Preferably, the enzymolysis temperature of the enzymolysis treatment is 45-65 ℃, and the enzymolysis time is 60-180 min.
Preferably, the enzyme inactivation is carried out by inactivating the enzyme in a boiling water bath, and the enzyme is inactivated by the extracting solution in the boiling water bath for more than 20 min.
Preferably, the rotation speed of the centrifugation in the step 3) is 4500rpm or more, and the time of the centrifugation is 8min or more; the rotating speed of the centrifugation in the step 4) is more than 3000rpm, and the time of the centrifugation is more than 5 min.
Preferably, the alcohol precipitation is to add three times of volume of absolute ethanol into the supernatant to precipitate the polysaccharide, and collect the precipitate.
Preferably, the washing is to wash the precipitate with absolute ethyl alcohol for more than 2 times; the drying is carried out in a vacuum oven at 50-70 ℃ for 40-60 h.
Preferably, the extraction rate of the gracilaria tenuistipitata polysaccharide extracted by ultrasonic-assisted composite enzymolysis is 25.01-38.60 wt% by phenol-sulfuric acid method.
A Gracilaria tenuistipitata polysaccharide is extracted by the method; the gracilaria tenuistipitata polysaccharide has a hydroxyl radical clearance rate of 50-66%; wherein
The test method is as follows: taking 0.5mL of the solution with the concentration of 1.2 mg/mL-1The sample solution was put into a 7mL centrifuge tube with a cap, 1mL of a phosphate buffer solution having a pH of 7.4 was added to the tube at a concentration of 0.1M, and 0.5mL of 0.75 mmol/L was further added-1After shaking the phenanthroline solution evenly, 0.5mL of 0.75 mmol.L is added-1Rapidly shaking ammonium ferrous sulfate solution, adding 0.5mL solution of 0.01% hydrogen peroxide, sealing tube, shaking, keeping at 37 deg.C for 60min, cooling to room temperature, measuring absorbance at 510nm with distilled water as reference, and recording absorbance Asample(ii) a Replacing the sample solution with distilled water, treating, measuring, and recording absorbance value as Ablank(ii) a Replacing the sample solution and hydrogen peroxide with distilled water, treating, and measuring with absorbance value A0。
The type of the fine gracilaria used in the invention is not particularly limited, and the fine gracilaria powder can be purchased directly or can be automatically crushed and decolored, and the powder for extraction is sieved by a 100-mesh sieve.
The bromelain, the cellulase and the pectinase are all plant-derived enzymes and are safe and reliable to use, wherein the bromelain is a pure natural plant protease prepared from parts such as skin, stem and core of pineapple fruits through a biological technology, has the molecular weight of 33000 and the isoelectric point of 9.55, belongs to sulfhydryl protease, has the active center of sulfhydryl (-SH), can perform various biochemical reactions such as protein hydrolysis and the like, and is a natural, non-toxic, harmless, sanitary and safe protease which can be widely applied to the industries such as food, medicine, biology and the like. The bromelain structure belongs to glycoprotein, the optimum pH (100000U) is about 7 generally, and the optimum working temperature is 55 ℃.
Cellulase (beta-1, 4-glucan-4-glucan hydrolase) is a general name of a group of enzymes for degrading cellulose to generate glucose, is not a monomer enzyme, is a multi-component enzyme system with a synergistic effect, is a complex enzyme, mainly comprises exo-beta-glucanase, endo-beta-glucanase, beta-glucosidase and the like, and also has xylanase with high activity. Acting on cellulose and products derived from cellulose. The cellulase has very important significance in the aspects of converting insoluble cellulose into glucose, destroying cell walls in fruit and vegetable juice so as to improve the yield of the fruit juice and the like, and is widely applied to feed, alcohol, textile, food and the like. The optimum pH value of the cellulase is generally 4.5-6.5, and the working temperature is 40-60 ℃.
Pectinase is a multi-enzyme complex that breaks down pectin and typically includes protopectinase, pectin methyl ester hydrolase, and pectinase. The pectin is completely decomposed by the combined action of the two components. Converting natural pectic substance into water soluble pectin under the action of protopectinase; the pectin is catalyzed by pectin methyl ester hydrolase to remove methyl ester group to generate pectic acid; pectic acid is degraded by pectic acid hydrolase and pectic acid lyase to give galacturonic acid. The pectinase is mainly used for juicing and clarifying fruit and vegetable juice beverages and fruit wine, and has a good effect of decomposing pectin. The optimum pH value of the cellulase is generally 3.0-6.0, the working temperature is 15-55 ℃, and the optimum action temperature is 50 ℃.
More importantly, the invention discovers that the multiple dispersion of the fine gracilaria powder and the multiple crushing of gracilaria cells are realized by utilizing the single-step enzymolysis of ultrasonic and multi-enzyme compounding, particularly the bromelain and the second component enzyme (cellulase or pectinase) are cooperatively matched, so that the extraction rate of the fine gracilaria polysaccharide is improved, the composition of the fine gracilaria polysaccharide is improved, and the antioxidant activity of the fine gracilaria polysaccharide is improved. Wherein the extraction rate of the gracilaria tenuistipitata polysaccharide reaches more than 25 wt%, the hydroxyl radical clearance rate of the gracilaria tenuistipitata polysaccharide reaches more than 54%, and the two indexes simultaneously far exceed the prior art. Probably because the structures of the bromelain, the cellulase and the pectinase are close, and the optimal enzymolysis pH is close, the multienzyme complex system has good solubility in a similar buffer system and is easy to generate efficient enzymolysis synergistic effect at a proper pH point. And the structures of the bromelain, the cellulase and the pectinase are similar to the cellulose structure of the hedgerow powder, and the enzymes are easily adsorbed on the surface and in loose cavities of the hedgerow powder under the action of a solvent in a buffer system. In addition, most of the proteins in the hedgerow powder are in a form of glycoprotein in a chemical bonding mode, and are combined with cellulose and agarose to form a form of glycoprotein, most of high-molecular saccharides exist in the form of cellulose and agarose in the hedgerow powder, and in order to improve the extraction rate of hedgerow polysaccharide, multi-site enzyme digestion is carried out to complete the dissociation of peptide bonds, cellulose glycosidic bonds and agarose glycosidic bonds, so that the molecular weights of the glycoprotein, the cellulose and the agarose are remarkably reduced, and the solubility in a buffer system is increased. The invention further improves the action speed of the enzyme and the hedgerow powder by means of the action of the ultrasonic wave, and efficiently delivers the bromelain, the cellulase and the pectinase which are well dissolved at low concentration to the reaction sites, thereby greatly improving the enzymolysis efficiency and optimizing the molecular weight composition.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) the invention adopts an ultrasonic-assisted composite enzymolysis technology to prepare the gracilaria tenuistipitata polysaccharide, and the method utilizes ultrasonic and multi-enzyme composite single-step enzymolysis to perform multiple dispersion on fine gracilaria tenuistipitata powder and multiple crushing on gracilaria tenuistipitata cells, thereby not only shortening the extraction time, but also greatly improving the extraction rate (the extraction rate is 25.01-38.60 wt%) of the gracilaria tenuistipitata polysaccharide, and the method has mild process conditions and simple process.
(2) The gracilaria tenuistipitata polysaccharide obtained by the method has strong oxidation resistance, has a good scavenging effect on free radicals, and has a hydroxyl radical scavenging rate of 54.94-65.54% (the polysaccharide concentration is 1.2 mg/mL)-1) Can be used as a natural antioxidant with high nutritive and health-care values.
Detailed Description
For a better understanding of the present invention, the present invention is further explained below with reference to examples, but the embodiments of the present invention are not limited thereto.
Unless otherwise specified, the experimental procedures or conditions used in the following examples are conventional procedures, conventional conditions, or procedures and conditions recommended by the manufacturer of the apparatus.
In the following examples, the extraction rate was measured by the phenol-sulfuric acid method. The phenol-sulfuric acid method was prepared and measured according to the method proposed by Cuesta et al (j. microbiological. methods.,2003,52(1), 69-73). The specific operation is as follows: taking 2mL of the properly diluted extracting solution, adding 1mL of 5 wt% phenol solution into a test tube, shaking uniformly, adding 5mL of 98% sulfuric acid, shaking uniformly, standing at room temperature for 30min, measuring absorbance on an ultraviolet-visible spectrophotometer, and drawing a standard curve by taking glucose dried at 105 ℃ for 2h as a reference to calculate the extraction rate, wherein the extraction rate is calculated by the content of the glucose.
In the following examples, the clearance of hydroxyl radicals is measured and the measurement is carried out by the method of the reference (science and technology in food industry, 2005,26(3),67-69) with a concentration of 0.5mL and 1.2 mg/mL-1The sample solution was put into a 7mL centrifuge tube with a cap, and 1mL of a phosphate buffer solution (0.1M) having a pH of 7.4 was added thereto, followed by 0.5mL of 0.75 mmol/L-1After shaking the phenanthroline solution evenly, 0.5mL of 0.75 mmol.L is added-1Rapidly shaking ammonium ferrous sulfate solution, adding 0.5mL of 0.01% (w/w) hydrogen peroxide solution, sealing tube, shaking, keeping at 37 deg.C for 60min, cooling to room temperature, measuring absorbance at 510nm with distilled water as reference, and recording absorbance Asample(ii) a Replacing the sample solution with distilled water, treating, measuring, and recording absorbance value as Ablank(ii) a Replacing the sample solution and hydrogen peroxide with distilled water, treating, and measuring with absorbance value A0. The hydroxyl radical clearance rate is calculated by the formula (I):
example 1
Taking 1.0g of gracilaria tenuistipitata powder, adding 40mL of prepared disodium hydrogen phosphate-citric acid buffer solution with the pH value of 5.5, stirring on a magnetic stirrer to ensure that the powder fully absorbs water and is uniformly mixed, then placing the powder in an ultrasonic cleaner which is heated to 55 ℃, setting the ultrasonic time to be 40min and the ultrasonic power to be 150W, and carrying out ultrasonic treatment.
After the ultrasonic treatment is finished, 1mL of bromelain solution (15 mg. mL) is added according to the dosage of 3 wt% of compound enzyme-1) And 1mL cellulase solution (15 mg. mL)-1) Then immediately transferring the mixture into a constant-temperature water bath at 55 ℃ for enzymolysis for 120 min. Here Bromelain, Chitino chemical products Co., Ltd. in Zhengzhou province (the same sources of Bromelain were used in the following examples); cellulase CELLUCLAST 1.5L, Novoxin (China) Biotech limited (the same cellulase source is used in the examples below).
And after the enzymolysis is finished, transferring the mixture into a boiling water bath kettle for 20min, then immediately centrifuging for 10min under the condition of 5000rpm, collecting supernatant, adding 3 times of volume of absolute ethyl alcohol into the supernatant to precipitate polysaccharide, then centrifuging for 5min under the condition of 3000rpm, after the centrifugation is finished, collecting ethanol precipitate, washing the ethanol precipitate for 3 times with absolute ethyl alcohol, and then drying in a vacuum oven at 50-60 ℃ for 48h to obtain a solid, namely the gracilaria tenuistipitata polysaccharide.
The extraction rate is 28.36 +/-3.84% by adopting a phenol-sulfuric acid method.
The clearance rate of hydroxyl free radical is 64.00 percent (polysaccharide concentration is 1.2 mg/mL)-1)。
Detailed description of the documentThe best oxidation resistance index of the gracilaria verrucosa polysaccharide is 1.25 mg/mL-1And hydroxyl radical clearance of 51.54% (food industry science and technology, 2005,26(3), 67-69).
Example 2
Taking 1.0g of gracilaria tenuistipitata powder, adding 100mL of prepared disodium hydrogen phosphate-citric acid buffer solution with the pH value of 5.5, stirring on a magnetic stirrer to ensure that the powder fully absorbs water and is uniformly mixed, then placing the powder in an ultrasonic cleaner which is heated to 55 ℃, setting the ultrasonic time to be 40min and the ultrasonic power to be 150W, and carrying out ultrasonic treatment.
After the ultrasonic treatment is finished, 1mL of bromelain solution (10 mg. mL) is added according to the dosage of 3 wt% of compound enzyme-1) And 1mL cellulase solution (20 mg. mL)-1) Then immediately transferring the mixture into a constant-temperature water bath at 55 ℃ for enzymolysis for 120 min.
And after the enzymolysis is finished, transferring the mixture into a boiling water bath kettle for 20min, then immediately centrifuging for 10min under the condition of 5000rpm, collecting supernatant, adding 3 times of volume of absolute ethyl alcohol into the supernatant to precipitate polysaccharide, then centrifuging for 5min under the condition of 3000rpm, after the centrifugation is finished, collecting ethanol precipitate, washing the ethanol precipitate for 3 times with absolute ethyl alcohol, and then drying in a vacuum oven at 50-60 ℃ for 48h to obtain a solid, namely the gracilaria tenuistipitata polysaccharide.
The extraction rate is 38.52 + -3.93% by phenol-sulfuric acid method.
The clearance rate of hydroxyl free radical is 63.59% (polysaccharide concentration is 1.2 mg/mL)-1)。
Example 3
Taking 1.0g of gracilaria tenuistipitata powder, adding 80mL of prepared disodium hydrogen phosphate-citric acid buffer solution with the pH value of 5.5, stirring on a magnetic stirrer to ensure that the powder fully absorbs water and is uniformly mixed, then placing the powder in an ultrasonic cleaner which is heated to 55 ℃, setting the ultrasonic time to be 20min and the ultrasonic power to be 150W, and carrying out ultrasonic treatment.
After the ultrasonic treatment is finished, 1mL of bromelain solution (12.5 mg. mL) is added according to the dosage of 3 wt% of compound enzyme-1) And 1mL of cellulase solution (17.5 mg. multidot.mL)-1) Then immediately transferring the mixture into a constant-temperature water bath at 55 ℃ for enzymolysis,the time is 120 min.
And after the enzymolysis is finished, transferring the mixture into a boiling water bath kettle for 20min, then immediately centrifuging for 10min under the condition of 5000rpm, collecting supernatant, adding 3 times of volume of absolute ethyl alcohol into the supernatant to precipitate polysaccharide, then centrifuging for 5min under the condition of 3000rpm, after the centrifugation is finished, collecting ethanol precipitate, washing the ethanol precipitate for 3 times with absolute ethyl alcohol, and then drying in a vacuum oven at 50-60 ℃ for 48h to obtain a solid, namely the gracilaria tenuistipitata polysaccharide.
The extraction rate is 33.16 + -1.78% by phenol-sulfuric acid method.
The clearance rate of hydroxyl radical is 64.96% (polysaccharide concentration is 1.2 mg. mL)-1)。
Example 4
Taking 1.0g of gracilaria tenuistipitata powder, adding 60mL of prepared disodium hydrogen phosphate-citric acid buffer solution with the pH value of 5.5, stirring on a magnetic stirrer to ensure that the powder fully absorbs water and is uniformly mixed, then placing the powder in an ultrasonic cleaner which is heated to 55 ℃, setting the ultrasonic time to be 40min and the ultrasonic power to be 90W, and carrying out ultrasonic treatment.
After the ultrasonic treatment is finished, 1mL of bromelain solution (10 mg. mL) is added according to the dosage of 3 wt% of compound enzyme-1) And 1mL cellulase solution (20 mg. mL)-1) Then immediately transferring the mixture into a constant-temperature water bath at 55 ℃ for enzymolysis for 120 min.
And after the enzymolysis is finished, transferring the mixture into a boiling water bath kettle for 20min, then immediately centrifuging for 10min under the condition of 5000rpm, collecting supernatant, adding 3 times of volume of absolute ethyl alcohol into the supernatant to precipitate polysaccharide, then centrifuging for 5min under the condition of 3000rpm, after the centrifugation is finished, collecting ethanol precipitate, washing the ethanol precipitate for 3 times with absolute ethyl alcohol, and then drying in a vacuum oven at 50-60 ℃ for 48h to obtain a solid, namely the gracilaria tenuistipitata polysaccharide.
The extraction rate is 25.01 +/-3.82% by adopting a phenol-sulfuric acid method.
The clearance rate of hydroxyl free radical is 54.94% (polysaccharide concentration is 1.2 mg/mL)-1)。
Example 5
Taking 1.0g of gracilaria tenuistipitata powder, adding 60mL of prepared disodium hydrogen phosphate-citric acid buffer solution with the pH value of 5.5, stirring on a magnetic stirrer to ensure that the powder fully absorbs water and is uniformly mixed, then placing the powder in an ultrasonic cleaner which is heated to 55 ℃, setting the ultrasonic time to be 40min and the ultrasonic power to be 120W, and carrying out ultrasonic treatment.
After the ultrasonic treatment is finished, 1mL of bromelain solution (10 mg. mL) is added according to the dosage of 3 wt% of compound enzyme-1) And 1mL cellulase solution (20 mg. mL)-1) Then immediately transferring the mixture into a constant-temperature water bath at 55 ℃ for enzymolysis for 120 min.
And after the enzymolysis is finished, transferring the mixture into a boiling water bath kettle for 20min, then immediately centrifuging for 10min under the condition of 5000rpm, collecting supernatant, adding 3 times of volume of absolute ethyl alcohol into the supernatant to precipitate polysaccharide, then centrifuging for 5min under the condition of 3000rpm, after the centrifugation is finished, collecting ethanol precipitate, washing the ethanol precipitate for 3 times with absolute ethyl alcohol, and then drying in a vacuum oven at 50-60 ℃ for 48h to obtain a solid, namely the gracilaria tenuistipitata polysaccharide.
The extraction rate is 30.62 +/-0.99 percent by adopting a phenol-sulfuric acid method.
The clearance rate of hydroxyl free radical is 61.72 percent (polysaccharide concentration is 1.2 mg/mL)-1)。
Example 6
Taking 1.0g of gracilaria tenuistipitata powder, adding 60mL of prepared disodium hydrogen phosphate-citric acid buffer solution with the pH value of 5.5, stirring on a magnetic stirrer to ensure that the powder fully absorbs water and is uniformly mixed, then placing the powder in an ultrasonic cleaner which is heated to 55 ℃, setting the ultrasonic time to be 40min and the ultrasonic power to be 90W, and carrying out ultrasonic treatment.
After the ultrasonic treatment is finished, 1mL of bromelain solution (10 mg. mL) is added according to the dosage of 3 wt% of compound enzyme-1) And 1mL cellulase solution (20 mg. mL)-1) Then immediately transferring the mixture into a constant-temperature water bath at 55 ℃ for enzymolysis for 180 min.
And after the enzymolysis is finished, transferring the mixture into a boiling water bath kettle for 20min, then immediately centrifuging for 10min under the condition of 5000rpm, collecting supernatant, adding 3 times of volume of absolute ethyl alcohol into the supernatant to precipitate polysaccharide, then centrifuging for 5min under the condition of 3000rpm, after the centrifugation is finished, collecting ethanol precipitate, washing the ethanol precipitate for 3 times with absolute ethyl alcohol, and then drying in a vacuum oven at 50-60 ℃ for 48h to obtain a solid, namely the gracilaria tenuistipitata polysaccharide.
The extraction rate is 37.17 +/-4.51 percent by adopting a phenol-sulfuric acid method.
The clearance rate of hydroxyl free radical is 55.85 percent (polysaccharide concentration is 1.2 mg/mL)-1)。
Example 7
Taking 1.0g of gracilaria tenuistipitata powder, adding 60mL of prepared disodium hydrogen phosphate-citric acid buffer solution with the pH value of 4.5, stirring on a magnetic stirrer to ensure that the powder fully absorbs water and is uniformly mixed, then placing the powder in an ultrasonic cleaner which is heated to 45 ℃, setting the ultrasonic time to be 40min and the ultrasonic power to be 150W, and carrying out ultrasonic treatment.
After the ultrasonic treatment is finished, 1mL of bromelain solution (3.33 mg. mL) is added according to the dosage of 1 wt% of compound enzyme-1) And 1mL of cellulase solution (6.67 mg. multidot.mL)-1) Then immediately transferring the mixture into a constant-temperature water bath at 45 ℃ for enzymolysis for 60 min.
And after the enzymolysis is finished, transferring the mixture into a boiling water bath kettle for 20min, then immediately centrifuging for 10min under the condition of 5000rpm, collecting supernatant, adding 3 times of volume of absolute ethyl alcohol into the supernatant to precipitate polysaccharide, then centrifuging for 5min under the condition of 3000rpm, after the centrifugation is finished, collecting ethanol precipitate, washing the ethanol precipitate for 3 times with absolute ethyl alcohol, and then drying in a vacuum oven at 50-60 ℃ for 48h to obtain a solid, namely the gracilaria tenuistipitata polysaccharide.
The extraction rate is 34.51 +/-1.14 percent by adopting a phenol-sulfuric acid method.
The clearance rate of hydroxyl free radical is 65.54 percent (polysaccharide concentration is 1.2 mg/mL)-1)。
Example 8
Taking 1.0g of gracilaria tenuistipitata powder, adding 60mL of prepared disodium hydrogen phosphate-citric acid buffer solution with the pH value of 4.5, stirring on a magnetic stirrer to ensure that the powder fully absorbs water and is uniformly mixed, then placing the powder in an ultrasonic cleaner which is heated to 65 ℃, setting the ultrasonic time to be 40min and the ultrasonic power to be 150W, and carrying out ultrasonic treatment.
After the ultrasonic treatment is finished, 1mL of bromelain solution (10 mg. mL) is added according to the dosage of 3 wt% of compound enzyme-1) And 1mL of pectinase solution (20 mg. mL)-1Pectinase Pectinase C80max, DSM group in the Netherlands), immediately transferring to a constant temperature water bath of 65 ℃ for enzymolysis for 60 min.
And after the enzymolysis is finished, transferring the mixture into a boiling water bath kettle for 20min, then immediately centrifuging for 10min under the condition of 5000rpm, collecting supernatant, adding 3 times of volume of absolute ethyl alcohol into the supernatant to precipitate polysaccharide, then centrifuging for 5min under the condition of 3000rpm, after the centrifugation is finished, collecting ethanol precipitate, washing the ethanol precipitate for 3 times with absolute ethyl alcohol, and then drying in a vacuum oven at 50-60 ℃ for 48h to obtain a solid, namely the gracilaria tenuistipitata polysaccharide.
The extraction rate is 37.83 +/-0.40% by adopting a phenol-sulfuric acid method.
The clearance rate of hydroxyl free radical is 65.49% (polysaccharide concentration is 1.2 mg/mL)-1)。
Example 9
Taking 1.0g of gracilaria tenuistipitata powder, adding 60mL of prepared disodium hydrogen phosphate-citric acid buffer solution with the pH value of 5.5, stirring on a magnetic stirrer to ensure that the powder fully absorbs water and is uniformly mixed, then placing the powder in an ultrasonic cleaner which is heated to 65 ℃, setting the ultrasonic time to be 40min and the ultrasonic power to be 150W, and carrying out ultrasonic treatment.
After the ultrasonic treatment is finished, 1mL of bromelain solution (16.7 mg. mL) is added according to the dosage of 5 wt% of compound enzyme-1) And 1mL of cellulase solution (33.3 mg. multidot.mL)-1) Then immediately transferring the mixture into a constant-temperature water bath at 65 ℃ for enzymolysis for 60 min.
And after the enzymolysis is finished, transferring the mixture into a boiling water bath kettle for 20min, then immediately centrifuging for 10min under the condition of 5000rpm, collecting supernatant, adding 3 times of volume of absolute ethyl alcohol into the supernatant to precipitate polysaccharide, then centrifuging for 5min under the condition of 3000rpm, after the centrifugation is finished, collecting ethanol precipitate, washing the ethanol precipitate for 3 times with absolute ethyl alcohol, and then drying in a vacuum oven at 50-60 ℃ for 48h to obtain a solid, namely the gracilaria tenuistipitata polysaccharide.
The extraction rate is 38.60 +/-1.13% by adopting a phenol-sulfuric acid method.
The clearance rate of hydroxyl free radical is 63.67 percent (polysaccharide concentration is 1.2 mg/mL)-1)。
Example 10
Taking 1.0g of gracilaria tenuistipitata powder, adding 60mL of prepared disodium hydrogen phosphate-citric acid buffer solution with the pH value of 5.5, stirring on a magnetic stirrer to ensure that the powder fully absorbs water and is uniformly mixed, then placing the powder in an ultrasonic cleaner which is heated to 65 ℃, setting the ultrasonic time to be 30min and the ultrasonic power to be 150W, and carrying out ultrasonic treatment.
After the ultrasonic treatment is finished, 1mL of bromelain solution (16.7 mg. mL) is added according to the dosage of 5 wt% of compound enzyme-1) And 1mL of cellulase solution (33.3 mg. multidot.mL)-1) Then immediately transferring the mixture into a constant-temperature water bath at 65 ℃ for enzymolysis for 60 min.
And after the enzymolysis is finished, transferring the mixture into a boiling water bath kettle for 20min, then immediately centrifuging for 10min under the condition of 5000rpm, collecting supernatant, adding 3 times of volume of absolute ethyl alcohol into the supernatant to precipitate polysaccharide, then centrifuging for 5min under the condition of 3000rpm, after the centrifugation is finished, collecting ethanol precipitate, washing the ethanol precipitate for 3 times with absolute ethyl alcohol, and then drying in a vacuum oven at 50-60 ℃ for 48h to obtain a solid, namely the gracilaria tenuistipitata polysaccharide.
The extraction rate is 38.20 + -1.08% by phenol-sulfuric acid method.
The clearance rate of hydroxyl free radical is 64.90 percent (polysaccharide concentration is 1.2 mg/mL)-1)。
Example 11
Taking 1.0g of gracilaria tenuistipitata powder, adding 60mL of prepared disodium hydrogen phosphate-citric acid buffer solution with the pH value of 5.5, stirring on a magnetic stirrer to ensure that the powder fully absorbs water and is uniformly mixed, then placing the powder in an ultrasonic cleaner which is heated to 65 ℃, setting the ultrasonic time to be 10min and the ultrasonic power to be 150W, and carrying out ultrasonic treatment.
After the ultrasonic treatment is finished, 1mL of bromelain solution (16.7 mg. mL) is added according to the dosage of 5 wt% of compound enzyme-1) And 1mL of cellulase solution: (33.3mg·mL-1) Then immediately transferring the mixture into a constant-temperature water bath at 65 ℃ for enzymolysis for 60 min.
And after the enzymolysis is finished, transferring the mixture into a boiling water bath kettle for 20min, then immediately centrifuging for 10min under the condition of 5000rpm, collecting supernatant, adding 3 times of volume of absolute ethyl alcohol into the supernatant to precipitate polysaccharide, then centrifuging for 5min under the condition of 3000rpm, after the centrifugation is finished, collecting ethanol precipitate, washing the ethanol precipitate for 3 times with absolute ethyl alcohol, and then drying in a vacuum oven at 50-60 ℃ for 48h to obtain a solid, namely the gracilaria tenuistipitata polysaccharide.
The extraction rate is 33.19 +/-1.90% by adopting a phenol-sulfuric acid method.
The clearance rate of hydroxyl radical is 59.63% (polysaccharide concentration is 1.2 mg. mL)-1)。
It should be understood that the above embodiments are only used for illustrating the technical solutions of the present invention, and are not used for limiting the protection scope of the present invention. Various changes or modifications may be made by those skilled in the art, and equivalents of such various changes or modifications are also within the scope of the invention as defined by the appended claims.
Claims (9)
1. The method for extracting the gracilaria tenuistipitata polysaccharide by ultrasonic-assisted composite enzymolysis is characterized by comprising the following steps of:
1) adding the decolorized fine gracilaria powder into a buffer solution, uniformly stirring, and performing ultrasonic treatment;
2) after the ultrasonic treatment is finished, adding a complex enzyme for enzymolysis; the compound enzyme is bromelain and a second component enzyme, the second component enzyme is cellulase or pectinase, and the mass ratio of the bromelain to the second component enzyme is 1: 1-1: 2; the using amount of the complex enzyme is 1-5 wt% of the fine gracilaria powder;
3) after enzymolysis, inactivating enzyme by adopting boiling water bath, separating an extracting solution by centrifugation and suction filtration, and collecting supernatant;
4) and carrying out alcohol precipitation, centrifugation, washing and drying on the extracting solution to obtain the gracilaria tenuistipitata polysaccharide extract.
2. The method for extracting Gracilaria tenuistipitata polysaccharide by ultrasonic-assisted composite enzymolysis according to claim 1, wherein the buffer solution is citric acid-disodium hydrogen phosphate buffer solution, the pH is 4.5-6.5, and the concentration of disodium hydrogen phosphate is 0.05 +/-0.005 mol x L-1The mass ratio of the volume of the buffer solution to the powder is 40: 1-100: 1.
3. The method for extracting Gracilaria tenuistipitata polysaccharide by ultrasonic-assisted composite enzymolysis according to claim 1, wherein the water bath temperature during the ultrasonic treatment is 45-65 ℃, the ultrasonic power is 90-150W, and the ultrasonic time is 10-40 min.
4. The method for extracting Gracilaria tenuistipitata polysaccharide by ultrasonic-assisted composite enzymolysis according to claim 1, wherein the enzymolysis temperature of the enzymolysis treatment is 45-65 ℃, and the enzymolysis time is 60-180 min.
5. The method for extracting Gracilaria tenuistipitata polysaccharide by ultrasonic-assisted composite enzymolysis according to claim 1, wherein the enzyme inactivation is carried out by inactivating the enzyme in a boiling water bath, and the extraction solution is subjected to inactivation in the boiling water bath for more than 20 min.
6. The method for extracting Gracilaria tenuissima polysaccharide by ultrasonic-assisted composite enzymolysis according to claim 1, wherein the rotation speed of the centrifugation in step 3) is above 4500rpm, and the time of the centrifugation is above 8 min; the rotating speed of the centrifugation in the step 4) is more than 3000rpm, and the time of the centrifugation is more than 5 min.
7. The method for extracting Gracilaria tenuistipitata polysaccharide by ultrasonic-assisted composite enzymolysis according to claim 1, wherein the alcohol precipitation is that three times of volume of absolute ethanol is added into the supernatant to precipitate the polysaccharide, and the precipitate is collected.
8. The method for extracting Gracilaria tenuistipitata polysaccharide by ultrasonic-assisted composite enzymolysis according to claim 1, wherein the washing is to wash the precipitate with absolute ethanol for more than 2 times; the drying is carried out in a vacuum oven at 50-70 ℃ for 40-60 h.
9. The method for extracting Gracilaria tenuistipitata polysaccharide by ultrasonic-assisted compound enzymolysis according to claim 1, wherein the extraction rate of the Gracilaria tenuistipitata polysaccharide by ultrasonic-assisted compound enzymolysis is 25.01-38.60 wt% as determined by phenol-sulfuric acid method.
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