A method for simultaneously preparing rubusoside and tea polyphenols from sweet tea

Abstract

The present invention provides a method for simultaneously preparing rubusoside and tea polyphenols from sweet tea. The rubusoside and tea polyphenols extracted and produced from sweet tea by the method provided by the present invention have high purity and high yield, and the process route is simple, suitable for actual production, and has strong industrialization capability. The results show that the content of rubusoside prepared by the method is ≥99% and the yield is 90%; the rubusoside prepared by the method is white in color, and no independent decolorization process is required in the entire process; the content of tea polyphenols produced by the method is ≥97% and the yield is ≥87%.

Description

Technical Field

[0001] The invention belongs to the technical field of natural product extraction, and particularly relates to a method for simultaneously preparing rubusoside and tea polyphenols from sweet tea. Background Art

[0002] Sweet tea is a perennial deciduous shrub of the genus Rubus in the Rosaceae family. It is a new species discovered in recent years. It mainly grows in the southern tropical zone. In my country, it is mainly distributed in Guangxi, Yunnan, Sichuan and Hunan. Its leaves are sweet and often used as tea, hence the name "sweet tea". It is reported that sweet tea has a variety of health benefits. Japanese research has found that sweet tea has a significant effect on the prevention and treatment of pollen allergies and other rhinitis, dermatitis, etc.; long-term drinking of sweet tea by patients with hypertension, diabetes and obesity has a good health care effect, and it is also an ideal drink for high-temperature workers; in addition, sweet tea also has the effects of clearing heat, moistening the lungs, removing phlegm and relieving cough. The reason why sweet tea has these multiple health functions is mainly because sweet tea is rich in nutrients and contains a variety of active ingredients, including rubusoside and tea polyphenols.

[0003] The main sweet ingredient of sweet tea is rubusoside, also known as rubusoside and sweetleaf rubusoside, which is a tetracyclic diterpene glycoside formed by the combination of steviol and glucose, and is similar to stevioside in chemical structure. The purity of its sweetness is close to that of sucrose, and its relative sweetness is 150 times that of a 2% sucrose solution. As a non-sucrose natural sweetener with high sweetness, low calories and pure taste, rubusoside is increasingly valued and is the most studied effective active ingredient in sweet tea for extraction and purification, and great progress has been made.

[0004] Sweet tea contains a large amount of tea polyphenols. Most of the tea polyphenols are also extracted during the extraction of rubusoside. These tea polyphenols are the main source of bitterness in the crude extract of sweet tea. Generally, they are discharged and wasted with the waste liquid when preparing rubusoside. Sweet tea polyphenols are highly effective antioxidants, and also have multiple functions such as anti-allergy, antibacterial and antiviral, and immunity enhancement. They are active substances with great development potential.

[0005] At present, the research on sweet tea mainly focuses on the extraction and separation of rubusoside, and its high-content rubusoside has been industrialized, but there is little research on sweet tea polyphenols. Sweet tea polyphenols are generally directly wasted with the wastewater during the preparation process of rubusoside. Therefore, how to prepare high-content sweet tea polyphenols while preparing rubusoside is of practical significance to improve the added value of sweet tea.

[0006] There are some public information on the preparation of rubusoside and tea polyphenols in the prior art, as follows.

[0007] CN105166198 discloses a method for preparing high-purity rubusoside, which is obtained by crushing raw materials, water extraction, membrane filtration and concentration, drying, alcohol crystallization twice, and drying. This process does not involve the preparation of tea polyphenols.

[0008] CN102702284 discloses a production process for high-purity rubusoside, which is obtained by extraction, concentration, flocculation, centrifugal separation, macroporous resin chromatography, ion exchange resin chromatography, desalting resin chromatography, composite decolorization, concentration, and drying. This process does not involve the preparation of tea polyphenols, and the process is extremely complicated, requiring targeted removal of decolorization and desalination, and undergoing three chromatography processes, and the production cost is extremely high.

[0009] CN108752231 discloses a method for extracting theanine from sweet tea and extracting rubusoside and tea polyphenols at the same time, which is through leakage leaching, macroporous adsorption resin adsorption chromatography (ethanol desorption, nanofiltration, crystallization to obtain rubusoside), polyamide resin chromatography (gradient ethanol desorption to obtain tea polyphenols), ion exchange resin chromatography (inorganic salt elution to obtain theanine). This process does not have a targeted decolorization treatment for the separation and purification of sweet glycosides, so the obtained rubusoside product must have a light yellow color, and the tea polyphenols have not been decolorized, so the product will also have color. More importantly, the "rubusoside nanofiltration" in this technical solution is dissolved with 50-99% low-carbon alcohol, and the high-concentration low-carbon alcohol is used for nanofiltration operation. The nanofiltration membrane is a high-precision organic membrane, which itself is an organic matter. According to industry common sense, conventional organic membranes are not suitable for organic solvents such as ethanol and methanol, and expensive special membranes must be used; at the same time, nanofiltration will produce high temperature, which is easy to cause the temperature to be too high during the nanofiltration process and exceed the boiling point of ethanol or methanol, which brings safety hazards.

[0010] CN114053315 discloses a method for comprehensively extracting rubusoside, total polyphenols and total flavonoids from sweet tea and its application, wherein rubusoside is obtained by water extraction, macroporous adsorption resin chromatography, gradient ethanol aqueous solution elution, high ethanol eluent, anion exchange resin, concentration and crystallization; wherein water washing and low ethanol eluent are combined and concentrated, flocculated and precipitated, pH value is adjusted, macroporous adsorption resin chromatography, ethanol aqueous solution elution, ethyl acetate extraction, concentration and drying to obtain total polyphenols of sweet tea; supernatant is adsorbed by macroporous resin, desorbed by ethanol aqueous solution, adsorbed and eluted by polyamide resin, concentrated, concentrated and dried by ethyl acetate to obtain total flavonoids of sweet tea. The process is extremely cumbersome, and rubusoside and tea polyphenols are obtained after three chromatography processes, and large-scale production is relatively difficult.

[0011] CN113637038 discloses a method for extracting bitter-free rutabaga and sweet tea polyphenols from sweet tea leaves, which is to use pulse electric field and ultrasound to coordinate hot water tank extraction, ultrafiltration and nanofiltration membrane filtration, anion exchange resin separation, and the effluent and water washing liquid are subjected to macroporous adsorption resin chromatography, alcohol water desorption, gel chromatography column chromatography, alcohol water desorption, concentration and drying to obtain bitter-free rutabaga; anion exchange resin separation dilute alkali desorption, pH adjustment, merging, ultrafiltration, special macroporous adsorption resin chromatography, desorption liquid merging and concentration to obtain sweet tea polyphenols. This process involves 5 chromatography processes, the operation process is extremely complicated, and large-scale production is relatively difficult.

[0012] CN110078775 discloses an environmentally friendly production method of high-content rubusoside and rubusoside polyphenols, which is obtained by crushing the raw materials, flocculating, desalting and decolorizing with anion and cation exchange resins, adsorbing with macroporous adsorption resins, and gradient eluting with ethanol to obtain rubusoside; the flocculated residue is treated with acid, extracted, dehydrated with sodium sulfate, cooled and crystallized, and dried to obtain the product. This process involves flocculation, which will cause a considerable portion of rubusoside to remain in the flocculated residue, resulting in a low yield. At the same time, it undergoes three chromatography steps, and the process is relatively cumbersome.

[0013] Although the above methods can prepare rubusoside and tea polyphenols at the same time, most of them need to undergo multiple chromatography to obtain rubusoside and tea polyphenols. Some of the process steps are extremely complicated, involving multiple membrane filtration or extraction, and even require special production equipment, making industrialization difficult. Therefore, a method for preparing high-purity rubusoside and tea polyphenols is needed, which does not require sophisticated and expensive production equipment, does not require complicated production processes, and can achieve simultaneous preparation of high-purity rubusoside and tea polyphenols with fewer separation processes, which has practical significance for large-scale production. Summary of the invention

[0014] In view of this, the technical problem to be solved by the present invention is to provide a method for simultaneously preparing rubusoside and tea polyphenols from sweet tea. The method provided by the present invention extracts and produces rubusoside and tea polyphenols from sweet tea with high purity and high yield. At the same time, the process route is simple, fits actual production, and has strong industrialization capability.

[0015] The present invention provides a method for simultaneously preparing rubusoside and tea polyphenols from sweet tea, comprising the following steps:

[0016] A) adjusting the hot water extract of sweet tea to an acidic pH, adding a flocculant to flocculate, and then separating the solid and liquid, concentrating and filtering to obtain a concentrated clear liquid;

[0017] B) adding a precipitant to the concentrated clear solution under continuous stirring to obtain a turbid solution, wherein the precipitant is selected from basic zinc chloride or basic calcium chloride;

[0018] C) loading the turbid liquid onto an activated carbon column under stirring, and after loading, driving the column with water, combining the driven liquid with the effluent; then, desorbing the activated carbon column, and concentrating, crystallizing I, washing crystals I, drying, and pulverizing the desorbed liquid to obtain tea polyphenols;

[0019] D) The water-dried liquid and the effluent from the upper column are combined and loaded onto a macroporous adsorption resin chromatography column. After the loading is completed, the water-dried column is loaded and the chromatography column is desorbed. The desorbed liquid is concentrated, crystallized II, washed II, dried, and crushed to obtain rubusoside.

[0020] Preferably, the sweet tea is selected from dried sweet tea leaves and / or stems;

[0021] The mass ratio of the sweet tea to hot water is 1:(15-40), preferably 1:(20-30);

[0022] The leaching temperature for preparing the hot water extract is 70-95° C., preferably 80-90° C., the number of leaching times is 2-5 times, preferably 3-4 times, and the time for each leaching is 1-4 hours, preferably 2-3 hours.

[0023] Preferably, in step A), the pH is adjusted to an acidic pH value of 4 to 6, and the pH adjuster used is hydrochloric acid;

[0024] The flocculant is selected from chitosan;

[0025] The added amount of the flocculant is 0.03-0.05% of the weight of the extract.

[0026] Preferably, in step A), the concentration is performed to a soluble solid content of 7 to 15%;

[0027] The filtration is carried out by an ultrafiltration membrane, and the membrane has a molecular weight cut-off of 5000 to 10000Da.

[0028] Preferably, in step B), the stirring speed of the continuous stirring is 10 to 30 r / min;

[0029] The amount of the precipitant added is 2-6% of the weight of the concentrated clear liquid;

[0030] In the process of obtaining the turbid liquid, the temperature of the liquid is 30-50° C., the pH is 4-6, and the stirring time is continued for 1.5-3 hours.

[0031] Preferably, in step C), the turbid liquid is stirred and loaded onto an activated carbon column, the column liquid remains turbid, the stirring speed is 20-40 r / min, and the column loading time does not exceed 12 h; the particle size of the activated carbon is 200-2000 μm;

[0032] During the process of loading the activated carbon column, the column temperature of the loading liquid and the activated carbon is maintained at 30-50°C. After the loading is completed, the temperature is naturally cooled to room temperature;

[0033] The diameter-to-height ratio of the activated carbon column is 1:4-10, and the ratio of the column volume to the weight of the sweet tea raw material is 1:0.3-1 (ml / g).

[0034] The column loading speed is 0.5-1.5 BV / h.

[0035] The water-driving volume is 2-4 BV, and the water-driving flow rate is 1-1.5 BV / h.

[0036] Preferably, in step C), the elution is desorption with an acidic ethanol aqueous solution, the acidic ethanol aqueous solution has an ethanol volume fraction of 55-70%, a pH value of 1-5, a volume of 3-6 BV, and the acid is hydrochloric acid;

[0037] The concentration is performed until the soluble solids are 30-45%;

[0038] The crystallization I is to add alcohol and chloroform to the concentrated solution, stir evenly and then place it at low temperature to stand for crystallization;

[0039] The alcohol is ethanol or methanol;

[0040] The volume ratio of the concentrated solution obtained by concentrating the alcohol and the desorption liquid is 1:0.05-0.2.

[0041] The volume ratio of the chloroform to the desorption liquid obtained after concentration is 1:0.01-0.1.

[0042] The standing temperature is -10 to 10°C and the time is 4 to 12 hours;

[0043] After the crystallization, the crystals are collected through a 50-200 μm filter bag;

[0044] The crystal washing I is to wash the crystals with an alcohol-chloroform mixed solution at -10 to 10°C, the volume ratio of the alcohol to chloroform is 1:10 to 20, and the alcohol is ethanol or methanol.

[0045] Preferably, in step D), the macroporous adsorption resin model is any one of D101, AB-8, LX-T28, and LX-T81;

[0046] The chromatographic column has a diameter-to-height ratio of 1:3-7, and a ratio of column volume to sweet tea raw material weight of 1:0.5-2 (ml / g);

[0047] The column loading speed is 0.5-1.0 BV / h.

[0048] Preferably, in step D), the water-driving volume is 2 to 4 BV, and the water-driving flow rate is 1 to 1.5 BV / h;

[0049] The desorption is desorption with an alcohol-water solution;

[0050] The alcohol is ethanol or methanol or a combination of both;

[0051] The volume fraction of alcohol in the alcohol-water solution is 55-70%

[0052] The usage amount of the alcohol water is 2.5-4 BV, and the flow rate is 0.5-1.5 BV / h.

[0053] Preferably, in step D), the concentration is performed to a soluble solid content of 45-60%;

[0054] The crystallization II is to add alcohol and dichloromethane to the concentrated solution, stir evenly and then place it at low temperature to stand for crystallization;

[0055] The alcohol is ethanol or methanol, and the volume ratio of the alcohol to the concentrated solution is 1:0.05-0.2;

[0056] The volume ratio of the dichloromethane to the concentrated liquid is 1:0.05-0.1;

[0057] The temperature of the low temperature condition is -10 to 10°C, and the standing time is 4 to 8 hours;

[0058] After the crystallization, the crystals are collected through a 50-200 μm filter bag;

[0059] The crystal washing II is to wash the crystal with a mixed solution of alcohol and dichloromethane at -10 to 10°C, and the volume ratio of the alcohol to dichloromethane is 1:1 to 2.

[0060] Compared with the prior art, the present invention provides a method for simultaneously preparing rubusoside and tea polyphenols from sweet tea, comprising the following steps: A) adjusting the hot water extract of sweet tea to an acidic pH, adding a flocculant to flocculate, and then performing solid-liquid separation, concentration and filtration to obtain a concentrated clear liquid; B) adding a precipitant to the concentrated clear liquid under continuous stirring to obtain a turbid liquid, wherein the precipitant is selected from basic zinc chloride or basic calcium chloride; C) applying the turbid liquid to an activated carbon column under stirring, and after the application of the column, water is driven out of the column, and the water-driven liquid is combined with the effluent; then, the activated carbon column is desorbed, and the desorbed liquid is concentrated, crystallized I, washed crystals I, dried, and crushed to obtain tea polyphenols; D) combining the water-driven liquid and the effluent on a macroporous adsorption resin chromatography column, and after the application of the column, water is driven out of the column, and the chromatography column is desorbed, and the desorbed liquid is concentrated, crystallized II, washed crystals II, dried, and crushed to obtain rubusoside. The invention uses hot water to extract sweet tea raw materials rich in rubusoside and tea polyphenols, adjusts the acidity of the extract to improve the stability of tea polyphenols, then adds a flocculant to remove most of the protein and tannic acid and other substances, filters to obtain a clear liquid, and then concentrates the clear liquid and filters to further remove insoluble impurities; then adjusts the concentrated clear liquid to be slightly acidic to be more conducive to the dissolution of the basic salt, then maintains a relatively high constant temperature, adds a basic salt precipitation agent, and performs precipitation under a continuous stirring condition, so that the tea polyphenols maintain fine precipitation particles and are always suspended in the precipitation turbid liquid; then, the precipitation turbid liquid is loaded onto an activated carbon column under a stirring state, and the constant temperature is maintained, and the fine tea polyphenol particles are adsorbed on the activated carbon column (the water driven liquid and the effluent are combined to prepare rubusoside later), and the activated carbon column is eluted with acidic alcohol to obtain tea polyphenols with higher purity, then the eluate is concentrated to remove the alcohol, and an alcohol chloroform solution is added to perform low-temperature crystallization I, and finally the crystals are washed I, dried and crushed to obtain high-purity tea polyphenols. The water driven liquid and the effluent of the activated carbon column are combined and loaded onto a macroporous adsorption resin chromatography column, and then driven with water, desorbed with an alcohol aqueous solution, and concentrated. After adding alcohol dichloromethane for crystallization II, the crystals are washed II, dried, and crushed to obtain high-purity rubusoside.

[0061] The results show that the content of rubusoside prepared by the method is ≥99% and the yield is 90%; the rubusoside prepared by the method is white in color, and no independent decolorization process is required in the entire process; the content of tea polyphenols produced by the method is ≥97% and the yield is ≥87%; the entire process of the present invention is simple, has wide adaptability, and is suitable for industrial production. DETAILED DESCRIPTION

[0062] The present invention provides a method for simultaneously preparing rubusoside and tea polyphenols from sweet tea, comprising the following steps:

[0063] A) adjusting the hot water extract of sweet tea to an acidic pH, adding a flocculant to flocculate, and then separating the solid and liquid, concentrating and filtering to obtain a concentrated clear liquid;

[0064] B) adding a precipitant to the concentrated clear solution under continuous stirring to obtain a turbid solution, wherein the precipitant is selected from basic zinc chloride or basic calcium chloride;

[0065] C) loading the turbid liquid onto an activated carbon column under stirring, and after loading, driving the column with water, combining the driven liquid with the effluent; then, desorbing the activated carbon column, and concentrating, crystallizing I, washing crystals I, drying, and pulverizing the desorbed liquid to obtain tea polyphenols;

[0066] D) The water-dried liquid and the effluent from the upper column are combined and loaded onto a macroporous adsorption resin chromatography column. After the loading is completed, the water-dried column is loaded and the chromatography column is desorbed. The desorbed liquid is concentrated, crystallized II, washed II, dried, and crushed to obtain rubusoside.

[0067] The invention uses sweet tea as raw material, wherein the sweet tea is selected from dried sweet tea leaves and / or stems.

[0068] The invention firstly performs hot water extraction on sweet tea and solid-liquid separation to obtain an extract.

[0069] The mass ratio of the sweet tea to hot water is 1:(15-40), preferably 1:(20-30); the amount of hot water used is the total mass of the hot water extraction, and the water used for extraction is added in batches according to the number of extractions.

[0070] The leaching temperature for preparing the hot water extract is 70-95° C., preferably 80-90° C., the number of leaching times is 2-5 times, preferably 3-4 times, and the time for each leaching is 1-4 hours, preferably 2-3 hours.

[0071] The solid-liquid separation is firstly through a filter screen of 80-200 meshes, and then centrifuged in a horizontal screw centrifuge or a butterfly centrifuge, and the clear liquid is taken to obtain an extract.

[0072] Next, the extract is flocculated. Before flocculation, the pH of the extract needs to be adjusted to 4-6, and the pH regulator used is hydrochloric acid. Flocculation under acidic conditions can keep the tea polyphenols stable and not degraded under acidic conditions; at the same time, the weak acid conditions are conducive to the solubility and precipitation of the subsequent basic salt.

[0073] Then, a flocculant is added for flocculation, wherein the flocculant is selected from chitosan; the amount of the flocculant added is 0.03-0.05% of the weight of the extract, preferably 0.03%, 0.04%, 0.05%, or any value between 0.03% and 0.05%.

[0074] The specific method is: add the flocculant directly into the extract, stir evenly, and let it stand for 5 to 10 hours.

[0075] After flocculation is completed, solid-liquid separation, concentration and filtration are carried out to obtain a concentrated clear liquid.

[0076] Wherein, the solid-liquid separation is centrifugation in a horizontal screw centrifuge or a butterfly centrifuge to obtain a clear liquid.

[0077] The concentration is to concentrate the clear liquid to a soluble solid content of 7-15%, preferably 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or any value between 7% and 15%;

[0078] The filtration is carried out using an ultrafiltration membrane, the ultrafiltration membrane having a membrane cutoff molecular weight of 5000 to 10000 Da, preferably 5000 Da, 6000 Da, 7000 Da, 8000 Da, 9000 Da, 10000 Da, or any value between 5000 and 10000 Da.

[0079] Under the condition of continuous stirring, a precipitant is added to the concentrated clear liquid to obtain a turbid liquid, wherein the precipitant is selected from basic zinc chloride or basic calcium chloride.

[0080] The stirring speed of the continuous stirring is 10 to 30 r / min, preferably 10, 20, 30, or any value between 10 and 30 r / min;

[0081] The amount of the precipitant added is 2-6% of the weight of the concentrated clear solution, preferably 2%, 3%, 4%, 5%, 6%, or any value between 2% and 6%;

[0082] In the process of obtaining the turbid liquid, the temperature of the liquid is 30-50°C, preferably 30, 40, 50, or any value between 30 and 50°C, the pH is 4-6, preferably 4, 5, 6, or any value between 4 and 6, and the stirring time is 1.5-3h, preferably 1.5, 2, 2.5, 3, or any value between 1.5 and 3h.

[0083] Basic salts are best soluble under weak acid conditions, and compared with ordinary normal salts, basic salts have excellent precipitation effects on tea polyphenols. Precipitation under continuous stirring can ensure that tea polyphenols are precipitated in the form of extremely small particles and always suspended in the stirring solution without agglomerating into large precipitates, which provides a prerequisite for subsequent column loading.

[0084] The turbid liquid is stirred, and the activated carbon column is applied, and the column liquid always remains turbid, the stirring speed is 20 to 40 r / min, preferably 20, 30, 40, or any value between 20 and 40 r / min, and the column time does not exceed 12 hours; the particle size of the activated carbon is 200 to 2000 μm, preferably 200, 500, 1000, 1500, 2000, or any value between 200 and 2000 μm;

[0085] During the process of loading the activated carbon column, the column temperature of the loading liquid and the activated carbon is maintained at 30-50°C, preferably 30, 35, 40, 45, 50, or any value between 30-50°C. After the loading is completed, the temperature is naturally cooled to room temperature;

[0086] The diameter-to-height ratio of the activated carbon column is 1:4-10, preferably 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, or any value between 1:4-10, the ratio of column volume to sweet tea raw material weight is 1:0.3-1 (ml / g), preferably 1ml:0.3g, 1ml:0.5g, 1ml:0.7g, 1ml:0.9g, 1ml:1.0g, or any value between 1:0.3-1 (ml / g). The column loading speed is 0.5-1.5BV / h, preferably 0.5BV / h, 1.0BV / h, 1.5BV / h, or any value between 0.5-1.5BV / h.

[0087] The turbid liquid is loaded onto an activated carbon column under stirring conditions. Under continuous stirring conditions, the tea polyphenols precipitated by the precipitant are suspended in the stirred solution in the form of extremely small particles. After the solution with the suspended tea polyphenol small particles is loaded onto the activated carbon column, the tea polyphenol small particles can smoothly pass through the chromatography column and be adsorbed on the activated carbon, thereby achieving effective separation of tea polyphenols.

[0088] After the column is loaded, the column is driven with water, and the driven liquid is combined with the effluent; the driven volume of the water is 2 to 4 BV, preferably 2, 3, 4, or any value between 2 and 4 BV, and the driven flow rate of the water is 1 to 1.5 BV / h, preferably 1, 1.1, 1.2, 1.3, 1.4, 1.5, or any value between 1 and 1.5 BV / h.

[0089] Then, the activated carbon column is desorbed, and the desorbed liquid is concentrated, crystallized I, washed, dried, and crushed to obtain tea polyphenols.

[0090] Wherein, the elution is desorption with an acidic ethanol aqueous solution, the acidic ethanol aqueous solution has an ethanol volume fraction of 55-70%, preferably 55%, 60%, 65%, 70%, or any value between 55-70%, a pH value of 1-5, preferably 1, 2, 3, 4, 5, or any value between 1-5, a volume of 3-6 BV, preferably 3, 4, 5, 6, or 3-6 BV, and the acid is hydrochloric acid;

[0091] The concentration is performed to a soluble solid content of 30-45%, preferably 30%, 35%, 40%, 45%, or any value between 30% and 45%;

[0092] The crystallization I is to add alcohol and chloroform to the concentrated solution, stir evenly and then place it at low temperature to stand for crystallization.

[0093] The alcohol is ethanol or methanol; the volume ratio of the alcohol to the concentrated solution obtained after concentration of the desorption liquid is 1:0.05-0.2, preferably 1:0.05, 1:0.1, 1:0.15, 1:0.2, or any value between 1:0.05-0.2. The volume ratio of the chloroform to the concentrated solution obtained after concentration of the desorption liquid is 1:0.01-0.1, preferably 1:0.01, 1:0.03, 1:0.05, 1:0.07, 1:0.09, 1:0.1, or any value between 1:0.01-0.1.

[0094] The standing temperature is -10 to 10°C, preferably -10, -5, 0, 5, 10, or any value between -10 and 10°C, and the standing time is 4 to 12 hours, preferably 4, 6, 8, 10, 12, or any value between 4 and 12 hours;

[0095] After the crystallization I, the crystals are collected through a 50-200 μm filter bag; the crystal washing I is to elute the crystals with an alcohol-chloroform mixed solution at -10 to 10°C, the volume ratio of the alcohol to chloroform is 1:10 to 20, preferably 1:10, 1:15, 1:20, or any value between 1:10 and 20, and the alcohol is ethanol or methanol.

[0096] The purpose of drying is to remove the solution, including but not limited to any one or combination of vacuum drying, microwave drying and boiling drying; the drying temperature is 70-90°C, preferably 70, 80, 90, or any value between 70 and 90°C.

[0097] Combine the water driven liquid and the effluent from the upper column and apply it to the macroporous adsorption resin chromatography column. The macroporous adsorption resin model is any one of D101, AB-8, LX-T28, and LX-T81;

[0098] The chromatographic column has a diameter-to-height ratio of 1:3-7, preferably 1:3, 1:4, 1:5, 1:6, 1:7, or any value between 1:3-7, and a ratio of column volume to raw material weight of 1:0.5-2 (ml / g), preferably 1:0.5 ml / g, 1:1.5 ml / g, 1:2 ml / g, or any value between 1:0.5-2 (ml / g);

[0099] The column loading speed is 0.5 to 1.0 BV / h, preferably 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, or any value between 0.5 and 1.0 BV / h.

[0100] After loading the column, water is used to drive the column and desorb the column to obtain a desorbed liquid.

[0101] Wherein, the water rush volume is 2 to 4 BV, preferably 2, 3, 4, or any value between 2 and 4 BV, and the water rush flow rate is 1 to 1.5 BV / h, preferably 1, 1.1, 1.2, 1.3, 1.4, 1.5, or any value between 1 and 1.5 BV / h. ;

[0102] The desorption is performed by desorption with an alcohol aqueous solution; the alcohol is ethanol or methanol or a combination of the two; the volume fraction of the alcohol in the alcohol aqueous solution is 55-70%, preferably 55%, 60%, 65%, 70%, or any value between 55% and 70%;

[0103] The usage amount of the alcohol water is 2.5-4BV, preferably 2.5, 3, 3.5, 4, or any value between 2.5-4BV, and the flow rate is 0.5-1.5BV / h, preferably 0.5, 1, 1.5, or any value between 0.5-1.5BV / h.

[0104] Finally, the desorption liquid is concentrated, crystallized II, washed II, dried and crushed to obtain rubusoside.

[0105] The concentration is for the purpose of removing alcohol and water, including but not limited to any one of single-effect vacuum evaporation concentration, multi-effect vacuum evaporation concentration, and rotary evaporation concentration.

[0106] The concentration is to concentrate until the soluble solid content is 45-60%, preferably 45%, 50%, 55%, 60%, or any value between 45% and 60%;

[0107] The crystallization II is to add alcohol and dichloromethane to the concentrated solution, stir evenly and then place it at low temperature to stand for crystallization;

[0108] The alcohol is ethanol or methanol, and the volume ratio of the alcohol to the concentrated solution is 1:0.05-0.2, preferably 1:0.05, 1:0.1, 1:0.15, 1:0.2, or any value between 1:0.05 and 0.2.

[0109] The volume ratio of dichloromethane to the concentrated solution is 1:0.05-0.1, preferably 1:0.05, 1:0.05, 1:0.05, 1:0.05, 1:0.05, 1:0.05, 1:0.05;

[0110] The temperature of the low temperature condition is -10 to 10°C, and the standing time is 4 to 8 hours;

[0111] The crystals are collected by passing through a 50-200 μm filter bag after the crystallization II;

[0112] The crystal washing II is to wash the crystal with a mixed solution of alcohol and dichloromethane at -10 to 10°C, and the volume ratio of the alcohol to dichloromethane is 1:1 to 2.

[0113] The purpose of drying is to remove moisture, including but not limited to any one or combination of vacuum drying, microwave drying and boiling drying; the drying temperature is 70-90°C, preferably 70, 80, 90, or any value between 70 and 90°C.

[0114] The invention extracts sweet tea rich in rubusoside and tea polyphenols with hot water, adjusts the acidity of the extract to improve the stability of tea polyphenols, then adds a flocculant to remove most of the protein and tannic acid, filters to obtain a clear liquid, concentrates the clear liquid and filters to further remove insoluble impurities; then adjusts the concentrated clear liquid to slightly acidic to be more conducive to the dissolution of the basic salt, then adds a basic salt precipitant under a relatively high constant temperature, and precipitates under a continuous stirring condition so that the tea polyphenols maintain fine precipitation particles and are always suspended in the precipitation turbid liquid; then, the precipitation turbid liquid is loaded onto an activated carbon column under a stirring state and kept at a constant temperature, and the fine tea polyphenol particles are adsorbed on the activated carbon column (the water driven liquid and the effluent are combined to prepare rubusoside later), and the activated carbon column is eluted with acidic alcohol to obtain tea polyphenols with higher purity, then the eluate is concentrated to remove the alcohol, and an alcohol chloroform solution is added to perform low-temperature crystallization I, and finally the crystals are washed I, dried and crushed to obtain high-purity tea polyphenols. The water driven liquid and the effluent of the activated carbon are combined and loaded onto a macroporous adsorption resin chromatography column, and then driven with water, desorbed with an alcohol aqueous solution, and concentrated. After adding alcohol dichloromethane for crystallization II, the crystals are washed II, dried, and crushed to obtain high-purity rubusoside.

[0115] The results show that the content of rubusoside prepared by the method is ≥99% and the yield is 90%; the rubusoside prepared by the method is white in color, and no independent decolorization process is required in the entire process; the content of tea polyphenols produced by the method is ≥97% and the yield is ≥87%; the entire process of the present invention is simple, has wide adaptability, and is suitable for industrial production.

[0116] In order to further understand the present invention, the method for simultaneously preparing rubusoside and tea polyphenols from sweet tea provided by the present invention is described below in conjunction with examples, and the protection scope of the present invention is not limited by the following examples.

[0117] Detection method for tea polyphenols: Detection shall be carried out in accordance with GB / T 8313-2018 Detection method for tea polyphenols and catechins in tea.

[0118] Detection method of rubusoside: refer to DBS45 / 067-2020 Local Food Safety Standard for Determination of Rubusoside Content in Sweet Tea for detection.

[0119] Unless otherwise specified, the raw materials or chemical reagents used in the embodiments of the present invention are obtained through conventional commercial channels.

[0120] Example 1

[0121] (1) Extraction: Take 100g of sweet tea leaves and use 30 times hot water at 90℃ for 4 times extraction, each time for 2h. After filtering through a 100-mesh filter, perform butterfly separation to obtain 2740g of extract.

[0122] (2) Flocculation: The pH value of the extract was adjusted to 4.5 with dilute hydrochloric acid, and then 1.1 g of chitosan was added. After stirring evenly and standing at room temperature for 8 h, butterfly separation was performed to collect the clear liquid, which was then concentrated to a soluble solid content of 10.4%. After passing through an ultrafiltration membrane with a molecular weight cutoff of 8000 Da, a concentrated clear liquid was obtained.

[0123] (3) Precipitation: Under the condition of stirring speed of 30 r / min, maintaining the liquid temperature at 45°C and pH at 4.5, add 5% of the weight of the concentrated clear liquid basic zinc chloride and continue stirring for 2.5 h.

[0124] (4) Chromatography I: Under continuous stirring at a speed of 30 r / min, the column was loaded onto an activated carbon chromatography column with a volume of 100 ml and a diameter-to-height ratio of 1:6 at a speed of 1 BV / h. After loading, the column was washed with 2 BV of pure water at a flow rate of 1.5 BV / h, and the column effluent and the water-washing liquid were collected and combined for use. Desorption was performed with acidic ethanol with a pH of 2 and an ethanol volume fraction of 65% at a flow rate of 1 BV / h. The desorption volume was 5 BV. The desorbed liquid was collected and concentrated to a soluble solid content of 40%. Then, ethanol and chloroform were added at a volume ratio of 1:0.1 and 1:0.05 to the concentrated liquid, respectively. After stirring evenly, the mixture was allowed to stand at 0°C for 10 hours. After crystallization I was completed, the crystals were collected through a 150 μm filter bag, and the crystals were washed with a solution with a volume ratio of ethanol to chloroform of 1:15 and a temperature of -10°C. Finally, high-content tea polyphenols were obtained after vacuum drying.

[0125] (5) Chromatography II: Combine the collected upper column effluent and water-dripping liquid in step (4), and apply them to a D101 macroporous adsorption resin chromatography column with a volume of 50 ml and a diameter-to-height ratio of 1:6 at a column speed of 1BV / h. After the column is completed, use 2BV of pure water to drive the column at a flow rate of 1.5BV / h. After the column is completed, use a 60% alcohol aqueous solution at a flow rate of 1BV / h to desorb, and the desorption volume is 3BV. Collect the desorbed liquid and concentrate it to a soluble solid content of 47%, then add ethanol and dichloromethane at a volume ratio of 1:0.05 to the concentrated liquid respectively, stir evenly, and let it stand at 0°C for 7h. After the crystallization II is completed, collect the crystals through a 200μm filter bag, and wash the crystals II with a solution with a volume ratio of ethanol to dichloromethane of 1:2 and a temperature of -10°C, and finally vacuum dry to obtain high-content rubusoside.

[0126] Examples 2 to 3 and Comparative Examples 1 to 8

[0127] Referring to the preparation method of Example 1, rubusoside and tea polyphenols were prepared according to the preparation process in Table 1.

[0128] Table 1

[0129]

[0130]

[0131]

[0132] In Table 1, the stirring time before column loading is the continuous stirring time, that is, the precipitation time.

[0133] Taking Example 1 or Example 2 as a control, the differences in Comparative Examples 1-8 are respectively adjusted in terms of the precipitant (zinc chloride), the precipitant (calcium chloride), the stirring time before column loading, the column model, the eluent for elution, the solution added to Crystal I, the solution added to Crystal II, and the crystal washing solution. Specifically:

[0134] Comparative Example 1: According to the process steps of Example 1, only the precipitant is changed to zinc chloride, and other conditions remain unchanged;

[0135] Comparative Example 2: According to the process steps of Example 2, only the precipitant is changed to calcium chloride, and other conditions remain unchanged;

[0136] Comparative Example 3: According to the process steps of Example 1, only the stirring time before column loading was changed to 5 min, and other conditions remained unchanged;

[0137] Comparative Example 4: According to the process steps of Example 1, the activated carbon column in Example 1 was replaced by a D101 column, and other conditions remained unchanged;

[0138] Comparative Example 5: According to the process steps of Example 1, the elution liquid in the step of loading the activated carbon into the column in Example 1 is replaced with alkaline ethanol, wherein the base is sodium hydroxide, the pH is 10, and other conditions remain unchanged;

[0139] Comparative Example 6: According to the process steps of Example 1, the crystallization solution in the crystallization I step of Example 1 is replaced with ethanol, wherein the volume ratio of ethanol to the concentrated solution is 3:0.1, and the crystal washing I solution is replaced with the same volume of ethanol, and other conditions remain unchanged;

[0140] Comparative Example 7: According to the process steps of Example 1, the crystallization solution in the step of crystallization II in Example 1 is replaced by ethanol, wherein the volume ratio of ethanol to the concentrated solution is 2:0.05, and other conditions remain unchanged;

[0141] Comparative Example 8: According to the process steps of Example 1, the crystal washing solution in the crystal washing step II in Example 1 is replaced by ethanol, and other conditions remain unchanged.

[0142] After the above process, the products prepared in Examples 1 to 3 and Comparative Examples 1 to 8 were tested, and the results are shown in Table 2.

[0143] Table 2

[0144]

[0145] From the preparation process provided in Table 1 and the test data in Table 2, it can be seen that 1) the adjustment of the precipitant basic salt to inorganic salt has a great influence on the precipitation effect of tea polyphenols, such as: zinc chloride precipitation is incomplete, resulting in a low yield; calcium chloride precipitation effect is too strong, directly resulting in precipitation agglomeration and clogging of the activated carbon column, so that the experiment cannot be continued. 2) Too short precipitation time will also lead to incomplete precipitation of tea polyphenols, resulting in a low yield. 3) The macroporous adsorption resin represented by D101 replaces the activated carbon column, which has no difference in adsorption of tea polyphenols and rubusoside, directly resulting in the inability to effectively separate tea polyphenols and rubusoside. 4) Alkaline ethanol is used to replace acidic ethanol to desorb the activated carbon column, resulting in degradation of tea polyphenols, and incomplete elution of impurities, which leads to low tea polyphenol content and yield. 5) Ethanol is used to replace the mixture of ethanol and chloroform to crystallize tea polyphenols, and its crystallization is incomplete, and the yield is low. 6) When ethanol is used instead of a mixture of ethanol and dichloromethane to crystallize and wash rubusoside, the crystallization is incomplete and impurities attached to the crystals are not washed away completely, resulting in low content and yield.

[0146] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for simultaneously preparing rubusoside and tea polyphenols from sweet tea, characterized in that: The following steps are involved: A) adjusting the hot water extract of sweet tea to an acidic pH, adding a flocculant to flocculate, and then separating the solid and liquid, concentrating and filtering to obtain a concentrated clear liquid; the flocculant is selected from chitosan, and the pH value of adjusting the pH to an acidic pH is 4-6; the filtration is performed by an ultrafiltration membrane, and the membrane has a molecular weight cutoff of 5000-10000Da; B) adding a precipitant to the concentrated clear solution under continuous stirring to obtain a turbid solution, wherein the precipitant is selected from basic zinc chloride or basic calcium chloride, and the stirring time is continuous for 1.5 to 3 hours; C) The turbid liquid is loaded onto an activated carbon column under stirring conditions, and after loading, the column is driven out with water, and the driven out liquid is combined with the effluent; then, the activated carbon column is desorbed, and the desorbed liquid is concentrated, crystallized I, washed crystals I, dried, and crushed to obtain tea polyphenols, wherein the desorption is performed by using an acidic ethanol aqueous solution, the ethanol volume fraction of the acidic ethanol aqueous solution is 55-70%, the solvents used for crystallization I are ethanol and chloroform, the particle size of the activated carbon is 200-2000 μm, the diameter-to-height ratio of the activated carbon column is 1:4-10, the pH value is 1-5, and the volume ratio of ethanol to chloroform is 1:10-20; D) The water-dried liquid and the effluent from the upper column are combined and loaded onto a macroporous adsorption resin chromatography column. After the loading is completed, the water-dried column is loaded, and the chromatography column is desorbed. The desorbed liquid is concentrated, crystallized II, washed crystals II, dried, and crushed to obtain rubusoside. The desorption is performed by desorption with an alcohol-water solution, the volume fraction of alcohol in the alcohol-water solution is 55-70%, the solvents used for crystallization II are ethanol and dichloromethane, the solvents used for washing crystals II are ethanol and dichloromethane, the volume ratio of ethanol to dichloromethane is 1:1-2, and the diameter-to-height ratio of the chromatography column is 1:3-7.

2. The method according to claim 1, characterized in that The sweet tea is selected from dried sweet tea leaves and / or stems; The mass ratio of the sweet tea to hot water is 1:(15-40); The leaching temperature for preparing the hot water extract is 70-95° C., the number of leaching times is 2-5 times, and the time for each leaching is 1-4 hours.

3. The method according to claim 2, characterized in that The mass ratio of the sweet tea to hot water is 1:(20-30); The leaching temperature for preparing the hot water extract is 80-90°C, the number of leaching times is 3-4 times, and the time for each leaching is 2-3 hours.

4. The method according to claim 1, characterized in that In step A), the pH adjusting agent used is hydrochloric acid; The added amount of the flocculant is 0.03-0.05% of the weight of the extract.

5. The method according to claim 1, characterized in that In step A), the concentration is performed to a soluble solid content of 7-15%.

6. The method according to claim 1, characterized in that In step B), the stirring speed of the continuous stirring is 10 to 30 r / min; The amount of the precipitant added is 2-6% of the weight of the concentrated clear liquid; In the process of obtaining the turbid liquid, the temperature of the liquid is 30-50°C and the pH is 4-6.

7. The method according to claim 1, characterized in that In step C), the turbid liquid is placed on the activated carbon column while being stirred, and the liquid on the column remains turbid at all times. The stirring speed is 20 to 40 r / min, and the loading time does not exceed 12 hours; During the process of loading the activated carbon column, the column temperature of the loading liquid and activated carbon is maintained at 30~50℃. After the loading is completed, the temperature is naturally cooled to room temperature; The ratio of column volume to sweet tea raw material weight is 1:0.3-1 (ml / g); the column loading speed is 0.5-1.5 BV / h; the water chasing volume is 2-4 BV, and the water chasing flow rate is 1-1.5 BV / h.

8. The method according to claim 1, characterized in that In step C), the volume is 3-6 BV, and the acid is hydrochloric acid; The concentration is to a soluble solid content of 30-45%; The crystallization I is to add ethanol and chloroform to the concentrated solution, stir evenly and then place it at low temperature to stand for crystallization; The volume ratio of the alcohol to the concentrated solution obtained by concentrating the desorption liquid is 1:0.05-0.2; the volume ratio of the chloroform to the concentrated solution obtained by concentrating the desorption liquid is 1:0.01-0.1; the standing temperature is -10-10°C, and the time is 4-12 hours; After the crystallization, the crystals are collected through a 50-200 μm filter bag; The crystal washing I is to wash the crystals with an alcohol-chloroform mixed solution at -10 to 10°C.

9. The method according to claim 1, characterized in that: In step D), the macroporous adsorption resin is any one of D101, AB-8, LX-T28, and LX-T81; The ratio of column volume to sweet tea raw material weight is 1:0.5~2 (ml / g); The column loading speed is 0.5-1.0 BV / h.

10. The method according to claim 1, characterized in that In step D), the water-driving volume is 2-4 BV, and the water-driving flow rate is 1-1.5 BV / h; The usage amount of the alcohol water is 2.5-4 BV, and the flow rate is 0.5-1.5 BV / h.

11. The method according to claim 1, characterized in that: In step D), the concentration is performed until the soluble solids are 45-60%; The crystallization II is to add ethanol and dichloromethane to the concentrated solution, stir evenly and then place it at low temperature to stand for crystallization; The volume ratio of the alcohol to the concentrated liquid is 1:0.05-0.2; The volume ratio of the dichloromethane to the concentrated liquid is 1:0.05-0.1; The temperature of the low temperature condition is -10~10°C, and the standing time is 4~8h; After the crystallization, the crystals are collected through a 50-200 μm filter bag; The crystal washing II is to wash the crystals with a mixed solution of alcohol and dichloromethane at -10 to 10°C.

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