CN-121627933-B - Separation and purification method and application of golden flower Tibetan tea polysaccharide

Abstract

The invention discloses a method for separating and purifying polysaccharide of Tibetan golden flower tea and application thereof, belonging to the technical field of Tibetan golden flower tea, comprising the steps of crushing and sieving Tibetan golden flower tea, adding ethanol for extraction and centrifuging to obtain precipitate; adding water into the precipitate to regulate pH, adding complex enzyme for enzymolysis, centrifuging to collect supernatant, vacuum concentrating, precipitating with ethanol, and centrifuging to obtain crude polysaccharide extract. Adding water to regulate pH, adding papain for enzymolysis, heating, maintaining temperature, adding compound solvent, centrifuging to obtain upper water phase, petroleum ether treating, compound adsorbent adsorbing, dialyzing to obtain refined crude polysaccharide, chromatography, gradient eluting, concentrating, dialyzing, and freeze drying to obtain the final product. According to the invention, through the multi-step collaborative purification processes of composite enzymolysis, composite solvent impurity removal, composite adsorbent adsorption, gradient elution and the like, proteins, liposoluble pigments, micromolecular impurities and salts in the Tibetan golden flower tea polysaccharide are effectively removed, and the purity and uniformity of the product are obviously improved.

Inventors

• YU MANYOU
• ZHU YONGQING
• GAN LU
• WU TAO
• XIA CHEN
• ZHAO JIAJUN
• ZHANG TING
• Xiang Zhuoya
• DENG JUNLIN
• HUANG QIAOLIAN
• YUAN YINGHAO
• TANG XIAOBO

Assignees

• 四川省农业科学院农产品加工研究所(四川省农业科学院食物与营养健康研究所)

Dates

Publication Date

20260505

Application Date

20260204

Claims (3)

1. 1. The method for separating and purifying the Tibetan golden flower tea polysaccharide is characterized by comprising the following steps of: s1, crushing and sieving the golden flower Tibetan tea to obtain golden flower Tibetan tea powder, adding ethanol, extracting and centrifuging, and collecting precipitate; s2, adding water into the sediment of the S1, regulating the pH to 5.0-5.5, and adding compound enzyme, wherein the compound enzyme is pectase, cellulase and papain with the mass ratio of 1.5-2.3:1.4-2.1:0.8-1.2, and performing enzymolysis to obtain enzymolysis liquid; s3, extracting the enzymolysis liquid for 3 hours in a 55 ℃ water bath, carrying out ultrasonic treatment once every 10min, stirring for 1 time every 20min, centrifuging to collect supernatant extract after the extraction is finished, carrying out repeated extraction on the precipitate residues according to the steps, and combining the two extracts; S4, concentrating the supernatant extractive solution in vacuum, cooling to obtain concentrated solution A, adding absolute ethanol, precipitating with ethanol at 4deg.C for 8 hr, centrifuging, and collecting precipitated solid to obtain crude polysaccharide extract; S5, adding water into the crude polysaccharide extract, stirring, adjusting the pH to 6.0-6.5, adding papain according to 1% of the mass of the crude polysaccharide extract, carrying out enzymolysis, heating to 78-82 ℃, preserving heat, cooling, adding a compound solvent, oscillating and centrifuging, collecting an upper water phase, repeating for 3 times, and merging the water phases, wherein the compound solvent consists of chloroform and n-butanol in a volume ratio of 4-6:1; S6, adding petroleum ether with the same volume into the upper water phase, uniformly mixing for 10-15min, centrifuging for 5-8min at 4000-5000r/min, collecting the lower water phase, adding a composite adsorbent according to 12-18% of the volume of the lower water phase, oscillating and adsorbing for 8-12h at 25-30 ℃, filtering and collecting filtrate, adding the filtrate into a dialysis bag, dialyzing for 48-60h in the pure water, changing the pure water for 1 time every 10-12h, vacuum rotary evaporating and concentrating to 1/5-1/4 of the original volume, adding absolute ethanol with 3-4 times of the volume for 6-8h, centrifuging and collecting precipitate, and drying at 50-60 ℃ to obtain refined crude polysaccharide; The composite adsorbent consists of modified diatomite and macroporous resin AB-8 in a mass ratio of 1.8-2.4:1; S7, dissolving refined crude polysaccharide in pure water to prepare polysaccharide mother liquor of 15-30mg/mL, centrifuging at 9000-10000r/min for 8-10min, taking supernatant, balancing the column body of SEPHACRYL S-400HR separation column with pure water until effluent pH=7.0+ -0.2, loading the supernatant at a flow rate of 2-3mL/min, eluting with pure water for 2-3 times of column volume to obtain pre-chromatographic liquid, and combining main peak eluents of polysaccharide; S8, through a pure water balance DEAE SEPLIFE FF exchange column until effluent pH=7.0+/-0.2, loading the pre-chromatographic liquid at a flow rate of 1-2mL/min, eluting with 2-3 times of column volume pure water, and then sequentially adopting 0.05-0.1M, 0.1-0.2M, 0.2-0.3M and 0.3-0.4M NaCl solution for gradient elution, wherein the flow rate is kept at 3-4mL/min; s9, collecting all eluents, combining eluents corresponding to main peaks, concentrating in vacuum to obtain concentrated solution B, dialyzing the concentrated solution B in pure water to obtain dialysis concentrated solution, and freeze-drying to obtain the golden flower Tibetan tea polysaccharide; The preparation method of the modified diatomite comprises the steps of taking natural diatomite, adding distilled water with the volume being 2-3 times of that of the natural diatomite, stirring for 20-30min at 25-30 ℃, ball milling for 1.5-2h at 300-400r/min to obtain diatomite particles, weighing distilled water according to the weight being 2-3 times of that of the natural diatomite, adding methionine with the weight being 1-1.5% of that of the diatomite at the speed being 1-2mL/min at the speed being 150-200r/min, continuously stirring for 15-20min to obtain methionine aqueous solution, adding the diatomite particles into the methionine aqueous solution at the speed being 0.5-1.0g/min, improving the stirring speed by 20-50r/min, and dispersing for 30-40min at 25-30 ℃ to obtain a dispersion; Adding titanate coupling agent with the mass of 0.6-0.8% of natural diatomite into the dispersion liquid at the speed of 1-2mL/min, heating to 60-65 ℃ at the temperature of 2-5 ℃/min after dripping, reacting for 1.5-2h at the constant temperature of 150-200r/min, cooling to room temperature, filtering to collect filter cakes, washing the filter cakes with distilled water, washing the filter cakes with water which is 1-1.5 times of the mass of the natural diatomite each time, stirring and washing for 10-15min, then carrying out suction filtration, repeatedly washing until the pH value of the washing liquid is 6.5-7.5, drying the washed filter cakes at the temperature of 60-65 ℃ and the vacuum degree of 0.06-0.08MPa for 4-6h, and crushing, and sieving the filter cakes with a 200-mesh sieve to obtain the modified diatomite.
2. 2. A Tibetan tea polysaccharide of golden flower prepared by the separation and purification method of claim 1.
3. 3. Use of the golden flower Tibetan tea polysaccharide according to claim 2 in the preparation of a medicament for treating type 2 diabetes.

Description

Separation and purification method and application of golden flower Tibetan tea polysaccharide Technical Field The invention relates to the technical field of golden flower Tibetan tea, in particular to a golden flower Tibetan tea polysaccharide separation and purification method and application. Background The type II diabetes is taken as a global high-incidence chronic metabolic disease, long-term use of chemical synthesis drugs in treatment is easy to accompany side effects, so that a natural blood sugar-reducing active ingredient becomes a research hot spot, tea polysaccharide is taken as a natural active substance with homology of medicine and food, good potential is shown in the aspect of sugar metabolism regulation, and polysaccharide substances formed by fermenting the golden flower Tibetan tea by microorganisms have further development value due to unique fermentation conversion characteristics. In the field of preparation of the golden flower Tibetan tea polysaccharide, the existing extraction and purification process mostly adopts a single water extraction and alcohol precipitation combined simple impurity removal step, and separation pertinence of protein, fat-soluble pigment, small molecular impurities and the like in the polysaccharide is insufficient, so that the purity and uniformity of the obtained product are poor, and the hypoglycemic activity is poor, so that the application of the golden flower Tibetan tea polysaccharide in the field of hypoglycemic functional products is limited. Based on the above, the invention designs a method for separating and purifying the Tibetan tea polysaccharide of golden flower and application thereof to solve the problems. Disclosure of Invention Aiming at the defects existing in the prior art, the invention provides a method for separating and purifying Tibetan golden flower tea polysaccharide, which comprises the following steps: s1, crushing and sieving the golden flower Tibetan tea to obtain golden flower Tibetan tea powder, adding ethanol, extracting and centrifuging, and collecting precipitate; S2, adding water into the sediment of the S1, adjusting the pH to 5.0-5.5, adding complex enzyme, and performing enzymolysis to obtain enzymolysis liquid; S3, after the enzymolysis liquid is extracted, centrifugally collecting supernatant extract; s4, concentrating the supernatant extractive solution in vacuum, cooling to obtain concentrated solution A, precipitating with ethanol, centrifuging, and collecting precipitated solid to obtain crude polysaccharide extract; S5, adding water into the crude polysaccharide extract, stirring, adjusting the pH to 6.0-6.5, adding papain, performing enzymolysis, heating to 78-82 ℃ and preserving heat, cooling, adding a composite solvent, oscillating and centrifuging, and collecting an upper water phase; S6, adding petroleum ether into the upper water phase, mixing uniformly, centrifugally collecting the lower water phase, adding a composite adsorbent, oscillating and adsorbing, filtering and collecting filtrate, dialyzing, concentrating in vacuum, precipitating with ethanol, centrifugally drying to obtain refined crude polysaccharide; S7, dissolving refined crude polysaccharide in water to prepare polysaccharide mother solution, centrifuging to obtain supernatant, loading the supernatant, eluting to obtain pre-chromatographic liquid, and combining main peak eluents of polysaccharide; S8, loading and eluting the pre-chromatographic liquid, and then adopting NaCl solution with the concentration of 0.05-0.1M, 0.1-0.2M, 0.2-0.3M and 0.3-0.4M to perform gradient elution in sequence; and S9, collecting all eluents, combining eluents corresponding to main peaks, concentrating in vacuum to obtain concentrated solution B, dialyzing the concentrated solution B to obtain dialysis concentrated solution, and freeze-drying to obtain the Tibetan golden flower tea polysaccharide. Further, in S2, the compound enzyme is pectase, cellulase and papain with a mass ratio of 1.5-2.3:1.4-2.1:0.8-1.2. Further, the composite adsorbent consists of modified diatomite and macroporous resin AB-8 in a mass ratio of 1.8-2.4:1. Further, the preparation method of the modified diatomite comprises the steps of taking natural diatomite, adding distilled water with the volume being 2-3 times of that of the natural diatomite, stirring for 20-30min at 25-30 ℃, ball milling for 1.5-2h at 300-400r/min to obtain diatomite particles, weighing distilled water according to the weight being 2-3 times of that of the natural diatomite, adding methionine with the weight being 1-1.5% of that of the diatomite at the speed being 1-2mL/min at 150-200r/min, continuously stirring for 15-20min to obtain methionine aqueous solution, adding the diatomite particles into the methionine aqueous solution at the speed being 0.5-1.0g/min, increasing the stirring speed by 20-50r/min, and dispersing for 30-40min at 25-30 ℃ to obtain a dispersion; Adding titanate coupling agent with the mass