CN115120642B - Method for simultaneously preparing rubusoside, total amino acid and polysaccharide from sweet tea - Google Patents
Method for simultaneously preparing rubusoside, total amino acid and polysaccharide from sweet tea Download PDFInfo
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Abstract
The invention discloses a method for simultaneously preparing rubusoside, total amino acid and polysaccharide from sweet tea, which comprises the steps of enzymolysis extraction, preparation of total amino acid, preparation of sweet tea polysaccharide and preparation of rubusoside. According to the method for preparing rubusoside, total amino acid and polysaccharide from sweet tea simultaneously, cellulose is used for hydrolysis, so that plant cell walls can be damaged, active ingredients are promoted to enter a solvent more easily, and the extraction efficiency is improved. The invention reasonably uses the separation medium, and the extracting solution is firstly adsorbed by the cation exchange resin, so that the recovery rate and the yield of the amino acid can be greatly improved. After the effluent liquid from the column and the water eluent after the amino acid is adsorbed by the cation exchange resin pass through the activated carbon column, exogenous pollutants such as pesticide residue heavy metal and the like can be removed by utilizing the adsorption effect of the activated carbon, and the activated carbon is a nonpolar adsorbent, so that polysaccharide can be primarily separated on one hand and rubusoside can be separated on the other hand when the adsorbent is used as a separation material.
Description
Technical Field
The invention relates to the technical field of plant extraction, in particular to a method for simultaneously preparing rubusoside, total amino acid and polysaccharide from rubus.
Background
Sweet tea (Rubus suavissimus S.Lee) is a perennial defoliated bush of Rubus genus of Rosaceae family, and is mainly distributed in Guangxi, yunnan, sichuan and Hunan etc. The sweet tea contains various effective components, mainly including proteins, total amino acids, vitamins, sweet tea polyphenols, total flavonoids, rubusoside, polysaccharides, minerals, etc. Wherein the protein and total amino acid account for 11.8% -12.6% of the dry sweet tea weight, and contains 8 total amino acids essential to human body, wherein the total amino acid content is about 4.5%; the rubusoside accounts for about 1.5% of dry weight of the sweet tea, and meanwhile, the sweet tea also contains abundant polysaccharide. The sweet tea total amino acid has the effects of resisting oxidation, improving immunity and the like, and the polysaccharide plays an important role in neuroprotection, liver protection and aging resistance, and the sweet tea glycoside is used as a non-saccharide natural sweetener, so that the sweet tea total amino acid has wider application prospect in the market.
Patent cn201810688181.X discloses a method for extracting theanine from sweet tea and simultaneously extracting rubusoside and tea polyphenol, wherein the method comprises the steps of sequentially passing water extraction and water extract through macroporous resin, polyamide resin and ion exchange resin, and collecting effluent liquid to respectively prepare 3 active ingredients of theanine, rubusoside and tea polyphenol. The method realizes the comprehensive utilization of sweet tea resources, has strong operability, low cost and small sewage discharge, and is suitable for industrial production.
Patent CN200910244874.0 discloses a method for separating sweet tea polysaccharide by ultrafiltration membrane, in the method, after sweet tea is extracted by water or water-containing alcohol, the extract is filtered by centrifugation and microfiltration membrane, and then is ultrafiltered by ultrafiltration membranes of two specifications, and the retentate is decompressed and concentrated to separate sweet tea polysaccharide from impurities. The method only aims at extracting polysaccharide, the raw materials are not reasonably utilized, and the rubusoside which is most widely applied is not reasonably utilized, so that a great amount of raw materials are wasted.
Patent CN201510700707.8 discloses a process for preparing rubusoside and rubus total polyphenol simultaneously, in the method, macroporous resin is used for enriching the extracting solution, flavone, polyphenol and rubusoside are separated in sections, 3 products can be obtained, and the rubus polyphenol is obtained while the main active ingredients of the rubus are extracted, so that the comprehensive utilization of the rubus is improved.
In summary, in the current technology, the extraction technology of total amino acids and polysaccharides of sweet tea is seen, and the preparation of total amino acids and polysaccharides in sweet tea is not reported at the same time of preparing sweet tea glycoside, so the invention provides a method for simultaneously preparing sweet tea glycoside, total amino acids and polysaccharides from sweet tea, and simultaneously preparing sweet tea glycoside, total amino acids and polysaccharides.
Disclosure of Invention
The invention aims to provide a method for simultaneously preparing rubusoside, total amino acids and polysaccharides from sweet tea, so as to solve the problems in the prior art.
In order to achieve the above purpose, the present invention provides the following technical solutions:
a method for simultaneously preparing rubusoside, total amino acids and polysaccharide from sweet tea comprises the following steps:
step one: enzymolysis extraction, namely adding a sweet tea coarse powder raw material into a buffer solution, adding an enzyme preparation, extracting, and separating extraction residues and filtrate for later use after the extraction is completed;
step two: preparing total amino acid, namely passing the filtrate in the first step through cation exchange resin, collecting column effluent, eluting by using water and alkali alcohol, collecting alkali alcohol eluent, and further purifying by a membrane separation technology to obtain total amino acid extract;
step three: preparing sweet tea polysaccharide, combining column effluent liquid and water eluent in the second step, eluting by using water and aqueous alcohol solution respectively through an activated carbon column, collecting water eluent, concentrating and precipitating with alcohol to prepare sweet tea polysaccharide;
step four: and (3) preparing rubusoside, namely concentrating the aqueous alcohol eluent in the step (III) to remove alcohol, preparing a water phase, then passing through a polyamide resin column, eluting with water and aqueous alcohol respectively, collecting the aqueous alcohol eluent, and drying to obtain the rubusoside extract.
As a further scheme of the invention: crushing sweet tea, sieving with a 10-24 mesh sieve, extracting under the conditions of a constant temperature oscillator or a constant temperature stirring tank, wherein the ratio of raw materials to buffer solution is 1:6-10, the extraction temperature is 50-65 ℃, the extraction time is 1-2h, the extraction times are 1-2 times, and the oscillation/stirring frequency is 60-150 times/min.
As still further aspects of the invention: the buffer solution in the first step 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, pectase and hemicellulase, and the addition amount of the enzyme preparation is 0.5-2% of the weight of the raw materials.
As still further aspects of the invention: and in the first step, a centrifugal machine is used for separation, and the rotating speed is 3000-5000r/min.
As still further aspects of the invention: the cation exchange resin in the second step is one or two of D001, HD52 and D061, the pretreatment method of the cation exchange resin is that pure water is used for washing until effluent liquid is colorless, 10% hydrochloric acid is used for soaking for 4 hours, after pure water is used for washing until pH is stable, 5% NaOH is used for soaking for 4 hours, pure water is used for washing until pH value is stable, the radial column ratio is 1:4-8, the dosage of column loading liquid is 4-8BV, the flow rate of column loading is 0.8-1.5BV/h, the dosage of water eluting liquid is 1.5-3BV, the eluting flow rate is 1-2BV/h, the pH value of the alkali alcohol is 8-9, the alkali used is ammonia water, sodium hydroxide and sodium bicarbonate alcohol is 60% -70% ethanol (v/v), the eluting flow rate is 1-2BV/h, and the dosage is 3-5BV.
As still further aspects of the invention: the ultrafiltration membrane adopted in the membrane separation technology in the step two is a clustered hollow fiber membrane or a coiled membrane, and the molecular weight cut-off is 1000-20000.
As still further aspects of the invention: in the third step, the activated carbon is selected from granular activated carbon and powdery activated carbon, wherein the powdery activated carbon is required to be mixed with diatomite for use in a ratio of 1:1-2, the powdery activated carbon is selected from one of coconut shells, fruit shells and wood activated carbon, the column loading 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 is 60% -75%, the elution flow rate is 0.8-2BV/h, and the elution amount is 3-6BV.
As still further aspects of the invention: the alcohol used in the alcohol precipitation in the step three is one of 95% ethanol and absolute ethanol, the ethanol is added into the solution until the ethanol content is 75% -85%, the times of alcohol precipitation are 1-3 times, the precipitation solvent is 75% -85% ethanol solution, and the alcohol precipitation time is 8-24 hours.
As still further aspects of the invention: the diameter column ratio of the polyamide column in the step four is 1:5-8, water is added to dilute the column feeding liquid until the concentration of rubusoside contained in the column feeding liquid is 1.5-4mg/ml, the flow rate of the column feeding liquid is 0.5-1.5BV/h, the column feeding 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 2-4BV.
As still further aspects of the invention: and in the fourth step, the drying mode is spray drying.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention uses cellulase to hydrolyze, which can destroy plant cell wall, promote active ingredient to enter into solvent more easily, improve extraction efficiency, save energy and reduce production cost.
2. The invention reasonably uses the separation medium, and the extracting solution is firstly adsorbed by the cation exchange resin, so that the recovery rate and the yield of the amino acid can be greatly improved.
3. After the effluent liquid from the column and the water eluent after the amino acid is adsorbed by the cation exchange resin pass through the activated carbon column, exogenous pollutants such as pesticide residue heavy metal and the like can be removed by utilizing the adsorption effect of the activated carbon, and the activated carbon is a nonpolar adsorbent, so that polysaccharide can be primarily separated on one hand and rubusoside can be separated on the other hand when the adsorbent is used as a separation material.
4. In the prior art, the rubusoside is obtained by combining a macroporous resin column with an anion-cation resin column and extracting and separating in other purification modes, the steps are complex, the loss rate is high, and the rubusoside extract with high content and no pesticide residue heavy metal can be obtained only by adding the polyamide column to the activated carbon column, so that the recovery rate is high, the steps are simple, and the operation is easy.
5. The invention provides a method for preparing three active ingredients simultaneously, which fully utilizes resources, does not involve the use of harmful reagents in the whole process, is environment-friendly, can recycle separation media, has short production period and simple industrial preparation, and can be widely applied to the prepared products.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
In the embodiment of the invention, the method for simultaneously preparing rubusoside, total amino acid and polysaccharide from sweet tea comprises the following steps:
step one: enzymolysis extraction, namely adding a sweet tea coarse powder raw material into a buffer solution, adding an enzyme preparation, extracting, and separating extraction residues and filtrate for later use after the extraction is completed;
step two: preparing total amino acid, namely passing the filtrate in the first step through cation exchange resin, discharging column effluent, eluting by using water and alkali alcohol, collecting alkali alcohol eluent, and further purifying by a membrane separation technology to obtain total amino acid extract;
step three: preparing sweet tea polysaccharide, combining column effluent liquid and water eluent in the second step, eluting by using water and aqueous alcohol solution respectively through an activated carbon column, collecting water eluent, concentrating and precipitating with alcohol to prepare sweet tea polysaccharide;
step four: and (3) preparing rubusoside, namely concentrating the aqueous alcohol eluent in the step (III) to remove alcohol, preparing a water phase, then passing through a polyamide resin column, eluting by using water and aqueous alcohol solution respectively, collecting the aqueous alcohol eluent, and drying to obtain the rubusoside extract.
Crushing sweet tea, sieving with a 10-24 mesh sieve, extracting under the conditions of a constant temperature oscillator or a constant temperature stirring tank, wherein the ratio of raw materials to buffer solution is 1:6-10, the extraction temperature is 50-65 ℃, the extraction time is 1-2h, the extraction times are 1-2 times, and the oscillation/stirring frequency is 60-150 times/min.
The buffer solution in the first step 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, pectase and hemicellulase, and the addition amount of the enzyme preparation is 0.5-2% of the raw material.
In the first step, a centrifugal machine is used for separation, and the rotating speed is 3000-5000r/min.
In the second step, the cation exchange resin is selected from one or two of D001, HD52 and D061, the method of pretreatment of the cation exchange resin is that pure water is used for washing until effluent liquid is colorless, 10% hydrochloric acid is used for soaking for 4 hours, after pure water is used for washing until pH is stable, 5% NaOH is used for soaking for 4 hours, pure water is used for washing until pH value is stable, the radial column ratio is 1:4-8, the dosage of column loading liquid is 4-8BV, the flow rate of column loading is 0.8-1.5BV/h, the dosage of water eluting liquid is 1.5-3BV, the eluting flow rate is 1-2BV/h, the pH value of alkali alcohol is 8-9, the alkali used is ammonia water, sodium hydroxide and sodium bicarbonate, alcohol is 60% -70% ethanol (v/v), the eluting flow rate is 1-2BV/h, and the dosage is 3-5BV.
The ultrafiltration membrane adopted in the membrane separation technology in the second step is a clustered hollow fiber membrane or a rolled membrane, and the molecular weight cut-off is 1000-20000.
In the third step, the activated carbon is selected from granular activated carbon and powdery activated carbon, wherein the powdery activated carbon is required to be mixed with diatomite for use in a ratio of 1:1-2, the powdery activated carbon is selected from one of coconut shells, fruit shells and wood activated carbon, the column loading 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 3-6BV.
And in the third step, the alcohol used in the alcohol precipitation is one of 95% ethanol and absolute ethanol, the ethanol is added into the solution until the ethanol content is 75% -85%, the times of alcohol precipitation are 1-3 times, the precipitation solvent is 75% -85% ethanol solution, and the alcohol precipitation time is 8-24 hours.
In the fourth step, the diameter column ratio of the polyamide is 1:5-8, water is added to dilute the column feeding liquid until the concentration of rubusoside contained in the column feeding liquid is 1.5-4mg/ml, the column feeding flow rate is 0.5-1.5BV/h, the column feeding amount is 3.5-6BV, the water eluting flow rate is 1-2BV/h, the eluting amount is 1-2.5BV, the aqueous alcohol solution is 70-85% ethanol solution, the eluting flow rate is 1-3BV/h, and the eluting amount is 2-4BV.
And in the fourth step, the drying mode is spray drying.
Embodiment one:
step one: taking 50g of crushed 24-mesh-sieve sweet tea leaves, sequentially adding 500mL of citric acid-sodium citrate buffer solution and 0.25g of cellulase, adjusting the pH to 5, placing the sweet tea leaves in a constant temperature oscillator at 50 ℃ for 60 times/min, keeping the sweet tea leaves for 1h, centrifuging the sweet tea leaves at 3000r/min after completion, and collecting supernatant.
Step two: loading the D001 cation exchange resin into a column according to a radial column ratio of 1:4, pretreating for later use, taking supernatant fluid in the step, passing through the cation exchange resin at a flow rate of 0.8BV/h, eluting with pure water of 1.5BV at a flow rate of 1BV/h after the sample loading is finished, and then eluting with 60% ethanol solution with pH value of 8 regulated by ammonia water at a flow rate of 1BV/h, wherein the eluting amount is 3BV, and collecting column effluent liquid, water eluent and alkali alcohol eluent respectively. Centrifuging the alkaline alcohol eluent at 5000r/min, taking supernatant, sequentially passing through hollow fiber membranes with molecular weight cut-off of 10000 and 1000, collecting concentrated solution, spray drying to obtain total amino acid, weighing to 2.1g, and measuring total amino acid content by ninhydrin method.
Measurement results: the total amino acid content in the powder was 89.9% and recovery was 91.25%.
Step three: and (3) after the column effluent and the water eluent in the step two are combined, the mixture passes through a pretreated granular activated carbon column with a radial column ratio of 1:6 at a flow rate of 1.5BV, the column loading amount is 5BV, the column loading flow rate is 0.8BV/h, water and 70% ethanol are respectively used for eluting after the adsorption is finished, the water eluent and the 70% ethanol are respectively used for eluting, the water eluent is 2BV, the eluting flow rate is 1BV/h, the 60% ethanol eluent is 3BV, the eluting flow rate is 0.8BV/h, and the water eluent and the 60% ethanol eluent are respectively collected. Concentrating the water eluent to 40% of the original liquid volume, adding 95% ethanol until the ethanol content in the solution is 75%, stirring uniformly, 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%, stirring uniformly, standing for 18h, filtering, collecting precipitate, drying to obtain 0.76g of polysaccharide, and measuring the polysaccharide content by a phenol-concentrated sulfuric acid method.
Measurement results: the polysaccharide content of the powder was 90.25%.
Step four: in the third step, 70% ethanol eluent is concentrated to remove alcohol, water is added to dilute the mixture until the content of rubusoside is 1.5mg/mL, the mixture passes through a polyamide resin column with a diameter-column ratio of 1:5 at a flow rate of 0.5mL/min, the sample loading amount is 3.5BV, water and 70% ethanol are respectively used for eluting after the adsorption is finished, the water eluent is 1BV, the flow rate is 1BV/min, the 70% ethanol is 2BV, the flow rate is 1BV/min,70% ethanol eluent is collected, and 0.98g of rubusoside is obtained through concentration and drying, and the content of rubusoside is measured through HPLC.
Measurement results: the rubusoside content is 89.26%.
Embodiment two:
step one: taking 1kg of crushed 10-mesh-sieve sweet tea leaves, sequentially adding 10L of acetic acid-sodium acetate buffer solution, 5g of cellulase and 6g of pectase, adjusting the pH to 6, placing the sweet tea leaves 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: loading the column of the D061 cation exchange resin at a radial column ratio of 1:6, pretreating for later use, taking the supernatant fluid obtained in the step one, passing through the cation exchange resin at a flow rate of 1.5BV/h, eluting with pure water of 3BV at a flow rate of 2BV/h after the sample loading is completed, eluting with an ethanol solution of 60% containing sodium hydroxide with pH of 9 at a flow rate of 2BV/h, and collecting column effluent, water eluent and alkali alcohol eluent respectively at an elution rate of 5BV. Centrifuging the alkaline alcohol eluent at 5000r/min, taking supernatant, sequentially passing through hollow fiber membranes with molecular weight cut-off of 20000 and 2000, collecting concentrated solution, spray drying to obtain total amino acid, weighing 40.6g, and measuring total amino acid content by ninhydrin method.
Measurement results: the total amino acid content in the powder was 87.8% and recovery was 93.25%.
Step three: and (3) after the column effluent and the water eluent in the step two are combined, the mixture passes through a pretreated powdered activated carbon column (powdered activated carbon: diatomite=1:1) with a radial column ratio of 1:8 at a flow rate of 1.5BV/h, the water and 70% ethanol are respectively used for eluting after the adsorption is finished, the water eluent is 4BV, the elution flow rate is 2BV/h, the 70% ethanol eluent is 6BV, the elution flow rate is 1.5BV/h, and the water eluent and the 70% ethanol eluent are respectively collected. Concentrating the water eluent to 40% of the stock solution, adding 95% ethanol until the ethanol content in the solution is 80%, stirring uniformly, 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%, stirring uniformly, standing for 12h, filtering, collecting precipitate, drying to obtain 20.38g of polysaccharide, and measuring the polysaccharide content by a phenol-concentrated sulfuric acid method.
Measurement results: the polysaccharide content of the powder was 92.16%.
Step four: concentrating 70% ethanol eluent in the third step to remove alcohol, adding water to dilute until the solution contains rubusoside at 4mg/mL, passing through a polyamide resin column with a diameter-column ratio of 1:8 at a flow rate of 1.5mL/min, eluting with water and 70% ethanol respectively after adsorption, wherein the water eluent is 2.5BV, the flow rate is 2BV/min, the 70% ethanol is 4BV, the flow rate is 3BV/min, collecting 70% ethanol eluent, concentrating and drying to obtain rubusoside at 19.38g, and determining the rubusoside content by HPLC.
Measurement results: the rubusoside content is 87.79%.
Embodiment III:
step one: 500kg of crushed 10-mesh-sieve sweet tea leaves are taken, 4000L of phosphoric acid-sodium phosphate buffer solution, 5kg of hemicellulose and 3.5kg of cellulase are sequentially added, the pH is regulated to 6, the mixture is placed in a 50 ℃ reflux stirring tank, the stirring speed is 60 times/min, the mixture is kept for 2 hours, and after completion, the mixture is centrifuged at 4000r/min, and the supernatant is collected.
Step two: loading the D001 cation exchange resin into a column according to a radial column ratio of 1:6, pretreating for later use, taking the supernatant fluid in the step one, passing through the cation exchange resin at a flow rate of 1BV/h, eluting with pure water of 2BV at a flow rate of 1.5BV/h after the sample loading is finished, eluting with an ethanol solution of 65% containing ammonia water with pH of 8.5 at a flow rate of 1.5BV/h, and collecting column effluent, water eluent and alkali alcohol eluent respectively at an elution amount of 4BV. Centrifuging the alkaline alcohol eluent at 4000r/min, taking supernatant, sequentially passing through hollow fiber membranes with cutoff molecular weights of 10000 and 1000, collecting concentrated solution, spray drying to obtain total amino acid, weighing 23.58kg, and measuring total amino acid content by ninhydrin method.
Measurement results: the total amino acid content in the powder was 90.18% and recovery was 90.18%.
Step three: and (3) after the column effluent and the water eluent in the step two are combined, the mixture passes through a pretreated granular activated carbon column with a diameter-column ratio of 1:8 at a flow rate of 1BV/h, the column loading amount is 7BV, the column loading flow rate is 1BV/h, water and 70% ethanol are respectively used for eluting after the adsorption is finished, the water eluent is 3BV, the eluting flow rate is 1.5BV/h, the 70% ethanol eluent is 5BV, the eluting flow rate is 1BV/h, and the water eluent and 60% ethanol eluent are respectively collected. Concentrating the water eluent to 40% of the stock solution, adding 95% ethanol until the ethanol content in the solution is 80%, stirring uniformly, 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%, stirring uniformly, standing for 24h, filtering, collecting precipitate, drying to obtain 10.26kg of polysaccharide, and measuring the polysaccharide content by a phenol-concentrated sulfuric acid method.
Measurement results: the polysaccharide content of the powder was 91.34%.
Step four, a step four is carried out; concentrating 70% ethanol eluent in the third step to remove alcohol, adding water to dilute the eluent until the content of rubusoside is 3.1mg/mL, passing through a polyamide resin column with a diameter-column ratio of 1:6 at a flow rate of 1mL/min, eluting with water and 70% ethanol respectively after adsorption, wherein the water eluent is 1.5BV, the flow rate is 1.5BV/min, the 70% ethanol is 3BV, the flow rate is 2BV/min, collecting 70% ethanol eluent, concentrating and drying to obtain 10.28kg of rubusoside, and determining the content of rubusoside by HPLC.
Measurement results: the rubusoside content is 88.87%.
The invention can be obtained through the first to third examples, and can extract and prepare rubusoside, total amino acid and polysaccharide at the same time to the maximum extent.
Although the present invention has been described with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described, or equivalents may be substituted for elements thereof, and any modifications, equivalents, improvements and changes may be made without departing from the spirit and principles of the present invention.
Claims (1)
1. A method for simultaneously preparing rubusoside, total amino acid and polysaccharide from sweet tea is characterized in that: the method comprises the following steps:
step one: adding the coarse powder raw materials of sweet tea into buffer solution, adding enzyme preparation, extracting, separating extraction slag and filtrate for later use, crushing sweet tea, sieving with a 10-24 mesh sieve, extracting in a constant-temperature oscillator or a constant-temperature stirring tank under the condition that the ratio of raw materials to the buffer solution is 1:6-10, the extraction temperature is 50-65 ℃, the extraction time is 1-2h, the extraction times are 1-2 times, the oscillation/stirring frequency is 60-150 times/min, 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, pectase and hemicellulase, the addition amount of the enzyme preparation is 0.5-2% of the weight of the raw materials, and the separation uses a centrifuge with the rotating speed of 3000-5000r/min;
step two: the preparation of total amino acid, the filtrate in the first step is passed through cation exchange resin, the effluent of the column is collected, then water and alkali alcohol are used for eluting, the alkali alcohol eluent is collected, further purified by membrane separation technology, so as to obtain the total amino acid extract, the cation exchange resin is selected from one or two of D001, HD52 and D061, the pretreatment method of the cation exchange resin is that the effluent is washed by pure water until colorless, and then is soaked by 10% hydrochloric acid for 4 hours, and then is washed by pure water until pH is stable, soaking with 5% NaOH for 4h, washing with pure water until the pH value is stable, wherein the diameter-column ratio is 1:4-8, the dosage of column liquid is 4-8BV, the column flow rate is 0.8-1.5BV/h, the dosage of water eluent is 1.5-3BV, the elution flow rate is 1-2BV/h, the pH value of the alkaline alcohol is 8-9, the used alkali is ammonia water, sodium hydroxide and sodium bicarbonate, the alcohol is 60% -70% ethanol (v/v), the elution flow rate is 1-2BV/h, the dosage is 3-5BV, and the ultrafiltration membrane selected by the membrane separation technology is a clustered hollow fiber membrane and a coiled membrane, and the molecular weight cut-off is 1000-20000;
step three: the preparation of sweet tea polysaccharide, wherein column effluent liquid and water eluent in the second step are combined, water and aqueous alcohol solution are respectively used for eluting through an activated carbon column, water eluent is collected and concentrated, alcohol precipitation is carried out to prepare sweet tea polysaccharide, the activated carbon is selected from granular activated carbon and powdery activated carbon, the powdery activated carbon is selected from one of coconut shell, fruit shell and wood activated carbon, the column loading 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 aqueous alcohol is 60% -75%, the elution flow rate is 0.8-2BV/h, the elution amount is 3-6BV, the alcohol used in alcohol precipitation is 95% ethanol and absolute ethanol, the number of times is 1-3, the precipitation solvent is 75% -85% ethanol solution, and the alcohol precipitation time is 8-24h;
step four: and (3) preparing rubusoside, namely concentrating and dealcoholizing the aqueous alcohol eluent in the step III, preparing a water phase, then passing through a polyamide resin column, eluting with water and aqueous alcohol solution respectively, collecting the aqueous alcohol eluent, and drying to obtain rubusoside extract, wherein the diameter-column ratio of the polyamide column is 1:5-8, the aqueous alcohol eluent is added to dilute the column liquid until the rubusoside concentration is 1.5-4mg/ml, the column 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, the elution amount is 2-4BV, and the drying mode is spray drying.
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| CN101709094A (en) * | 2009-12-17 | 2010-05-19 | 天津工业大学 | Method for separating sweet tea polysaccharide by ultrafiltration membranes |
| CN108752231A (en) * | 2018-06-28 | 2018-11-06 | 湖南华诚生物资源股份有限公司 | The method extracted theanine from Sweet tea and extract Rubusoside and tea polyphenols simultaneously |
| CN113214413A (en) * | 2021-06-02 | 2021-08-06 | 成都农业科技中心 | Sweet tea polysaccharide extract with high antioxidant activity and extraction method thereof |
| CN113512081A (en) * | 2021-09-09 | 2021-10-19 | 湖南绿蔓生物科技股份有限公司 | Method for separating trilobatin and phlorizin from lithocarpus litseifolius leaves |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101709094A (en) * | 2009-12-17 | 2010-05-19 | 天津工业大学 | Method for separating sweet tea polysaccharide by ultrafiltration membranes |
| CN108752231A (en) * | 2018-06-28 | 2018-11-06 | 湖南华诚生物资源股份有限公司 | The method extracted theanine from Sweet tea and extract Rubusoside and tea polyphenols simultaneously |
| CN113214413A (en) * | 2021-06-02 | 2021-08-06 | 成都农业科技中心 | Sweet tea polysaccharide extract with high antioxidant activity and extraction method thereof |
| CN113512081A (en) * | 2021-09-09 | 2021-10-19 | 湖南绿蔓生物科技股份有限公司 | Method for separating trilobatin and phlorizin from lithocarpus litseifolius leaves |
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