Green and safe tea dietary polysaccharide extraction preparation method and application thereof

Abstract

The invention relates to tea polysaccharide, in particular to an extraction and preparation method and application of green and safe tea dietary polysaccharide. The extraction preparation method comprises the following steps: crushing and sieving; washing with alcohol to remove impurities; performing water extraction and alcohol precipitation; protein enzymolysis; and carrying out polyamide column chromatography and starch removal to obtain the tea dietary polysaccharide. According to the method disclosed by the invention, the tea dietary polysaccharide is extracted and prepared by combining high-concentration ethanol degreasing and decolorizing, water extraction and alcohol precipitation, papain enzymolysis and polyamide column chromatography combined deproteinization, decolorization and polyphenol removal, amylase enzymolysis and starch removal and other technologies, and the method is free of toxic organic reagent pollution and residue, green and safe.

Description

TECHNICAL FIELD

[0001] The present application relates to tea polysaccharide, and in particular to a green and safe tea dietary polysaccharide extraction and preparation method and application thereof. BACKGROUND

[0002] Polysaccharide is the third macromolecular substance with complex structure and biological function after nucleic acid and protein. Tea polysaccharide is another macromolecular active substance with various health functions in addition to polyphenol and polypeptide in tea. Tea polysaccharide has application value in antioxidant, blood sugar reduction, immune regulation, blood lipid reduction, weight loss and the like. Therefore, the development and application of polysaccharide extraction and preparation technology have gradually become a research hotspot in the field of deep processing and functional food.

[0003] At present, the extraction process of tea polysaccharide mainly includes crushing, degreasing, water extraction and alcohol precipitation, decolorization and deproteinization, removal of small molecule impurities, drying and the like. The degreasing is usually carried out by using petroleum ether to remove by reflux washing, and since petroleum ether has certain toxicity and volatile residue, the existing technology generally uses anhydrous ethanol to replace (CN 202010512897.1). Water extraction and alcohol precipitation is the most common method with low cost and little influence on the structure of polysaccharide, although there are acid extraction, alkali extraction, ultrasonic-assisted extraction, steam explosion extraction, ultrahigh pressure extraction, enzyme-assisted extraction, microwave-assisted extraction and the like, and these technologies can improve the yield of polysaccharide, but these technologies will damage the glycosidic bond of polysaccharide, change the structure, molecular weight and monosaccharide composition of polysaccharide, and affect the application value of tea polysaccharide (CN 202010512897.1, CN 202210291498.6, CN 202210466052.2, CN 202310554562.X). Supercritical carbon dioxide extraction technology is also gradually introduced into the extraction of tea polysaccharide, although it has the technical advantages of no impurity residue, integrated extraction and separation, and little influence on the structure, but it has high cost and great application difficulty (CN 201811282333.2). Common impurity removal methods include activated carbon adsorption decolorization, hydrogen peroxide decolorization, macroporous resin adsorption decolorization, Sevage (chloroform:n-butanol = 4:1) method deproteinization, trichloroacetic acid (Trichloroacetic acid, CTA) method deproteinization, protease enzymolysis deproteinization and the like (CN 202210466052.2, CN 202310554562.X), which can effectively decolorize and deproteinize, but these methods are difficult to completely remove impurities, and have problems of large polysaccharide loss, toxic organic reagent residue and the like. Although there is a report on the use of polyamide column chromatography for decolorization and deproteinization (CN 202110123087.1), which can simultaneously complete the impurity removal operation of decolorization and deproteinization, but the single processing capacity is small, the time consumption is long, and the efficiency is low, which is a problem of large-scale application. The methods for removing small molecule impurities include dialysis and ultrafiltration, which have the problem of long time consumption. In addition, there are also studies on the use of DEAE ion exchange column chromatography and dextran gel chromatography for separation and purification of tea polysaccharide (CN 202210436409.2), but these technologies are technical means for in-depth research of tea polysaccharide, and can obtain tea polysaccharide with high purity, good uniformity and available structure analysis related research, however, the yield is very low, which leads to low practical popularization and application value.

[0004] Although less than 1% of starch polysaccharide is contained in dry tea, but in the preparation process of tea polysaccharide, due to the low extraction rate, a large amount of starch polysaccharide is enriched. However, the existing tea polysaccharide preparation process does not involve a starch removal process. SUMMARY

[0005] To at least solve one of the above problems in the prior art, the present application provides a green and safe tea dietary polysaccharide extraction and preparation method and application thereof.

[0006] A green and safe tea dietary polysaccharide extraction and preparation method comprises the following steps:

[0007] S1, crushing and sieving: grinding and sieving tea leaves to collect tea powder;

[0008] S2, alcohol washing and impurity removing: washing the tea powder with 80%-90% ethanol, filtering, and drying the filter residue to obtain impurity-removed tea powder;

[0009] S3, water extraction and alcohol precipitation: extracting the impurity-removed tea powder with water, centrifuging to take supernatant, concentrating, precipitating with ethanol, centrifuging to discard supernatant, washing and dehydrating, and drying the precipitate to obtain tea dietary crude polysaccharide;

[0010] S4, enzymatic protein hydrolysis: redissolving the tea dietary crude polysaccharide with pure water, adding papain for enzymatic hydrolysis, centrifuging, and collecting supernatant;

[0011] S5, polyamide column chromatography: wet loading the supernatant, adsorbing and equilibrating, pure water elution, and constant flow collection of eluate; using colorimetric method to determine and analyze the 420nm absorbance of the collected eluate, combining the collected eluate before the inflection point, reducing pressure concentration, pure water dialysis of the concentrated solution at room temperature, centrifuging the dialysate to take supernatant, reducing pressure concentration, alcohol precipitation overnight, centrifuging to discard supernatant, washing and dehydrating, and drying the precipitate to obtain tea dietary impurity-removed polysaccharide;

[0012] In the inflection point, the target component and impurity component in the eluate change as follows: the anthrone color developing OD value of polysaccharide suddenly decreases; the coomassie brilliant blue color developing OD value of protein suddenly increases; the Folin-phenol color developing OD value of polysaccharide first decreases and then increases; and the OD value at 420nm of pigment first decreases and then increases;

[0013] S6, starch removing: redissolving the tea dietary impurity-removed polysaccharide with pure water, adding amylase for enzymatic hydrolysis, cooling the enzymatic hydrolysis product at room temperature, centrifuging to take supernatant, room temperature dialysis, centrifuging the dialysate to take supernatant, concentrating, alcohol precipitation, centrifuging, dehydrating and drying to obtain (non-starch) tea dietary polysaccharide.

[0014] Specifically, in step S1, the selected tea leaves include but are not limited to green tea, black tea and dark tea. The used tea green leaves include but are not limited to one bud and two to five leaves of Yinghong No. 9 summer and autumn tea tree fresh leaves.

[0015] Specifically, in step S1, the tea leaves are crushed by a sample grinder, and sieved through 20 mesh and 100 mesh screen nets, and the tea powder between 20-100 mesh is collected. The sieved 20-100 mesh tea powder can not only improve the alcohol washing and impurity removing effect and the crude polysaccharide extraction rate, but also reduce the filtration time cost caused by too fine tea powder

[0016] Specifically, in step S2, the tea powder is washed twice with 80%-90% ethanol; in the first alcohol washing, the tea powder is mixed with 80%-90% ethanol at a material-liquid ratio (m / m) of 1:(15-20); the washing is performed at a temperature of 60-80°C for 30-50 min; in the second alcohol washing, the tea powder is mixed with 80%-90% ethanol at a material-liquid ratio (m / m) of 1:(13-18); the washing is performed at a temperature of 60-80°C for 30-50 min.

[0017] The washing can be performed under water bath conditions. After the alcohol washing, the tea powder can be filtered through a 60-100 mesh sieve. The filtered residue can be dried at 60-70°C.

[0018] The present application finds that washing the tea powder with 80%-90% ethanol can avoid the loss of polysaccharides caused by impurity removal with low-concentration ethanol or water, and can also reduce the residual caffeine and economic cost caused by impurity removal with anhydrous ethanol.

[0019] Specifically, in step S3, the impurity-removed tea powder is mixed with water (e.g. pure water) at a material-liquid ratio (m / m) of 1:(15-20); the extraction is performed at a temperature of 70-90°C for 1-2 h. The extraction can be usually performed twice. In the second extraction, the material-liquid ratio (m / m) can be 1:(13-18).

[0020] Specifically, in step S3, 50-100 g of the impurity-removed tea powder is mixed with pure water at a material-liquid ratio (m / m) of 1:(15-20), and extracted at 70-90°C for 1-2 h under water bath stirring, filtered through a Buchner funnel with a 60-100 mesh sieve, and the tea residue is squeezed with a grinding rod to squeeze out the adsorbed extractive liquid, the residue is extracted twice with pure water at a material-liquid ratio (m / m) of 1:(13-18), the extractive liquids are combined, cooled to room temperature, centrifuged at 4000-5000 rpm for 5-10 min, and the supernatant is collected, the supernatant is concentrated to 1 / 15-1 / 20 of the original volume by rotary evaporation under reduced pressure (vacuum degree -0.95 to -0.1 MPa) at 50-60°C, cooled to room temperature, 3-4 times the volume of 95%-99.7% ethanol is added, stirred with a glass rod, sealed with plastic wrap, and alcohol precipitated at room temperature for 8-14 h, centrifuged at 4000-5000 rpm for 5-10 min, the supernatant is poured off, 3-4 times the volume of 80% ethanol is added, the precipitate is resuspended by stirring with a glass rod, the supernatant is removed by centrifugation, the resuspension is repeated twice, and the same volume of anhydrous ethanol is added and resuspended by stirring twice, and the precipitate is dried in an oven at 50-60°C to obtain tea dietary crude polysaccharides.

[0021] Specifically, in step S4, the enzymatic hydrolysis is performed at a temperature of 40-50°C for 3-5 h. The enzymatic hydrolysis can be performed under water bath conditions. After the enzymatic hydrolysis, the mixture is cooled to room temperature, centrifuged at 4000-5000 rpm for 5-10 min, and the supernatant is collected.

[0022] Specifically, the enzyme activity of papain is 200-800 U / mg.

[0023] Specifically, in step S4, the tea dietary crude polysaccharide is dissolved in pure water to prepare a 10-20 mg / mL solution; and papain (800 U / mg) is added in a proportion of 0.03%-0.08% (m / v, g / mL) for enzymolysis. The concentration of the tea dietary crude polysaccharide solution has a great influence on the effect of impurity removal and purification. If the concentration is too low, the single treatment capacity of the polyamide column chromatography is reduced; if the concentration is too high, the polyamide powder cannot effectively adsorb impurities such as proteins, polyphenols and pigments, affecting the effect of impurity removal and purification. Within the concentration range, the tea dietary crude polysaccharide solution can not only improve the single treatment capacity (loading capacity) of the tea dietary crude polysaccharide, but also does not affect the effect of impurity removal and purification.

[0024] Specifically, in step S4, 5-18 g of the dietary crude polysaccharide is weighed and dissolved in 40-50℃ preheated pure water to prepare a 10-20 mg / mL crude extract solution, and papain (800 U / mg) is added in a proportion of 0.03-0.08% (m / v, g / mL), and the solution is subjected to enzymolysis at 40-50℃ for 3-5 h, cooled to room temperature, and centrifuged at 4000-5000 rpm for 5-10 min, and the supernatant is collected.

[0025] The present application finds that the effective eluent is collected at the inflection point of the polyamide column chromatography process, the volume of the eluent and the introduction of impurities are reduced, the operation time is greatly shortened, the efficiency is improved, and the application value of the technology is improved. Research has found that there is an inflection point in the polyamide column chromatography process, and the content of proteins, pigments and polyphenols in the eluent after the inflection point increases, and the content of polysaccharides decreases, which affects the effect of impurity removal. Therefore, collecting the effective eluent before the inflection point can achieve good impurity removal effect, and only 1-1.3 column volumes of eluent need to be collected, which reduces the volume of the ineffective eluent, including the volume of the eluent without sugar and the volume of the eluent with oligosaccharides. The prior art needs to collect 3-5 column volumes of eluent.

[0026] Specifically, in step S5, the adsorption equilibrium time is 20-40 min, for example, 30 min.

[0027] Specifically, in step S5, the collection liquid before the inflection point is 1-1.3 column volumes of eluent, preferably 1 column volume of eluent.

[0028] Specifically, in step S5, the supernatant is gently added to the top of the column bed of the chromatographic column by a long pipette, the lower end switch is opened, and the supernatant is completely adsorbed in the chromatographic column. After 30 min of adsorption equilibrium, a peristaltic pump is connected to the upper end of the chromatographic column to control the flow rate at 3 mL / min, the lower end outlet is connected to a fraction collector, and the setting is 3.3 min / tube. The eluate is collected at 1-1.3 column volumes. The collected liquid is analyzed by 420 nm absorbance determination, and the collected liquids before the inflection point are combined. The concentrated liquid is concentrated at 45-55°C under reduced pressure to about 1 / 15-1 / 20 of the original volume. The concentrated liquid is transferred to a 3000-4000 Da dialysis bag using a pipette, 80-100 times the volume of the concentrated liquid of pure water is added, and dialysis is performed at room temperature (25-30°C) for 24-36 h, with the pure water being replaced at intervals of 2-3 h, 3-4 h, 5-6 h, and 11-12 h, respectively. The dialysate is centrifuged to obtain the supernatant, which is concentrated under reduced pressure to about 1 / 3-1 / 5 of the original volume. The concentrated liquid is alcohol precipitated overnight, centrifuged to remove water (anhydrous ethanol), dried at 45-55°C, and tea dietary impurity-removed polysaccharides are obtained.

[0029] Specifically, in step S5, the polyamide column is filled with 100-200 mesh polyamide powder, and the column can be filled by wet method.

[0030] Specifically, in step S5, the polyamide column has a specification of 400 mL (33 mm x 47 cm) to 2.2 L (55 mm x 93 cm), and the tea dietary crude polysaccharide loading amount is 2-18 g, preferably 15-18 g. The present method can increase the single processing amount (loading amount) of tea dietary crude polysaccharides from 2 g to 15-18 g, expand the single processing amount, and improve the impurity removal efficiency.

[0031] In some specific embodiments, in step S5, the polyamide column has a volume of 2.2 L, and the tea dietary crude polysaccharide loading amount is 15-18 g.

[0032] The polyamide column chromatography technology has good decolorization and deproteinization effect, no pollution and residue, and less polysaccharide loss, but has problems of small processing amount, long time consumption, and low efficiency. The present application uses the technology of combining protease and polyamide column chromatography to remove protein, color, and polyphenol, expands the volume of the chromatographic column, increases the single processing amount, uses the inflection point of the polyamide column chromatography process to reduce the eluate volume, increases the dialysis water replacement frequency, reduces the dialysis time, greatly shortens the operation time, and increases the efficiency.

[0033] Specifically, in step S5, the reduced pressure concentration can reduce the volume of the eluate, increase the concentration of impurities therein, and thus facilitate subsequent dialysis. Dialysis with pure water can remove small molecular impurities such as polyphenol, flavonoid, nucleotide, amino acid, and oligosaccharide.

[0034] Specifically, in step S6, the amylase is a high-temperature-resistant alpha-amylase (CAS: 9001-19-8, 40 U / mg).

[0035] Specifically, in step S6, the temperature of enzymolysis is 90-100℃, and the time of enzymolysis is 20-40 min. The enzymolysis can be carried out under water bath conditions.

[0036] Specifically, in step S6, the supernatant after centrifugation is dialyzed with pure water for 24-36 h.

[0037] Specifically, in step S6, the tea dietary impurity polysaccharide is redissolved with pure water to prepare a solution of 5-10 mg / mL; the thermostable alpha-amylase (CAS: 9001-19-8, 40 U / mg) is prepared into a solution of 50-100 mg / mL; 100 μL of the thermostable alpha-amylase solution is added to 10 mL of the tea dietary impurity polysaccharide solution.

[0038] Specifically, in step S6, 100 μL (50-100 mg / mL) of the thermostable alpha-amylase (CAS: 9001-19-8, 40 U / mg) is added to 10 mL (5-10 mg / mL) of the mixture, and the mixture is incubated in a water bath at 90-100℃ for 20-40 min with stirring. After cooling to room temperature, centrifugation is carried out at 4000-5000 rpm for 5-10 min, the supernatant is dialyzed with pure water for 24-36 h, the dialysate is concentrated by centrifugation, alcohol precipitation is carried out, and dehydration is carried out by centrifugation to obtain (non-starch) tea dietary polysaccharide.

[0039] The method of the present application utilizes the enzymolysis of amylase to decompose starch polysaccharide in tea dietary impurity polysaccharide, and then removes dextrin fragments generated by the enzymolysis through dialysis to obtain non-starch tea dietary polysaccharide. It is found that the use of thermostable alpha-amylase for enzymolysis can completely remove starch polysaccharide, and improve the uniformity of non-starch polysaccharide in tea dietary polysaccharide. Compared with non-enzymolysis, the enzymolysis greatly reduces the influence of starch polysaccharide on the purity of non-starch dietary polysaccharide in tea. Other methods for removing starch from polysaccharide have high technical requirements (high-pressure homogenization treatment), high equipment costs (microwave-assisted enzymolysis), affect the structure of non-starch polysaccharide (acid-base treatment), or waste polysaccharide (hydrogen peroxide treatment or ethanol fractionation precipitation). The specificity, efficiency, applicability, and environmental friendliness of these existing starch removal methods are not as good as the amylase enzymolysis treatment of the present application.

[0040] The (non-starch) tea dietary polysaccharide prepared by the present application has high total sugar content (87%-92%), high content of uronic acid and neutral sugar active substances (>50%), low content of protein (<2.0%), polyphenol (<1.0%), and pigment (420 nm, OD value <0.1) impurities, and clear active substance basis.

[0041] The present application also includes (non-starch) tea dietary polysaccharide prepared by the above method.

[0042] The application also includes the use of the (non-starch) tea leaf dietary polysaccharide prepared by the above method in the preparation of health food or medicine for weight loss, sugar reduction or fat reduction.

[0043] The method of the application adds a starch removal process, which is beneficial to avoid starch enrichment in the extraction process, avoid the false positive phenomenon of tea polysaccharide with particularly high total sugar and neutral sugar content, improve the uniformity of tea leaf dietary polysaccharide, obtain non-starch tea leaf dietary polysaccharide, and effectively eliminate the influence of starch on the functional application value of tea leaf dietary polysaccharide. The tea polysaccharide prepared by the prior art will enrich starch, which has a significant influence on the uniformity and application value of tea leaf dietary polysaccharide. The tea polysaccharide containing starch and the tea leaf dietary polysaccharide not containing starch have certain differences in composition and efficacy. The tea leaf non-starch dietary polysaccharide prepared by the application has higher application value.

[0044] The introduction and residue of organic reagents exist safety hazards in the active application of tea leaf dietary polysaccharide. The application uses high-concentration ethanol to remove fat and color, water extraction and alcohol precipitation, papain enzymolysis, and polyamide column chromatography to remove protein, color and polyphenol, and starch enzyme to remove starch, etc. to extract and prepare tea leaf dietary polysaccharide, which is free of toxic organic reagent pollution and residue, and is green and safe. The tea leaf dietary polysaccharide prepared by the application has very low content of impurities such as protein, polyphenol and pigment, and high content of uronic acid. The application analyzes the weight loss, fat reduction and sugar reduction effects of tea leaf non-starch dietary polysaccharide from the animal level, which has high application value. BRIEF DESCRIPTION OF DRAWINGS

[0045] Figure 1 Figure 1 is a diagram of the intervention scheme for the weight loss and sugar reduction experiment of the tea leaf dietary polysaccharide of the application.

[0046] Figure 2 Figure 2 is a diagram of the change of mouse weight during the intervention process of the tea leaf dietary polysaccharide of the application.

[0047] Figure 3 Figure 3 is a diagram of the weight loss and sugar reduction effect of the tea leaf dietary polysaccharide of the application.

[0048] Figure 4 Figure 4 is a diagram of the fat reduction effect of the tea leaf dietary polysaccharide of the application.

[0049] Figure 5 Figure 5 is a diagram of the inflection point and effective elution range in the polyamide column chromatography process of the application. DETAILED DESCRIPTION

[0050] In order to make the above objectives, features and advantages of the present application more obvious and easy to understand, the specific embodiments of the present application are described in detail below. In the following description, a large number of specific details are set forth in order to facilitate a thorough understanding of the present application. However, the present application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without departing from the scope of the present application, so the present application is not limited to the specific implementation disclosed below.

[0051] The technical solutions of the present application, if not specifically stated, are conventional solutions in the art, and the reagents or raw materials used, if not specifically stated, are purchased from commercial channels or are already disclosed.

[0052] Example 1

[0053] The present embodiment provides a green and safe tea (green tea) dietary polysaccharide extraction and preparation method, comprising:

[0054] S1, crushing and sieving: green tea is crushed by a sample grinder, and sieved through 20 mesh and 100 mesh screens, and tea (green tea) powder between 20-100 meshes is collected;

[0055] S2, alcohol washing and impurity removal: 80 g of tea (green tea) powder is weighed, washed with 85% ethanol for 2 times; the first time is washed at a solid-liquid ratio (m / m) of 1:20, stirred and washed at 80°C for 50 min, and filtered through a 60 mesh screen; the second time is washed at a solid-liquid ratio (m / m) of 1:18; the filter residue is dried at 70°C to obtain impurity-removed tea (green tea) powder;

[0056] S3, water extraction and alcohol precipitation: 70 g of impurity-removed tea (green tea) powder is weighed, and pure water is added at a solid-liquid ratio (m / m) of 1:20, stirred and extracted at 80°C for 1.5 h, filtered through a 60 mesh screen in a Buchner funnel, and the extracted liquid adsorbed in the tea residue is squeezed out with a grinding rod; the filter residue is extracted twice at a solid-liquid ratio (m / m) of 1:18, and the extracted liquids are combined; cooled to room temperature, centrifuged at 4000 rpm for 5 min, and the supernatant is collected; the supernatant is concentrated to 1 / 14 of the original volume by rotary evaporation under reduced pressure (vacuum degree -0.95 to -0.1 MPa) at 50-55°C, cooled to room temperature, added with 4 times the volume of 95% ethanol, stirred evenly with a glass rod, sealed with plastic wrap, and precipitated at room temperature for 10 h; centrifuged at 4500 rpm for 5 min, the supernatant was poured off, 4 times the volume of 80% ethanol was added, the precipitate was resuspended by stirring with a glass rod, the supernatant was removed by centrifugation, and the operation was repeated twice; the same volume of anhydrous ethanol was added and resuspended by stirring twice, and the precipitate was dried in a 60°C oven to obtain tea (green tea) dietary crude polysaccharide;

[0057] S4, enzymatic hydrolysis of protein: 15 g of tea (green tea) dietary crude polysaccharide was weighed and dissolved in 50℃ preheated pure water to prepare a 10 mg / mL crude extract solution. Papain (800 U / mg) was added at a ratio of 0.05% (m / v, g / mL), and enzymatic hydrolysis was performed at 50℃ for 3 h. After cooling to room temperature, centrifugation was performed at 4000 rpm for 6 min, and the supernatant was collected.

[0058] S5, polyamide column chromatography: the supernatant obtained in step S4 was gently added to the column bed of a polyamide column (55 mm x 93 cm, 2.2 L, filler: 100-200 mesh polyamide powder) using a long pipette. The lower end switch was opened, and the supernatant was completely adsorbed in the chromatography column. After 35 min of adsorption equilibrium, a peristaltic pump was connected to the upper end of the chromatography column to control the flow rate at 3.5 mL / min. The lower end outlet was connected to a fraction collector set at 4 min / tube, and 1-1.1 column volumes of eluate were collected. The collected liquid was analyzed by 420 nm absorbance determination, and the collected liquid before the inflection point (i.e., 1 column volume of eluate) was combined. The concentrated solution was transferred to a 3500 Da dialysis bag using a pipette, and 100 times the volume of the concentrated solution of pure water was added. Dialysis was performed at room temperature (25-30℃) for 24 h, and the pure water was replaced at intervals of 2 h, 3 h, 6 h, and 12 h. The dialysate was centrifuged to obtain the supernatant, which was concentrated under reduced pressure to 1 / 15 of the original volume. The concentrated solution was transferred to a 3500 Da dialysis bag using a pipette, and 100 times the volume of the concentrated solution of pure water was added. Dialysis was performed at room temperature (25-30℃) for 24 h, and the pure water was replaced at intervals of 2 h, 3 h, 6 h, and 12 h. The dialysate was centrifuged to obtain the supernatant, which was concentrated under reduced pressure to 1 / 3 of the original volume. The ethanol concentration of the concentrated solution was adjusted to 75% with anhydrous ethanol, and alcohol precipitation was performed for 12 h. After centrifugal dehydration, drying was performed at 50℃ to obtain tea (green tea) dietary impurity-removed polysaccharide;

[0059] In the inflection point eluate, the target component and the impurity component showed the following change rules: the OD value of the polysaccharide sulfuric acid anthrone coloration suddenly decreased; the OD value of the protein coomassie brilliant blue coloration suddenly increased; the OD value of the polysaccharide Folin phenol coloration first decreased and then increased; and the OD value of the pigment at 420 nm first decreased and then increased.

[0060] S6, starch removal: tea (green tea) dietary impurity-removed polysaccharide was weighed and reconstituted with pure water to a concentration of 5 mg / mL. 100 uL (100 mg / mL) of heat-resistant alpha-amylase (CAS: 9001-19-8, 40 U / mg) was added, and the mixture was stirred and incubated at 95℃ for 40 min. After cooling to room temperature, centrifugation was performed at 5000 rpm for 6 min. The supernatant was dialyzed against pure water for 24 h, and the dialysate was centrifuged, concentrated, and adjusted to an ethanol concentration of 75% with anhydrous ethanol. Alcohol precipitation, centrifugation, dehydration, and drying were performed to obtain (non-starch green tea) tea dietary polysaccharide.

[0061] Example 2

[0062] The present embodiment provides a green and safe tea (black tea) dietary polysaccharide extraction and preparation method, which comprises:

[0063] S1, crushing and sieving: black tea was crushed by a sample grinder, and sieved through 20 mesh and 100 mesh screens, and the tea (black tea) powder between 20-100 mesh was collected;

[0064] S2, alcohol washing: 100 g of tea (black tea) powder was washed with 80% ethanol twice, the first time with a solid-liquid ratio of 1:18 (m / m) at 70°C for 30 min, and the second time with a solid-liquid ratio of 1:16 (m / m); the filter residue was dried at 65°C to obtain the impurity-removed tea (black tea) powder;

[0065] S3, water extraction and alcohol precipitation: 100 g of the impurity-removed tea (black tea) powder was added with pure water at a solid-liquid ratio of 1:20 (m / m), and extracted at 80°C for 1 h; the extraction liquid was filtered through a Buchner funnel with a 60 mesh screen, and the adsorbed extraction liquid in the filter residue was squeezed out with a grinding rod; the filter residue was extracted twice with a solid-liquid ratio of 1:18 (m / m), and the extraction liquids were combined and cooled to room temperature; the supernatant was collected by centrifugation at 4500 rpm for 6 min; the supernatant was concentrated to 1 / 18 of the original volume by rotary evaporation under reduced pressure (vacuum degree -0.95 to -0.1 MPa) at 55-60°C, and cooled to room temperature; 4 times the volume of 95% ethanol was added, stirred with a glass rod, sealed with plastic wrap, and precipitated at room temperature for 11 h; the supernatant was removed by centrifugation at 4500 rpm for 7 min, and 3 times the volume of 80% ethanol was added; the precipitate was resuspended by stirring with a glass rod, and the supernatant was removed by centrifugation; the above steps were repeated twice, and the precipitate was resuspended by stirring with the same volume of anhydrous ethanol twice; the precipitate was dried in a 50°C oven to obtain tea (black tea) dietary crude polysaccharide;

[0066] S4, enzymatic protein hydrolysis: 18 g of tea (black tea) dietary crude polysaccharide was dissolved in preheated pure water at 45°C to prepare a 10 mg / mL crude extract solution; papain (800 U / mg) was added at a proportion of 0.08% (m / v, g / mL); the solution was hydrolyzed at 45°C for 4 h; the solution was cooled to room temperature and centrifuged at 4500 rpm for 7 min, and the supernatant was collected.

[0067] S5, polyamide column chromatography: the supernatant obtained in step S4 was gently added to the column bed of a polyamide column (55 mm x 93 cm, 2.2 L, 100-200 mesh polyamide powder) at the upper end of the column bed using a long pipette, the lower end switch was opened, and the supernatant was completely adsorbed in the column. After 40 min of adsorption equilibrium, a peristaltic pump was connected to the upper end of the column to control the flow rate at 2.8 mL / min, and a fraction collector was connected to the lower end outlet to set 4.5 min / tube. The eluate was collected for 1-1.2 column volumes. The collected liquid was analyzed by 420 nm absorbance determination, and the collected liquid before the inflection point (i.e. 1 column volume of eluate) was combined and concentrated to 1 / 17 of the original volume at 55°C under reduced pressure. The concentrated liquid was transferred to a 3000 Da dialysis bag using a pipette, 90 times the volume of the concentrated liquid of pure water was added, and dialysis was performed at room temperature (25-30°C) for 24 h, with the pure water being replaced at intervals of 3 h, 4 h, 6 h, and 11 h. The dialysate was centrifuged to obtain the supernatant, which was concentrated to 1 / 4 of the original volume under reduced pressure, and the ethanol concentration of the concentrated liquid was adjusted to 75% with anhydrous ethanol. The concentrated liquid was alcohol precipitated overnight, centrifuged to remove water, and dried at 50°C to obtain tea (black tea) dietary polysaccharide;

[0068] In the eluate at the inflection point, the target component and the impurity component change as follows: the OD value of the anthrone coloration of the polysaccharide suddenly decreases; the OD value of the coomassie brilliant blue coloration of the protein suddenly increases; the OD value of the foline coloration of the polysaccharide first decreases and then increases; and the OD value at 420 nm of the pigment first decreases and then increases.

[0069] S6, starch removal: tea (black tea) dietary polysaccharide was weighed, and the concentration was adjusted to 6 mg / mL by adding pure water. 100 uL (100 mg / mL) of heat-resistant alpha-amylase (CAS: 9001-19-8, 40 U / mg) was added, and the mixture was incubated at 95°C in a water bath for 30 min. After cooling to room temperature, the mixture was centrifuged at 4500 rpm for 6 min. The supernatant was dialyzed against pure water for 24 h, and the dialysate was centrifuged, concentrated, and adjusted to an ethanol concentration of 75% with anhydrous ethanol. The concentrated liquid was alcohol precipitated, centrifuged, dehydrated, and dried to obtain (non-starch black tea) tea dietary polysaccharide.

[0070] Example 3

[0071] The present embodiment provides a green and safe method for extracting and preparing tea (black tea) dietary polysaccharide, which comprises the following steps:

[0072] S1, crushing and sieving: the black tea was crushed by a sample grinder, and sieved through 20 mesh and 100 mesh sieves. The tea (black tea) powder between 20-100 mesh was collected;

[0073] S2, alcohol washing and impurity removal: 90 g of tea (black tea) powder was weighed, and washed with 85% ethanol for 2 times; the first time was washed with 70°C water bath stirring for 45 min at a ratio of 1:19 (m / m), and filtered with a 100 mesh screen; the second time was washed with 1:17 (m / m) ratio, and the filter residue was dried at 65°C to obtain the impurity-removed tea (black tea) powder;

[0074] S3, water extraction and alcohol precipitation: 90 g of the impurity-removed tea (black tea) powder was weighed, and 1:19 (m / m) pure water was added, and extracted with 80°C water bath stirring for 2 h; the extraction liquid was filtered with a 60 mesh screen in a Buchner funnel, and the extraction liquid adsorbed in the tea residue was squeezed out with a grinding rod; the filter residue was extracted twice with 1:17 (m / m) ratio, and the extraction liquid was combined; the extraction liquid was cooled to room temperature, and centrifuged at 5000 rpm for 5 min; the supernatant was collected, and the supernatant was concentrated to 1 / 17 of the original volume at 52-55°C under reduced pressure (vacuum degree -0.95 to -0.1 MPa); the supernatant was cooled to room temperature, and 4 times the volume of 95% ethanol was added; the mixture was stirred with a glass rod, sealed with plastic wrap, and alcohol precipitated at room temperature for 13 h; the mixture was centrifuged at 5000 rpm for 5 min, and the supernatant was poured off; 4 times the volume of 80% ethanol was added, and the precipitate was resuspended by stirring with a glass rod; the supernatant was removed by centrifugation, and the operation was repeated twice; the same volume of anhydrous ethanol was added, and the resuspension was repeated twice; the precipitate was dried in a 60°C oven to obtain tea (black tea) dietary crude polysaccharide;

[0075] S4, enzymatic protein: 16 g of tea (black tea) dietary crude polysaccharide was weighed, and dissolved in 40°C preheated pure water to prepare a 11 mg / mL crude extract solution; 0.06% (m / v, g / mL) papain (800 U / mg) was added, and the mixture was enzymatically hydrolyzed at 50°C water bath for 5 h; the mixture was cooled to room temperature, and centrifuged at 5000 rpm for 5 min; the supernatant was collected.

[0076] S5, polyamide column chromatography: the supernatant obtained in step S4 was gently added to the top of the column bed of a polyamide column (55 mm x 93 cm, 2.2 L, 100-200 mesh polyamide powder as filler) using a long pipette, the lower end switch was opened, and the supernatant was completely adsorbed in the column. After 35 min of adsorption equilibrium, a peristaltic pump was connected to the upper end of the column to control the flow rate at 2.5 mL / min, and a fraction collector was connected to the lower end outlet to set 5 min / tube. The eluate was collected for 1-1.3 column volumes. The collected liquid was analyzed by measuring the absorbance at 420 nm, and the collected liquid before the inflection point (i.e., 1 column volume of eluate) was combined. The concentrated liquid was concentrated under reduced pressure at 45°C to 1 / 16 of the original volume. The concentrated liquid was transferred to a 4000 Da dialysis bag using a pipette, 80 times the volume of the concentrated liquid of pure water was added, and dialysis was performed at room temperature (25-30°C) for 24 h. The pure water was replaced at intervals of 2.5 h, 3.5 h, 5.5 h, and 11.5 h, respectively. The dialysate was centrifuged to obtain the supernatant, which was concentrated under reduced pressure to 1 / 4 of the original volume. The ethanol concentration of the concentrated liquid was adjusted to 75% with anhydrous ethanol, and the concentrated liquid was alcohol-precipitated overnight. After centrifugal dehydration, the tea leaf (black tea) dietary impurity-removed polysaccharide was obtained by drying at 45°C.

[0077] In the eluate at the inflection point, the target component and the impurity component showed the following change rule: the OD value of the sulfuric acid anthrone coloration of the polysaccharide suddenly decreased; the OD value of the coomassie brilliant blue coloration of the protein suddenly increased; the OD value of the Folin-phenol coloration of the polysaccharide first decreased and then increased; and the OD value at 420 nm of the pigment first decreased and then increased.

[0078] S6, starch removal: the tea leaf (black tea) dietary impurity-removed polysaccharide was weighed and dissolved in pure water to a concentration of 6 mg / mL; 100 uL (70 mg / mL) of heat-resistant alpha-amylase (CAS: 9001-19-8, 40 U / mg) was added, and the mixture was stirred and incubated in a 100°C water bath for 20 min. After cooling to room temperature, the mixture was centrifuged at 5000 rpm for 5 min. The supernatant was dialyzed against pure water for 24 h, and the dialysate was centrifuged, concentrated, and adjusted to an ethanol concentration of 75% with anhydrous ethanol. The concentrated liquid was alcohol-precipitated, centrifuged, dehydrated, and dried to obtain the (non-starch black tea) tea leaf dietary polysaccharide.

[0079] Comparative Example 1

[0080] The difference between this comparative example and Example 1 is only that 20% ethanol was used for washing and removing impurities in step S2.

[0081] The results show that the removal effects of caffeine and catechin are good, but the yield of crude polysaccharide is significantly reduced, indicating that 20% ethanol washing and removing impurities will cause loss of polysaccharide, which is not conducive to the extraction and preparation of tea leaf dietary polysaccharide.

[0082] Comparative Example 2

[0083] The difference between this comparative example and Example 1 is only that anhydrous ethanol was used for washing and removing impurities in step S2.

[0084] The results show that the removal effect of catechin is better, but the removal effect of caffeine is poor, which indicates that the impurity removal by ethanol washing will cause caffeine residue and high economic cost, which is not conducive to the extraction and preparation of tea dietary polysaccharide.

[0085] Experiment 1

[0086] The main chemical composition analysis method is as follows:

[0087] (1) Neutral sugar content detection: 0.2-1.0 mg / L tea dietary polysaccharide solution was prepared, D-glucose was used as a standard, and sulfuric acid anthrone colorimetry was used to detect the neutral sugar content;

[0088] (2) Sugar acid content detection: 0.2-1.0 mg / L tea dietary polysaccharide solution was prepared, galacturonic acid was used as a standard, and sulfuric acid carbazole colorimetry was used to determine the sugar acid content;

[0089] (3) Total sugar content detection: 0.2-1.0 mg / L tea dietary polysaccharide solution was prepared, glucose was used as a standard, and sulfuric acid phenol colorimetry was used to detect the total sugar content;

[0090] (4) Pigment quantitative analysis: 0.2-1.0 mg / L tea dietary polysaccharide solution was prepared, and the absorbance was detected at 420 nm to quantify the pigment;

[0091] (5) Protein content determination: 0.2-1.0 mg / L tea dietary polysaccharide solution was prepared, and the protein content was determined according to the Coomassie brilliant blue method;

[0092] (6) Polyphenol content detection: 0.2-1.0 mg / L tea dietary polysaccharide solution was prepared, and the polyphenol content was detected by Folin phenol method.

[0093] The chemical composition analysis results of the (non-starch) tea dietary polysaccharide prepared in the above examples are shown in Table 1 below.

[0094] Table 1

[0095]

[0096] As can be seen from Table 1, the total sugar content of green tea, black tea and black tea non-starch dietary polysaccharide is 87%-92%, and the impurity content of protein, polyphenol and the like does not exceed 2.0%, which indicates that the non-starch tea dietary polysaccharide prepared by the present application has high purity and less impurities, and can be used as a main active ingredient for subsequent application analysis of weight loss, fat loss and sugar reduction function.

[0097] Experiment 2 Fat loss and sugar reduction efficacy evaluation

[0098] Select 6-7 weeks old C57BL / 6J male mice (purchased from Hunan Slike Jingda Experimental Animal Co., Ltd.), fed with high-fat feed (45%-60%) to induce obesity, while using different doses (100-400 mg / kg) of non-starch tea dietary polysaccharides for continuous intervention for 8 weeks, record and observe the changes of body weight, food intake, water intake and behavior characteristics of mice, and measure the fasting blood glucose of mice 4 days before the end of intervention. At the time of dissection, the fasting body weight, liver weight, epididymal fat weight, inguinal subcutaneous fat weight, and scapular brown fat weight were detected, and the important organ tissues were photographed and recorded.

[0099] The specific experimental scheme is shown in Figure 1 .

[0100] Among them, GTSDF, BTSDF, DTSDF are non-starch green tea dietary polysaccharides, non-starch red tea dietary polysaccharides and non-starch black tea dietary polysaccharides prepared in Examples 1-3.

[0101] Normal control feed TP23302 and high-fat feed TP23300 were purchased from Nantong Trelodge Feed Co., Ltd.

[0102] The changes of body weight of mice during the intervention are shown in Figure 2 . The results show that there is no significant difference in the initial body weight of mice in each group ( Figure 2 A), the body weight of mice in each dietary polysaccharide group increases slowly during the intervention ( Figure 2 B and Figure 2 E), and the body weight of mice in each dietary polysaccharide group after 8 weeks of intervention ( Figure 2 C), the body weight change value ( Figure 2 D) and the body weight change rate ( Figure 2 F) are significantly lower than those of the model group mice, and the low-dose black tea dietary polysaccharide group has the best weight loss effect.

[0103] The results of tea dietary polysaccharides for weight loss and blood glucose reduction are shown in Figure 3 . The results show that after 8 weeks of continuous intervention of dietary polysaccharides, the appearance and body type of mice are slender, the abdominal width is narrower, and the obesity degree is reduced ( Figure 3 A), which shows a certain weight loss effect. At the same time, the fasting body weight ( Figure 3 B), fasting blood glucose ( Figure 3 C) and Lee's index ( Figure 3 D) of each dietary polysaccharide intervention group are significantly lower than those of the high-fat model group, indicating that dietary polysaccharides have obvious weight loss and blood glucose reduction effects.

[0104] The results of tea dietary polysaccharides for weight loss are shown in Figure 4 . The results show that the appearance of epididymal fat and inguinal subcutaneous fat in each dietary polysaccharide intervention group is smaller in volume than that of the model group ( Figure 4A). After dietary polysaccharide intervention, the epididymal fat (A) in mice of each group Figure 4 B and C), inguinal subcutaneous fat ( Figure 4 D and E) and scapular brown fat ( Figure 4 The weight and weight index of F and G in the group were significantly lower than those in the model group, suggesting that dietary polysaccharides can significantly alleviate the accumulation of organ fat and have a significant fat-reducing effect.

[0105] Experiment 3: Inflection point and effective eluent range during polyamide column chromatography

[0106] The eluent collected in step S5 of Example 3 was analyzed, and the results are shown in [the table below]. Figure 5 .

[0107] The column volume was 2200 mL (55 mm × 93 cm), and 10 mL of eluent was collected in each collection tube. Near the 218th collection tube, the OD value of the anthrone sulfate staining for polysaccharides suddenly decreased, the OD value of the Coomassie Brilliant Blue staining for proteins suddenly increased, the OD value of the Folin-Ciocalteu staining for polysaccharides initially decreased and then increased, and the OD value of the pigments at 420 nm initially decreased and then increased. This suggests that there is an inflection point in the eluent collection process of crude tea polysaccharides in polyamide column chromatography, and this inflection point is approximately one column volume in size.

[0108] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A green and safe method for preparing dietary tea polysaccharides, characterized in that, It comprises the following steps: S1, crushing and sieving: grinding and sieving tea leaves to collect tea powder; S2, alcohol washing and impurity removing: washing the tea powder with 80%-90% ethanol, filtering, drying the filter residue to obtain impurity-removed tea powder; S3, water extraction and alcohol precipitation: extracting the impurity-removed tea powder with water, centrifuging to take supernatant, concentrating, precipitating with ethanol, centrifuging to discard supernatant, washing and dehydrating, drying the precipitate to obtain tea dietary crude polysaccharide; S4, enzymatic hydrolysis of protein: redissolving the tea dietary crude polysaccharide with pure water and adding papain for enzymatic hydrolysis; centrifuging to collect supernatant; S5, polyamide column chromatography: wet loading the supernatant, adsorbing and equilibrating, washing with pure water, collecting the washing liquid at constant flow; using colorimetric method to determine and analyze the 420nm absorbance of the collected washing liquid, and combining the collected liquid before the inflection point; concentrating under reduced pressure; dialyzing the concentrated liquid with pure water at room temperature; centrifuging the dialyzed liquid to take supernatant, concentrating under reduced pressure, precipitating with alcohol overnight, centrifuging to discard supernatant, washing and dehydrating, drying the precipitate to obtain tea dietary impurity-removed polysaccharide; wherein, the target component and impurity component in the washing liquid at the inflection point change as follows: the anthrone color developing OD value of polysaccharide suddenly decreases; the coomassie brilliant blue color developing OD value of protein suddenly increases; the Folin-phenol color developing OD value of polysaccharide first decreases and then increases; the 420nm OD value of pigment first decreases and then increases; S6, starch removing: redissolving the tea dietary impurity-removed polysaccharide with pure water, adding amylase for enzymatic hydrolysis; cooling the enzymatic hydrolysis product at room temperature, centrifuging to take supernatant, dialyzing at room temperature, centrifuging the dialyzed liquid to take supernatant, concentrating, precipitating with alcohol, centrifuging, dehydrating and drying to obtain tea dietary polysaccharide.

2. The green and safe method for extracting and preparing tea dietary polysaccharides according to claim 1, characterized in that, The tea powder in step S1 is 20-100 mesh.

3. The green and safe method for extracting and preparing tea dietary polysaccharides according to claim 1 or 2, characterized in that, In step S2, the tea powder is washed with 80%-90% ethanol for 2 times; in the first alcohol washing, the solid-liquid ratio (m / m) of the tea powder to 80%-90% ethanol is 1:(15-20); the temperature during washing is 60-80℃, and the time is 30-50min; in the second alcohol washing, the solid-liquid ratio (m / m) of the tea powder to 80%-90% ethanol is 1:(13-18); the temperature during washing is 60-80℃, and the time is 30-50min.

4. The process as claimed in any one of claims 1 to 3, wherein the process is carried out under green and safe conditions. In step S3, the solid-liquid ratio (m / m) of the impurity-removed tea powder to water is 1:(15-20); the extraction temperature is 70-90℃, and the time is 1-2h; preferably, the extraction is performed for 2 times, and the solid-liquid ratio (m / m) in the second extraction is 1:(13-18).

5. The process as claimed in any one of claims 1 to 4, wherein the process is carried out under green and safe conditions. In step S4, the enzymatic hydrolysis temperature is 40-50℃, and the time is 3-5h; Preferably, the enzyme activity of papain is 200-800U / mg; Preferably, in step S4, the tea dietary crude polysaccharide is redissolved with pure water to prepare a 10-20mg / mL solution; papain is added for enzymatic hydrolysis according to a proportion of 0.03%-0.08% (m / v, g / mL).

6. The process as claimed in any one of claims 1 to 5, wherein the process is carried out under green and safe conditions. In step S5, the adsorption and equilibration time is 20-40min; and / or, in step S5, the collected liquid before the inflection point is 1-1.3 column volumes of washing liquid, preferably 1 column volume of washing liquid.

7. The process as claimed in any one of claims 1 to 6, wherein the process is carried out under green and safe conditions. In step S5, the polyamide column is filled with 100-200 mesh polyamide powder; and / or, the volume of the polyamide column is 300 mL to 2.2 L, and the loading amount of the tea dietary crude polysaccharide is 2-18, preferably 15-18 g.

8. The process as claimed in any one of claims 1 to 7, wherein the process is carried out in the presence of a green tea leaf dietary polysaccharide extract. In step S6, the amylase is a thermostable alpha-amylase. Preferably, in step S6, the temperature for enzymolysis is 90-100℃, and the time for enzymolysis is 20-40 min. Preferably, in step S6, the tea dietary impurity-removed polysaccharide is re-dissolved in pure water to prepare a solution of 5-10 mg / mL; the thermostable alpha-amylase (CAS: 9001-19-8, 40 U / mg) is prepared into a solution of 50-100 mg / mL; and 100 μL of the thermostable alpha-amylase solution is added to every 10 mL of the tea dietary impurity-removed polysaccharide solution.

9. A tea dietary polysaccharide prepared by the method of any one of claims 1-8.

10. Use of the dietary polysaccharide of claim 9 in the preparation of a health food or a medicine for weight loss, blood sugar reduction, or fat reduction.

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