CN115120642A - Method for simultaneously preparing rubusoside, total amino acids and polysaccharide from rubus suavissimus - Google Patents
Method for simultaneously preparing rubusoside, total amino acids and polysaccharide from rubus suavissimus Download PDFInfo
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Abstract
The invention discloses a method for simultaneously preparing rubusoside, total amino acids and polysaccharide from rubus suavissimus, which comprises enzymolysis extraction, total amino acid preparation, rubus suavissimus polysaccharide preparation and rubus suavissimus glycoside preparation. According to the method for simultaneously preparing rubusoside, total amino acids and polysaccharide from sweet tea, cellulase is used for hydrolysis, so that the cell walls of plants can be damaged, effective components can be promoted to enter a solvent more easily, and the extraction efficiency is improved. The invention reasonably uses the separation medium, and the extract is firstly absorbed by the cation exchange resin, thereby greatly improving the recovery rate and yield of the amino acid. After the effluent liquid and the water eluent which are subjected to the amino acid adsorption by the cation exchange resin pass through the activated carbon column, the exogenous pollutants such as pesticide residue heavy metals and the like can be removed by utilizing the adsorption effect of the activated carbon, and the activated carbon is a nonpolar adsorbent and can be used as a separation material, so that the polysaccharide can be preliminarily separated, and the rubusoside can be separated.
Description
Technical Field
The invention relates to the technical field of plant extraction, in particular to a method for simultaneously preparing rubusoside, total amino acids and polysaccharide from rubus suavissimus.
Background
Sweet tea (Rubus suavissimus S.Lee) is perennial deciduous shrub of Rubus of Rosaceae, and is mainly distributed in Guangxi, Yunnan, Sichuan and Hunan. The sweet tea contains various effective components, mainly including protein, total amino acids, vitamins, sweet tea polyphenols, total flavonoids, sweet tea glycosides, polysaccharides, minerals, etc. Wherein the protein and the total amino acid account for 11.8 to 12.6 percent of the weight of the dry sweet tea, and the sweet tea contains 8 kinds of total amino acid which are necessary for human bodies, wherein the content of the total amino acid is about 4.5 percent; the rubusoside accounts for about 1.5% of the dry weight of the sweet tea leaves, and the sweet tea leaves also contain abundant polysaccharides. The sweet tea total amino acids have the effects of resisting oxidation, improving immunity and the like, the polysaccharide plays an important role in the aspects of nerve protection, liver protection and aging resistance, and the rubusoside serving as a non-saccharide natural sweetener has wider application prospect in the market.
Patent CN201810688181.X discloses a method for extracting theanine and simultaneously extracting rubusoside and tea polyphenol from sweet tea, which comprises the steps of carrying out water extraction, sequentially passing the water extract through macroporous resin, polyamide resin and ion exchange resin, and collecting effluent to respectively prepare 3 effective components of theanine, rubusoside and tea polyphenol. The method realizes the comprehensive utilization of sweet tea resources, has strong operability, low cost and little sewage discharge, and is suitable for industrial production.
Patent CN200910244874.0 discloses a method for separating sweet tea polysaccharide by an ultrafiltration membrane, in the method, after sweet tea is extracted by water or aqueous alcohol, leaching liquor is filtered by a centrifugal membrane and a microfiltration membrane, and then is ultrafiltered by ultrafiltration membranes with two specifications, and trapped fluid is subjected to reduced pressure concentration to separate sweet tea polysaccharide from impurities. The method only aims at extracting polysaccharide, raw materials are not reasonably utilized, and the sweet tea glycoside which is most widely applied is not reasonably utilized, so that a great deal of waste of the raw materials is caused.
Patent CN201510700707.8 discloses a process for simultaneously preparing rubusoside and rubusoside total polyphenol, in the method, macroporous resin is used for enriching the extract, and flavone, polyphenol and rubusoside are separated by stages, so that 3 products can be obtained.
In summary, in the current process, the extraction process of the sweet tea total amino acids and polysaccharides is seen, and the preparation of the sweet tea total amino acids and polysaccharides in the sweet tea leaves is not reported yet while the sweet tea glycosides are prepared, so the invention provides a method for simultaneously preparing the sweet tea glycosides, the total amino acids and the polysaccharides from the sweet tea, and simultaneously preparing the sweet tea glycosides, the total amino acids and the polysaccharides.
Disclosure of Invention
The present invention aims to provide a method for simultaneously preparing rubusoside, total amino acids and polysaccharide from rubus suavissimus to solve the problems of the background art.
In order to achieve the purpose, the invention provides the following technical scheme:
a method for simultaneously preparing rubusoside, total amino acids and polysaccharide from rubus suavissimus comprises the following steps:
the method comprises the following steps: performing enzymolysis extraction, namely adding the sweet tea coarse powder raw material into a buffer solution, adding an enzyme preparation, then extracting, and separating extraction residues and filtrate after extraction is finished, wherein the filtrate is reserved;
step two: preparing total amino acids, namely passing the filtrate obtained in the step one through cation exchange resin, collecting column effluent, then eluting by using water and alkali alcohol, collecting alkali alcohol eluent, and further purifying by using a membrane separation technology to obtain a total amino acid extract;
step three: preparing sweet tea polysaccharide, namely mixing the column effluent and the water eluent in the step two, passing through an active carbon column, eluting by using water and aqueous alcohol solution respectively, collecting water eluent, concentrating, and precipitating with ethanol to prepare the sweet tea polysaccharide;
step four: the preparation method of rubusoside comprises concentrating the aqueous alcohol eluate of step three to remove alcohol, making into water phase, passing through polyamide resin column, eluting with water and aqueous alcohol respectively, collecting the aqueous alcohol eluate, and drying to obtain rubusoside extract.
As a further scheme of the invention: in the first step, the sweet tea is ground and then sieved by a sieve of 10-24 meshes, the extraction condition is that the sweet tea is extracted in a constant temperature oscillator or a constant temperature stirring tank, the ratio of the raw materials to the buffer solution is 1:6-10, the extraction temperature is 50-65 ℃, the extraction time is 1-2h, the extraction frequency is 1-2 times, and the oscillation/stirring frequency is 60-150 times/min.
As a still further scheme of the invention: in the first step, the buffer solution is one of citric acid-sodium citrate, acetic acid-sodium acetate and phosphoric acid-sodium phosphate, the pH value is 5-6.5, the enzyme preparation is 1 or 2 of cellulase, pectinase and hemicellulase, and the addition amount of the enzyme preparation is 0.5-2% of the weight of the raw materials.
As a still further scheme of the invention: in the first step, a centrifugal machine is used for separation, and the rotating speed is 3000-.
As a still further scheme of the invention: and in the second step, one or two of D001, HD52 and D061 is selected as the cation exchange resin, the cation exchange resin is washed by pure water until the effluent liquid is colorless, then is soaked for 4 hours by 10 percent hydrochloric acid and washed by pure water until the pH value is stable, then is soaked for 4 hours by 5 percent NaOH and washed by pure water until the pH value is stable, the cation exchange resin can be used, the column diameter ratio is 1:4-8, the dosage of the upper column liquid is 4-8BV, the upper column flow rate is 0.8-1.5BV/h, the dosage of the water eluent is 1.5-3BV, the elution flow rate is 1-2BV/h, the pH value of the alkali alcohol is 8-9, the used alkali is ammonia water, sodium hydroxide and sodium bicarbonate, the alcohol is 60-70 percent ethanol (v/v), the elution flow rate is 1-2BV/h, and the dosage is 3-5 BV.
As a still further scheme of the invention: the ultrafiltration membrane selected by the membrane separation technology in the step two is a cluster hollow fiber membrane and a roll-type membrane, and the molecular weight cutoff is 1000-20000.
As a still further scheme of the invention: the activated carbon in the third step is selected from granular activated carbon and powdered activated carbon, wherein the powdered activated carbon needs to be mixed with diatomite for use at a ratio of 1:1-2, the powdered activated carbon is selected from one of coconut shells, fruit shells and wood activated carbon, the column loading ratio is 1:6-10, the column loading flow rate is 0.8-1.5BV/h, the column loading amount is 5-8BV, the water elution flow rate is 1-2BV/h, the elution amount is 2-4BV, the volume content of alcohol in the aqueous alcohol is 60-75%, the elution flow rate is 0.8-2BV/h, and the elution amount is 3-6 BV.
As a still further scheme of the invention: the alcohol used in the alcohol precipitation in the third step is one of 95% ethanol and absolute ethanol, the ethanol is added into the solution until the ethanol content is 75% -85%, the alcohol precipitation times are 1-3 times, the precipitation solvent is 75% -85% ethanol solution, and the alcohol precipitation time is 8-24 h.
As a still further scheme of the invention: the diameter-column ratio of the polyamide column in the fourth step is 1:5-8, water is added to dilute the column loading liquid until the concentration of rubusoside contained in the column loading liquid is 1.5-4mg/ml, the column loading flow rate is 0.5-1.5BV/h, the column loading amount is 3.5-6BV, the water elution flow rate is 1-2BV/h, the elution amount is 1-2.5BV, the aqueous alcohol is 70-85% ethanol solution, the elution flow rate is 1-3BV/h, and the elution amount is 2-4 BV.
As a still further scheme of the invention: the drying mode in the fourth step is spray drying.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention uses cellulase for hydrolysis, can destroy plant cell walls, promotes effective components to enter a solvent more easily, improves extraction efficiency, saves energy and reduces production cost.
2. The invention reasonably uses the separation medium, the extract is firstly absorbed by the cation exchange resin, and the recovery rate and the yield of the amino acid can be greatly improved.
3. After the effluent liquid and the water eluent which are subjected to the amino acid adsorption by the cation exchange resin pass through the activated carbon column, the exogenous pollutants such as pesticide residue heavy metals and the like can be removed by utilizing the adsorption effect of the activated carbon, and the activated carbon is a nonpolar adsorbent and can be used as a separation material, so that the polysaccharide can be preliminarily separated, and the rubusoside can be separated.
4. In the prior art, the rubusoside is extracted and separated by combining a macroporous resin column, an anion-cation resin column and other purification modes, the steps are complex, the loss rate is high, the rubusoside extract with high content and no pesticide residue heavy metal can be obtained only by adding an activated carbon column and a polyamide column, the recovery rate is high, the steps are simple, and the operation is easy.
5. The invention provides a method for simultaneously preparing three effective components, which fully utilizes resources, does not involve the use of harmful reagents in the whole process, is green and environment-friendly, can recycle separation media, has short production period and simple and easy industrial preparation, and the prepared product has wide application.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the embodiment of the invention, a method for simultaneously preparing rubusoside, total amino acids and polysaccharide from rubus suavissimus comprises the following steps:
the method comprises the following steps: performing enzymolysis extraction, namely adding the sweet tea coarse powder raw material into a buffer solution, adding an enzyme preparation, then extracting, and separating extraction residues and filtrate after extraction is finished, wherein the filtrate is reserved;
step two: preparing total amino acids, namely passing the filtrate obtained in the step one through cation exchange resin, discharging column effluent, then eluting by using water and alkali alcohol, collecting alkali alcohol eluent, and further purifying by using a membrane separation technology to obtain a total amino acid extract;
step three: preparing sweet tea polysaccharide, namely mixing the column effluent liquid and the water eluent in the step two, passing through an activated carbon column, eluting by using water and aqueous alcohol solution respectively, collecting water eluent, concentrating, and precipitating with ethanol to prepare the sweet tea polysaccharide;
step four: the rubusoside is prepared by concentrating the aqueous alcohol eluate of step three, removing alcohol, making into water phase, passing through polyamide resin column, eluting with water and aqueous alcohol solution, collecting the aqueous alcohol eluate, and drying to obtain rubusoside extract.
In the first step, the sweet tea is ground and then sieved by a sieve of 10-24 meshes, the extraction condition is that the sweet tea is extracted in a constant temperature oscillator or a constant temperature stirring tank, the ratio of the raw materials to the buffer solution is 1:6-10, the extraction temperature is 50-65 ℃, the extraction time is 1-2h, the extraction frequency is 1-2 times, and the oscillation/stirring frequency is 60-150 times/min.
In the first step, the buffer solution is one of citric acid-sodium citrate, acetic acid-sodium acetate and phosphoric acid-sodium phosphate, the pH is 5-6.5, the enzyme preparation is 1 or 2 of cellulase, pectinase and hemicellulase, and the addition amount of the enzyme preparation is 0.5-2% of the raw materials.
In the first step, a centrifuge is used for separation, and the rotating speed is 3000-.
In the second step, one or two of D001, HD52 and D061 is selected as the cation exchange resin, the cation exchange resin is pretreated by washing with pure water until the effluent is colorless, then soaking with 10% hydrochloric acid for 4h, washing with pure water until the pH value is stable, soaking with 5% NaOH for 4h, washing with pure water until the pH value is stable, the column diameter ratio is 1:4-8, the dosage of the upper column liquid is 4-8BV, the upper column flow rate is 0.8-1.5BV/h, the dosage of the water eluent is 1.5-3BV, the eluent flow rate is 1-2BV/h, the pH of the alkali alcohol is 8-9, the alkali used is ammonia water, sodium hydroxide and sodium bicarbonate, the alcohol is 60-70% ethanol (v/v), the eluent flow rate is 1-2BV/h, and the dosage is 3-5 BV.
The ultrafiltration membrane selected by the membrane separation technology in the step two is a cluster hollow fiber membrane and a roll-type membrane, and the molecular weight cutoff is 1000-20000.
In the third step, the active carbon is selected from granular active carbon and powdered active carbon, wherein the powdered active carbon is required to be mixed with diatomite in a ratio of 1:1-2 for use, the powdered active carbon is selected from one of coconut shells, fruit shells and wood active carbon, the column packing diameter column ratio is 1:6-10, the column loading flow rate is 0.8-1.5BV/h, the column loading amount is 5-8BV, the water elution flow rate is 1-2BV/h, the elution amount is 2-4BV, the volume content of alcohol in the aqueous alcohol solution is 60-75%, the elution flow rate is 0.8-2BV/h, and the elution amount is 3-6 BV.
The alcohol used in the alcohol precipitation in the third step is one of 95% alcohol and absolute alcohol, the alcohol is added into the solution until the content of the alcohol is 75% -85%, the number of the alcohol precipitation is 1-3, the precipitation solvent is 75% -85% alcohol solution, and the alcohol precipitation time is 8-24 h.
The diameter-column ratio of the polyamide in the fourth step is 1:5-8, water is added to dilute the column-loading solution until the concentration of rubusoside contained in the column-loading solution is 1.5-4mg/ml, the column-loading flow rate is 0.5-1.5BV/h, the column loading amount is 3.5-6BV, the water elution flow rate is 1-2BV/h, the elution amount is 1-2.5BV, the aqueous alcohol solution is 70-85% ethanol solution, the elution flow rate is 1-3BV/h, and the elution amount is 2-4 BV.
The drying mode in the fourth step is spray drying.
The first embodiment is as follows:
the method comprises the following steps: taking 50g of sweet tea leaves which are crushed and sieved by a 24-mesh sieve, sequentially adding 500mL of citric acid-sodium citrate buffer solution and 0.25g of cellulase, adjusting the pH to be 5, placing the mixture in a constant-temperature oscillator at 50 ℃ for 1h at the frequency of 60 times/min, centrifuging at 3000r/min after the completion, and collecting supernatant.
Step two: d001 cation exchange resin is filled into a column according to a column diameter ratio of 1:4, the column is pretreated for standby, supernatant obtained in the step one passes through the cation exchange resin at a flow rate of 0.8BV/h, the sample loading amount is 4BV, after the sample loading is finished, 1.5BV of pure water is used for elution at a flow rate of 1BV/h, then 60% ethanol solution with the pH value adjusted to 8 by ammonia water is used for elution, the flow rate is 1BV/h, the elution amount is 3BV, and column effluent liquid, water eluent and alkali alcohol eluent are respectively collected. Centrifuging the alkali alcohol eluent at 5000r/min, collecting supernatant, sequentially passing through hollow fiber membranes with molecular weight cutoff of 10000 and 1000, collecting concentrated solution, spray drying to obtain total amino acid, weighing 2.1g, and determining total amino acid content by adopting an indantrione method.
And (3) measuring results: the total amino acid content in the powder was 89.9%, with a recovery of 91.25%.
Step three: and (2) combining the column effluent liquid and the water eluent in the step two, passing the combined liquid through a pretreated granular activated carbon column with a diameter-column ratio of 1:6 at a flow rate of 1.5BV/h, wherein the column loading amount is 5BV, the column loading flow rate is 0.8BV/h, after adsorption is finished, respectively eluting the combined liquid by using water and 70% ethanol, the water eluent is 2BV, the elution flow rate is 1BV/h, the 60% ethanol eluent is 3BV, and the elution flow rate is 0.8BV/h, and respectively collecting water eluent and 60% ethanol eluent. Concentrating the water eluent to 40% of the volume of the stock solution, adding 95% ethanol until the ethanol content in the solution is 75%, uniformly stirring, standing for 12h, filtering, collecting precipitate, drying to obtain 1.12g of crude polysaccharide, dissolving the precipitate with 150mL of water, adding 95% ethanol until the ethanol concentration in the solution is 80%, uniformly stirring, standing for 18h, filtering, collecting precipitate, drying to obtain 0.76g of polysaccharide, and determining the polysaccharide content by a phenol-concentrated sulfuric acid method.
And (3) measuring results: the content of polysaccharide in the powder was 90.25%.
Step four: concentrating 70% ethanol eluate in step three to remove alcohol, adding water to dilute until rubusoside content is 1.5mg/mL, passing through polyamide resin column with diameter column ratio of 1:5 at flow rate of 0.5mL/min, loading amount is 3.5BV, after adsorption, eluting with water and 70% ethanol respectively, eluting with water at flow rate of 1BV/min and 70% ethanol at flow rate of 2BV/min, collecting 70% ethanol eluate, concentrating, drying to obtain 0.98g rubusoside, and determining rubusoside content by HPLC.
And (3) measuring results: the rubusoside content was 89.26%.
Example two:
the method comprises the following steps: taking 1kg of sweet tea leaves which are ground and sieved by a 10-mesh sieve, sequentially adding 10L of acetic acid-sodium acetate buffer solution, 5g of cellulase and 6g of pectinase, adjusting the pH to 6, placing the mixture in a constant temperature oscillator at 65 ℃ for 1.5h at the frequency of 100 times/min, centrifuging at 4000r/min after completion, and collecting supernatant.
Step two: d061 cation exchange resin is loaded into a column according to a diameter-column ratio of 1:6, the column is pretreated for later use, supernatant obtained in the first step passes through the cation exchange resin at a flow rate of 1.5BV/h, the sample loading amount is 8BV, after the sample loading is finished, pure water of 3BV is used for elution at a flow rate of 2BV/h, then 60% ethanol solution containing sodium hydroxide and having a pH value of 9 is used for elution, the flow rate is 2BV/h, the elution amount is 5BV, and column effluent, water eluent and alkaline alcohol eluent are collected respectively. Centrifuging the alkali alcohol eluent at 5000r/min, collecting supernatant, sequentially passing through hollow fiber membranes with molecular weight cutoff of 20000 and 2000, collecting concentrated solution, spray drying to obtain total amino acid, weighing 40.6g, and determining total amino acid content by ninhydrin method.
And (3) measuring results: the total amino acid content in the powder was 87.8% with a recovery of 93.25%.
Step three: and (2) combining the column effluent and the water eluent in the second step, passing through a pretreated powdered activated carbon column (powdered activated carbon: diatomite is 1:1) with a diameter-column ratio of 1:8 at a flow rate of 1.5BV/h, wherein the column loading is 8BV, after adsorption is finished, respectively eluting with water and 70% ethanol, wherein the water elution amount is 4BV, the elution flow rate is 2BV/h, the 70% ethanol elution amount is 6BV, and the elution flow rate is 1.5BV/h, and respectively collecting the water eluent and the 70% ethanol eluent. Concentrating the water eluent to 40% of the stock solution, adding 95% ethanol until the ethanol content in the solution is 80%, uniformly stirring, standing for 12h, filtering, collecting precipitate, drying to obtain 22.57g of crude polysaccharide, dissolving the precipitate with water, adding 95% ethanol until the ethanol concentration in the solution is 85%, uniformly stirring, standing for 12h, filtering, collecting precipitate, drying to obtain 20.38g of polysaccharide, and determining the polysaccharide content by a phenol-concentrated sulfuric acid method.
And (3) measuring results: the content of polysaccharide in the powder was 92.16%.
Step four: concentrating 70% ethanol eluate in step three to remove alcohol, diluting with water until the solution contains rubusoside 4mg/mL, passing through polyamide resin column with diameter column ratio of 1:8 at flow rate of 1.5mL/min, loading sample amount of 6BV, eluting with water and 70% ethanol respectively, eluting with water at flow rate of 2.5 BV/min and 70% ethanol at flow rate of 3BV/min, collecting 70% ethanol eluate, concentrating, drying to obtain 19.38g rubusoside, and determining rubusoside content by HPLC.
And (3) measuring results: the rubusoside content is 87.79%.
Example three:
the method comprises the following steps: taking 500kg of sweet tea leaves which are ground and sieved by a 10-mesh sieve, sequentially adding 4000L of phosphoric acid-sodium phosphate buffer solution, 5kg of hemicellulose and 3.5kg of cellulase, adjusting the pH to 6, placing the mixture into a reflux stirring tank at the temperature of 50 ℃, keeping the stirring speed for 2 hours at 60 times/min, centrifuging at 4000r/min after the stirring is finished, and collecting supernatant.
Step two: d001 cation exchange resin is filled into a column according to the diameter-column ratio of 1:6, the column is pretreated for standby, supernatant obtained in the first step passes through the cation exchange resin at the flow rate of 1BV/h, the sample loading amount is 6BV, after the sample loading is finished, 2BV of pure water is used for elution at the flow rate of 1.5BV/h, then 65 percent ethanol solution containing ammonia water with the pH of 8.5 is used for elution, the flow rate is 1.5BV/h, the elution amount is 4BV, and column effluent liquid, eluting water and alkaline alcohol eluent are collected respectively. Centrifuging the alkali alcohol eluent at 4000r/min, taking supernatant, sequentially passing the supernatant through a hollow fiber membrane with the molecular weight cutoff of 10000 and 1000, collecting concentrated solution, spray drying to obtain total amino acid, weighing 23.58kg, and determining the content of the total amino acid by adopting an indantrione method.
And (3) measuring results: the total amino acid content in the powder was 90.18% with a recovery of 90.18%.
Step three: and (2) combining the column effluent liquid and the water eluent in the step two, passing the combined column effluent liquid through a pretreated granular activated carbon column with a diameter-column ratio of 1:8 at a flow rate of 1BV/h, wherein the column loading amount is 7BV, the column loading flow rate is 1BV/h, after adsorption is finished, respectively eluting the combined column effluent liquid by using water and 70% ethanol, the water eluent is 3BV, the elution flow rate is 1.5BV/h, the 70% ethanol eluent is 5BV, and the elution flow rate is 1BV/h, and respectively collecting the water eluent and the 60% ethanol eluent. Concentrating the water eluent to 40% of the stock solution, adding 95% ethanol until the ethanol content in the solution is 80%, uniformly stirring, standing for 12h, filtering, collecting precipitate, drying to obtain 12.31kg of crude polysaccharide, dissolving the precipitate with water, adding 95% ethanol until the ethanol concentration in the solution is 80%, uniformly stirring, standing for 24h, filtering, collecting precipitate, drying to obtain 10.26kg of polysaccharide, and determining the polysaccharide content by a phenol-concentrated sulfuric acid method.
And (3) measuring results: the content of polysaccharide in the powder was 91.34%.
Step four; concentrating 70% ethanol eluate in step three to remove alcohol, adding water to dilute until rubusoside content is 3.1mg/mL, passing through polyamide resin column with diameter-column ratio of 1:6 at flow rate of 1mL/min, loading amount is 5BV, after adsorption, respectively eluting with water and 70% ethanol, collecting 70% ethanol eluate, concentrating and drying to obtain rubusoside 10.28kg, and determining rubusoside content by HPLC.
And (3) measuring results: the rubusoside content is 88.87%.
Through the first to the third embodiments, the invention can simultaneously extract and prepare rubusoside, total amino acids and polysaccharide to the utmost extent.
Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes in the embodiments and/or modifications of the invention can be made, and equivalents and modifications of some features of the invention can be made without departing from the spirit and scope of the invention.
Claims (10)
1. A method for simultaneously preparing rubusoside, total amino acids and polysaccharide from rubus suavissimus is characterized in that: the method comprises the following steps:
the method comprises the following steps: performing enzymolysis extraction, namely adding the sweet tea coarse powder raw material into a buffer solution, adding an enzyme preparation, then extracting, and separating extraction residues and filtrate after extraction is finished, wherein the filtrate is reserved;
step two: preparing total amino acids, namely passing the filtrate obtained in the step one through cation exchange resin, collecting column effluent, then eluting by using water and alkali alcohol, collecting alkali alcohol eluent, and further purifying by using a membrane separation technology to obtain a total amino acid extract;
step three: preparing sweet tea polysaccharide, namely mixing the column effluent liquid and the water eluent in the step two, passing through an activated carbon column, eluting by using water and aqueous alcohol solution respectively, collecting water eluent, concentrating, and precipitating with ethanol to prepare the sweet tea polysaccharide;
step four: the rubusoside is prepared by concentrating the aqueous alcohol eluate of step three, removing alcohol, making into water phase, passing through polyamide resin column, eluting with water and aqueous alcohol solution, collecting the aqueous alcohol eluate, and drying to obtain rubusoside extract.
2. The method for simultaneously preparing rubusoside, total amino acids and polysaccharide from rubus suavissimus according to claim 1, wherein: in the first step, the sweet tea is ground and then sieved by a sieve of 10-24 meshes, the extraction condition is that the sweet tea is extracted in a constant temperature oscillator or a constant temperature stirring tank, the ratio of the raw materials to the buffer solution is 1:6-10, the extraction temperature is 50-65 ℃, the extraction time is 1-2h, the extraction frequency is 1-2 times, and the oscillation/stirring frequency is 60-150 times/min.
3. The method for simultaneously preparing rubusoside, total amino acids and polysaccharide from rubus suavissimus according to claim 1, wherein: in the first step, the buffer solution is one of citric acid-sodium citrate, acetic acid-sodium acetate and phosphoric acid-sodium phosphate, the pH value is 5-6.5, the enzyme preparation is 1 or 2 of cellulase, pectinase and hemicellulase, and the addition amount of the enzyme preparation is 0.5-2% of the weight of the raw materials.
4. The method for simultaneously preparing rubusoside, total amino acids and polysaccharide from rubus suavissimus according to claim 1, wherein: in the first step, a centrifugal machine is used for separation, and the rotating speed is 3000-.
5. The method for simultaneously preparing rubusoside, total amino acids and polysaccharide from rubus suavissimus according to claim 1, wherein: and in the second step, one or two of D001, HD52 and D061 is selected as the cation exchange resin, the cation exchange resin is washed by pure water until the effluent liquid is colorless, then is soaked for 4 hours by 10 percent hydrochloric acid and washed by pure water until the pH value is stable, then is soaked for 4 hours by 5 percent NaOH and washed by pure water until the pH value is stable, the cation exchange resin can be used, the column diameter ratio is 1:4-8, the dosage of the upper column liquid is 4-8BV, the upper column flow rate is 0.8-1.5BV/h, the dosage of the water eluent is 1.5-3BV, the elution flow rate is 1-2BV/h, the pH value of the alkali alcohol is 8-9, the used alkali is ammonia water, sodium hydroxide and sodium bicarbonate, the alcohol is 60-70 percent ethanol (v/v), the elution flow rate is 1-2BV/h, and the dosage is 3-5 BV.
6. The method for simultaneously preparing rubusoside, total amino acids and polysaccharide from rubus suavissimus according to claim 1, wherein: the ultrafiltration membrane selected by the membrane separation technology in the step two is a cluster hollow fiber membrane and a roll-type membrane, and the molecular weight cutoff is 1000-20000.
7. The method for simultaneously preparing rubusoside, total amino acids and polysaccharide from rubus suavissimus according to claim 1, wherein: the activated carbon in the third step is selected from granular activated carbon and powdered activated carbon, the powdered activated carbon is selected from one of coconut shells, fruit shells and wood activated carbon, the column ratio of column diameter is 1:6-10, the flow rate of column loading is 0.8-1.5BV/h, the column loading amount is 5-8BV, the flow rate of water elution is 1-2BV/h, the elution amount is 2-4BV, the volume content of alcohol in the aqueous alcohol is 60-75%, the elution flow rate is 0.8-2BV/h, and the elution amount is 3-6 BV.
8. The method for simultaneously preparing rubusoside, total amino acids and polysaccharide from rubus suavissimus according to claim 1, wherein: the alcohol used in the alcohol precipitation in the third step is one of 95% ethanol and absolute ethanol, the ethanol is added into the solution until the ethanol content is 75% -85%, the alcohol precipitation times are 1-3 times, the precipitation solvent is 75% -85% ethanol solution, and the alcohol precipitation time is 8-24 h.
9. The method for simultaneously preparing rubusoside, total amino acids and polysaccharide from rubus suavissimus according to claim 1, wherein: the diameter-column ratio of the polyamide column in the fourth step is 1:5-8, water is added to dilute the column loading liquid until the concentration of rubusoside contained in the column loading liquid is 1.5-4mg/ml, the column loading flow rate is 0.5-1.5BV/h, the column loading amount is 3.5-6BV, the water elution flow rate is 1-2BV/h, the elution amount is 1-2.5BV, the aqueous alcohol is 70-85% ethanol solution, the elution flow rate is 1-3BV/h, and the elution amount is 2-4 BV.
10. The method for simultaneously preparing rubusoside, total amino acids and polysaccharide from rubus suavissimus according to claim 1, wherein: the drying mode in the fourth step is spray drying.
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| CN116284173A (en) * | 2023-01-31 | 2023-06-23 | 湖南华诚生物资源股份有限公司 | Method for simultaneously preparing rubusoside and tea polyphenol from sweet tea |
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| CN116284173A (en) * | 2023-01-31 | 2023-06-23 | 湖南华诚生物资源股份有限公司 | Method for simultaneously preparing rubusoside and tea polyphenol from sweet tea |
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