CN102954943A - Method for detection of tea polysaccharide in Pu'er tea or Pu'er tea extract - Google Patents
Method for detection of tea polysaccharide in Pu'er tea or Pu'er tea extract Download PDFInfo
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Abstract
Belonging to the field of food analysis, the invention relates to a method for detection of tea polysaccharide in Pu'er tea or a Pu'er tea extract. The detection method comprises the steps of: 1. preparing a glucose standard solution; 2. preparing a Pu'er tea or Pu'er tea extract sample into a detection solution; 3. measuring the absorbance of the standard reference solution and the detection solution by ultraviolet spectrophotometry, and drawing a standard curve; and 4. calculating the content of tea polysaccharide in the Pu'er tea or the Pu'er tea extract.
Description
Technical field:
The present invention relates to the detection method of tea polysaccharide in a kind of Pu'er tea or the Pu'er tea, belong to the food analysis field.
Background technology:
The Pu'er tea original producton location is mainly in Simao Diqu and Xishuangbanna in Yunnan.Along with expanding economy, the enhancing of people's health care consciousness, Pu'er tea is sweet because of mellow time of its flavour, and the fine quality of Chen Xiang uniqueness and its special health-care efficacy to human body play concern and the attention of the whole society, and product is subjected to consumer's favor deeply.
Modern study shows that the health cares such as tealeaves lipopenicillinase, fat-reducing, hypoglycemic are the results of Multiple components combined action, what wherein play a major role has two large class materials, one class is polyphenol compound (abbreviation Tea Polyphenols), and another kind of is liopopolysaccharides compound (abbreviation tea polysaccharide).
Experimental study indicates, the health-care effect of tea polysaccharide may be more remarkable in the Pu'er tea.The research Pu ' er Tea Polysaccharide hypolipemic function dose-effect relationship results such as Wu Wenhua show, compare with high fat control group, low dosage tea polysaccharide group (3 groups) mice serum triglyceride (TG), cholesterol (TC), LDL-C (LDL-C), HDL-C (HDL-C) level and AI value do not have significant difference.And middle and high dosage group (4,5 groups) mice serum TG, TC, LDL-C level have descended respectively 40.60%, 25.36%, 24.38%; 40.02%, 26.68%, 33.59%.Simultaneously, the HDL-C level has improved respectively 73.44%, 82.81%.But there is not notable difference between middle and high 2 dosage groups.Show that tea polysaccharide suppresses high fat diet lipid of mice rising possibility amount effect relationship.Wu Wenhua etc. (2007) have studied tea polysaccharide and the comparison of Tea Polyphenols hypolipemic function in the Pu'er tea.The result shows: compare with high fat control group, tea polysaccharide group (3 groups) mice serum TG, TC, LDL-C level have descended respectively 40.60%, 25.36%, 24.38%, and the HDL-C level has risen 73.44% simultaneously.And the above indices of Tea Polyphenols group (4 groups) lipid of mice does not all have significant difference.Show that tea polysaccharide suppresses the effect of high fat diet lipid of mice rising greater than Tea Polyphenols in the Pu'er tea.
Tea polysaccharide also has the significant blood sugar health care that reduces.Ni Dejiang etc. studies show that different teas polysaccharide all have the test-type diabetic mice and reduce extremely significantly the blood sugar effect.Under low, middle dosage, tea polysaccharide falls hypoglycemic effect and obviously is better than the green tea tea polysaccharide in oolong tea, black tea, black tea, the white tea.Japanese scholars clear water Cen husband has disclosed also that hypoglycemic effective constituent is water miscible component complex polysaccharide, i.e. tea polysaccharide in the tealeaves.
Because tea polysaccharide has good health-care efficacy in the Pu'er tea, thus Accurate Determining tea polysaccharide content to select raw material and estimate extract, purifying process is extremely important, but perplexing the mensuration of tea polysaccharide always.At present, the Fast Measurement polyoses content mainly is with By Anthrone Sulphuric acid method or phenolsulfuric acid method both at home and abroad.Owing to containing the pigment materials such as a large amount of thearubigins, theabrownin, theaflavin in the Pu'er tea, the chromogenic reaction during tea polysaccharide is detected can cause very large interference, causes testing result higher, can't accurately obtain tea polysaccharide content.Therefore, content for tea polysaccharide in Accurate Determining Pu'er tea or the Pu'er tea, reduce the interference of other materials as far as possible, the invention provides the detection method of tea polysaccharide content in a kind of fast detecting Pu'er tea or the Pu'er tea, but the content of the method Accurate Determining tea polysaccharide has overcome the defective of prior art.
Summary of the invention:
The invention provides a kind of detection method that detects tea polysaccharide in Pu'er tea or the Pu'er tea.
Detection method of the present invention adopts ultraviolet spectrophotometry.
Detection method of the present invention adopts the glucose standard solution in contrast, is mixed with the standard solution of variable concentrations, with uv-spectrophotometric Instrument measuring and drawing standard curve.
Detection method of the present invention comprises Pu'er tea or Pu'er tea processed obtaining detecting liquid, detects with the uv-spectrophotometric instrument, obtains absorbance data.
Detection method of the present invention comprises the gained absorbance data is appraised and decided calculating with typical curve, obtains tea polysaccharide content.
Therefore, detection method of the present invention may further comprise the steps:
Step 1, preparation glucose standard solution is in contrast;
Step 2 is separated Pu'er tea or Pu'er tea sample through alcohol precipitation, acid hydrolysis and polyamide column, be mixed with detection liquid; Thereby separate tea polysaccharide and pigment, make the measurement of the polysaccharide content result more accurate.
Step 3 is with the absorbance of determined by ultraviolet spectrophotometry standard reference material solution and detection liquid, drawing standard curve;
Step 4 is calculated tea polysaccharide content in Pu'er tea or the Pu'er tea sample.
Wherein, the glucose standard solution in the step 1 is the D-anhydrous dextrose; Preparation glucose standard solution in the step 1 may further comprise the steps: get D-anhydrous dextrose reference substance an amount of, accurately weighed (being accurate to 0.001g) is dissolved in water and dilutes and make the solution that contains 0.2mg among every 1ml, in contrast product solution.
The Pu'er tea sample ligand is made in the step 2 detected liquid and be may further comprise the steps:
Get Pu'er tea 2g, put in the 250ml flask, add respectively 10-30ml (preferred 20ml) boiling water lixiviate 2-4 time (preferred 3 times), each 5-20min (preferred 10min), merging filtrate.Add ethanol and be adjusted to ethanol content 60-90% (preferred 80%); Filter, be 60-90% (preferred 80%) ethanol washing filter residue 2-4 time (preferred 3 times) with hot concentration, 20-40ml/ time (preferred 30ml/ time) volatilizes solvent, and filter paper is put in the flask together with filter residue, add 20-80ml (preferred 50ml) water, add hot reflux 0.5-1.5h (preferred 1h), filter while hot, with 60-100 ℃ of hot wash, merging filtrate is settled to 100ml.The accurate 5ml of absorption extracts solution, adds hydrochloric acid solution, and the concentration of hydrochloric acid is 0.2-1.0mol/L (preferred 0.5mol/L) in the mixed liquor, at 30-70 ℃ (preferred 40 ℃) lower hydrolysis 15-60min (preferred 30min), transfers pH to neutral after the cooling; Polysaccharide extraction liquid after the acid hydrolysis is put polyamide chromatography post (among the 15cm * 2cm), with 60-100 ℃ of (preferred 70-90 ℃) hot water elution, to be collected filtrate, is settled to 100ml, as need testing solution.
The Pu'er tea sample ligand is made in the step 2 detected liquid and be may further comprise the steps:
Get Pu'er tea 1g, put in the 250ml flask, add 60-90% (preferred 80%) ethanol 20-80ml (preferred 50ml), 80-95 ℃ of (preferred 95 ℃) water-bath backflow 15-60min (preferred 30min), filter while hot, with hot 60-90% (preferred 80%) ethanol washing 2-4 time (preferred 3 times), 20-40ml/ time (preferred 30ml/ time) volatilizes solvent, and filter paper is put in the flask together with filter residue, add 20-80ml (preferred 50ml) water, add hot reflux 0.5-1.5h (preferred 1h), filter while hot, use hot wash, merging filtrate is settled to 100ml.The accurate 5ml of absorption extracts solution, adds hydrochloric acid solution, and the concentration of hydrochloric acid is 0.2-1.0mol/L (preferred 0.5mol/L) in the mixed liquor, at 30-70 ℃ (preferred 40 ℃) lower hydrolysis 15-60min (preferred 30min), transfers pH to neutral after the cooling; Polysaccharide extraction liquid after the acid hydrolysis is put polyamide chromatography post (among the 15cm * 2cm), with 60-100 ℃ of (preferred 70-90 ℃) hot water elution, to be collected filtrate, is settled to 100ml, as need testing solution.
Wherein, may further comprise the steps with determined by ultraviolet spectrophotometry in the step 3, with glucose standard solution solution with detect liquid and add respectively 5% phenol solution 1.0ml, the jolting mixing adds 5.0ml sulfuric acid, with the rapid jolting mixing of miniature whirlpool mixed instrument, placed 30 minutes under the room temperature, take the 1st pipe as blank, according to an appendix VB of Chinese Pharmacopoeia version in 2005 UV-VIS spectrophotometry, measure absorbance at 487nm wavelength place.
Wherein, the drafting of step 3 Plays curve may further comprise the steps:
Accurate absorption reference substance solution 0.0,0.1,0.2,0.3,0.4,0.5ml put respectively in the 10ml tool plug test tube respectively, add respectively two pure water to 1.0ml, each is accurate to add 5% phenol solution and (takes by weighing the 2.5g re-distilled phenol, be dissolved in water and be diluted to 50ml, shake up, and get final product.) 1.0ml, the jolting mixing, add 5.0ml sulfuric acid, with the rapid jolting mixing of miniature whirlpool mixed instrument, placed 30 minutes under the room temperature, take the 1st pipe as blank, according to UV-VIS spectrophotometry (appendix VB of Chinese Pharmacopoeia version in 2005), measure absorbance (in 1 hour, measuring complete) at 487nm wavelength place.Make typical curve with absorbance (Y) and quality (X).
Pu'er tea of the present invention is Pu'er tea and the goods thereof that can buy on any market, such as the tea cake, and piece tea, liquid tea beverage, instant tea etc.
Pu'er tea of the present invention is that any Pu'er tea extraction by prior art or new technology separated the Pu'er tea that obtains, as prepare the raw material of instant tea, and the raw material of preparation liquid tea beverage, preparation contains the food of Pu'er tea composition, medicine, the raw material of health products etc.Method comprises:
Method 1 is got Pu'er tea, adds water and acutely seethes with excitement and decoct to extract 1-5 time, decocting time 0.5-5 hour, add water general times 5-50 doubly, extract 40-300 order filters, filtrate nature or heat exchange are cooled to 30-55 ℃, and 10-30 ten thousand molecular weight membrane filtrations get membrane filtration liquid and film trapped fluid; The film trapped fluid is concentrated into the concentrate that solid content is 5-30%, and concentrate is once centrifugal, a centrifugate secondary centrifuging, and with membrane filtration liquid and secondary centrifuging liquid separately or merge and be concentrated into proportion 1.01-1.4/55-65 ℃, condensed cream is dry.
Method 2 is got Pu'er tea, and filling group circulated in countercurrent is extracted, and every group is comprised of the 2-5 tank; Every tank boiling decocts extracts 3-8 time, adds water 3-10 doubly, each extraction time 0.5-1.5h at every turn; The extract first time of every tank enters fluid reservoir, and the subsequent extracted liquid of every tank is successively as the extraction solvent of next tank; After adding new raw material, the tank of finishing extraction enters circulation, begin new extraction, extract 40-300 order filters, filtrate is concentrated into tealeaves (weight): concentrate (volume)=1: 0.5-10, concentrate is once centrifugal, one time centrifugate is passed through secondary centrifuging again, and secondary centrifuging liquid is concentrated into proportion 1.01-1.4/55-65 ℃, and condensed cream is dry.
Method 3 is got Pu'er tea, and filling group circulated in countercurrent is extracted, and every group is comprised of the 2-5 tank; Every tank boiling decocts extracts 3-8 time, adds water 3-10 doubly, each extraction time 0.5-1.5h at every turn; The extract first time of every tank enters fluid reservoir, and the subsequent extracted liquid of every tank is successively as the extraction solvent of next tank; Enter circulation after the tank of finishing extraction adds new raw material, begin new extraction, the filtration of extracting liquid filtering extract 60-300 order, filtrate nature or heat exchange are cooled to 30-55 ℃, and 10-30 ten thousand molecular weight membrane filtrations get membrane filtration liquid and film trapped fluid; The film trapped fluid is concentrated into the concentrate that solid content is 5-30%, and concentrate is once centrifugal, a centrifugate secondary centrifuging, and with membrane filtration liquid and secondary centrifuging liquid separately or merge and be concentrated into proportion 1.01-1.4/55-65 ℃, condensed cream is dry.
Method 4 is got Pu'er tea, and filling group circulated in countercurrent is extracted, and every group is comprised of the 2-5 tank; Every tank boiling decocts extracts 3-8 time, adds water 3-10 doubly, each extraction time 0.5-1.5h at every turn; The extract first time of every tank enters fluid reservoir, and the subsequent extracted liquid of every tank is successively as the extraction solvent of next tank; After adding new raw material, the tank of finishing extraction enters circulation, begin new extraction, extract 40-300 order filters, filtrate is concentrated into tealeaves (weight): concentrate (volume)=1: 2-1: 10, left standstill 8-16 hour under concentrate 4-7 ℃, supernatant is concentrated into proportion 1.01-1.4/55-65 ℃ with pulp board or filter paper press filtration or filter element filtering, filtrate, and condensed cream is dry.
Method 5 is got Pu'er tea Ultramicro-powder flour, adds water in 75-98 ℃ of lixiviate 1-4 time, adds water multiple 8-30 doubly, extraction time 1-3 hour; Continuous stirring in extraction and the blowing process, each extraction slurry is centrifugal with hydro-extractor, merges centrifugate, and centrifugate nature or heat exchange are cooled to 30-55 ℃, and 10-30 ten thousand molecular weight membrane filtrations get membrane filtration liquid and film trapped fluid; The film trapped fluid is concentrated into the concentrate that solid content is 5-30%, and concentrate is once centrifugal, a centrifugate secondary centrifuging, and with membrane filtration liquid and secondary centrifuging liquid separately or merge and be concentrated into proportion 1.01-1.4/55-65 ℃, condensed cream is dry.
Beneficial effect of the present invention is as follows:
The present invention adopts acid hydrolysis to separate the preparation method that two steps have been improved Pu'er tea and Pu'er tea sample with polyamide column, has effectively separated measurement result is produced the materials such as Tea Pigment that disturb, and has realized accurately determination of polysaccharide.
Adopt this law (being hydrolyzed the post method) determination data and directly adopt phenolsulfuric acid method (direct development process behind the alcohol precipitation: do not contain sample acid hydrolysis and polyamide column separating step, all the other operations all operate identically with the present invention, and the constant volume liquid that adds hot reflux behind the sample thief alcohol precipitation is measured) determination data relatively sees Table 1.Data show, directly adopt the phenolsulfuric acid method to detect owing to there being the interference of Tea Pigment to cause the result higher; Adopt this law to detect the interference of effectively having removed the materials such as Tea Pigment, the result is more accurate.
Table 1: the phenolsulfuric acid method detects tea polysaccharide content in the Pu'er tea
Description of drawings:
Fig. 1: glucose typical curve
Embodiment:
Further specify by the following examples the present invention, but not as limitation of the present invention.
Embodiment 1: tea polysaccharide assay in the Pu'er tea
One. instrument and reagent
1. instrument
Ultraviolet-visible spectrophotometer; Miniature whirlpool mixed instrument; Ten thousand/balance; Ultrasound Instrument.
2. test sample and reagent
Test sample: instant Pu'er tea
Reagent: re-distilled phenol; Sulfuric acid.
Reference substance: D-anhydrous dextrose, Nat'l Pharmaceutical ﹠ Biological Products Control Institute's (for assay).
Two. test method
1. the preparation of need testing solution
Puerh tea leaves need testing solution preparation method: get approximately 2g (being accurate to 0.001g), put in the 250ml flask, add respectively 20ml boiling water lixiviate 3 times, each 10min, merging filtrate.Add absolute ethyl alcohol and be adjusted to ethanol content 80%.Filter, with hot 80% ethanol washing filter residue 3 times (30Ml/ time), volatilize solvent, filter paper is put in the flask together with filter residue, adds 50Ml water, adds hot reflux 1h, filters while hot, uses hot wash, and merging filtrate is settled to 100Ml.The accurate 5Ml of absorption extracts solution, adds hydrochloric acid solution (concentration of hydrochloric acid is 0.5mol/L in the mixed liquor), at 40 ℃ of lower hydrolysis 30min, transfers pH to neutral after the cooling.Polysaccharide extraction liquid after the acid hydrolysis is put polyamide chromatography post (among the 15cm * 2cm), to be used the hot water elution, collects filtrate, be settled to 100Ml, as need testing solution.
2. the preparation of reference substance solution
Get D-anhydrous dextrose reference substance an amount of, accurately weighed (being accurate to 0.001g) is dissolved in water and dilutes and make the solution that contains 0.2mg among every 1ml, in contrast product solution.
3. the drafting of typical curve
Accurate absorption reference substance solution 0.0,0.1,0.2,0.3,0.4,0.5ml put respectively in the 10ml tool plug test tube respectively, add respectively two pure water to 1.0ml, each is accurate to add 5% phenol solution and (takes by weighing the 2.5g re-distilled phenol, be dissolved in water and be diluted to 50ml, shake up, and get final product.) 1.0ml, the jolting mixing, add 5.0ml sulfuric acid, with the rapid jolting mixing of miniature whirlpool mixed instrument, placed 30 minutes under the room temperature, take the 1st pipe as blank, according to UV-VIS spectrophotometry (appendix VB of Chinese Pharmacopoeia version in 2005), measure absorbance (in 1 hour, measuring complete) at 487nm wavelength place.Make typical curve with absorbance (Y) and quality (X), regression equation is:
Y＝a·X+b
In the formula:
Y is the absorbance of reference substance solution;
X is the quality (mg) of D-anhydrous dextrose;
A, b are constant.
4. the mensuration of need testing solution is calculated tea polysaccharide content in Pu'er tea or the Pu'er tea sample.
The accurate 1ml need testing solution of drawing under the drafting item according to typical curve, from " the phenol solution 1.0ml of accurate adding 5% ", with the method operation, is measured respectively absorbance at the 487nm place.
Data are processed
Be calculated as follows content:
In the formula:
C is the tea polysaccharide percentage composition in the sample;
A is the absorbance of sample;
W is sample weighting amount (g);
V is constant volume (Ml);
F is extension rate;
A, b are constant.
Three. test findings
1. the range of linearity is investigated
Accurate absorption D-anhydrous dextrose reference substance solution (concentration is 0.2032mg/ml) 0.0,0.1,0.2,0.3,0.4,0.5ml put respectively in the 10ml tool plug test tube, add respectively two pure water to 1.0ml, the phenol solution of each accurate adding 5% (takes by weighing the 2.5g re-distilled phenol, be dissolved in water and be diluted to 50ml, shake up, and get final product.) 1.0ml, the jolting mixing, add 5.0ml sulfuric acid, with the rapid jolting mixing of miniature whirlpool mixed instrument, placed 30 minutes under the room temperature, take the 1st pipe as blank, according to UV-VIS spectrophotometry (appendix VB of Chinese Pharmacopoeia version in 2005), measure absorbance (in 1 hour, measuring complete) at 487nm wavelength place.Make typical curve with absorbance (Y) and quality (X), regression equation is:
Y＝8.0339X-0.0332
In the formula:
Y is the absorbance of reference substance solution;
X is the quality (mg) of D-anhydrous dextrose.
2. precision test
Get same lot number (200911004-1) sample, get approximately 2g, accurately weighed, operate according to the need testing solution preparation method, press the assay method of need testing solution, in 487nm place METHOD FOR CONTINUOUS DETERMINATION absorbance 6 times, the RSD that calculates absorbance is 0.36% (n=6).The results are shown in Table 2.
Table 2 precision test
3. replica test
Get same lot number (200911004-1) sample, get approximately 2g (totally 6 parts), accurately weighed, according to the need testing solution preparation manipulation, press the assay method of need testing solution, measure respectively absorbance in the 487nm place, calculate tea polysaccharide content, RSD is 1.13% (n=6).The results are shown in Table 3.
Table 3 replica test
4. stability test
Get same lot number (200911004-1) sample, get approximately 2g, accurately weighed, operate according to the need testing solution preparation method, press the assay method of need testing solution, 0,2,4,6,8,10,12h measures its absorbance in the 487nm place, the RSD that calculates absorbance is 0.44% (n=6).The results are shown in Table 4.
Table 4 stability test
5. recovery test
Get same lot number (200911004-1) sample, get approximately 2g (totally 6 parts, tea polysaccharide content is 61.01mg/g), accurately weighed, according to the need testing solution preparation manipulation, after volatilizing 80% hot ethanol, add the D-anhydrous dextrose reference substance solution of 20mg, carry out the application of sample recovery experiment, press the assay method of need testing solution, measure respectively absorbance in the 487nm place, calculate tea polysaccharide content, recovery RSD is 3.65% (n=6).The results are shown in Table 5.
Table 5 recovery test
Embodiment 2: tea polysaccharide assay in the Pu'er tea
One. instrument and reagent
1. instrument
Ultraviolet-visible spectrophotometer; Miniature whirlpool mixed instrument; Ten thousand/balance; Ultrasound Instrument.
2. test sample and reagent
Test sample: instant Pu'er tea
Reagent: re-distilled phenol; Sulfuric acid.
Reference substance: D-anhydrous dextrose, Nat'l Pharmaceutical ﹠ Biological Products Control Institute's (for assay).
Three. test method
1. the preparation of need testing solution
The preparation method of Pu'er tea need testing solution: get approximately 1g (being accurate to 0.001g) of Pu'er tea powder, put in the 250ml flask, add 80% ethanol 50ml, 95 ℃ of water-bath backflow 30min filter while hot, with 3 times (30ml/ time) of hot 80% ethanol washing, volatilize solvent, filter paper is put in the flask together with filter residue, adds 50ml water, adds hot reflux 1h, filter while hot, use hot wash, merging filtrate is settled to 100ml.The accurate 5Ml of absorption extracts solution, adds hydrochloric acid solution (concentration of hydrochloric acid is 0.5mol/L in the mixed liquor), at 40 ℃ of lower hydrolysis 30min, transfers pH to neutral after the cooling.Polysaccharide extraction liquid after the acid hydrolysis is put polyamide chromatography post (among the 15cm * 2cm), to be used the hot water elution, collects filtrate, be settled to 100ml, as need testing solution.
2. the preparation of reference substance solution: with embodiment 1.
3. the drafting of typical curve: with embodiment 1.
4. the mensuration of need testing solution is calculated tea polysaccharide content in Pu'er tea or the Pu'er tea sample.
Three. test findings
Make typical curve with absorbance (Y) and quality (X), regression equation is: Y=8.0339X-0.0332
In the formula: Y is the absorbance of reference substance solution; X is the quality (mg) of D-anhydrous dextrose.
Absorbance A tea polysaccharide content (%)
Pu'er tea 0.294 8.15
Embodiment 3: tea polysaccharide assay in the Pu'er tea
One. instrument and reagent: with embodiment 1.
Two. method:
1. the preparation of need testing solution
Puerh tea leaves need testing solution preparation method: the Pu'er tea sample ligand is made detection liquid may further comprise the steps: get Pu'er tea 2g, put in the 250ml flask, add respectively 10ml boiling water lixiviate 4 times, each 5min, merging filtrate; Add ethanol and be adjusted to ethanol content 60%; Filtering, is 60% ethanol washing filter residue 2 times with hot concentration, 20ml/ time, volatilize solvent, and filter paper is put in the flask together with filter residue, adds 20ml water, adds hot reflux 0.5h, filters while hot, and with 60 ℃ of hot washes, merging filtrate is settled to 100ml; The accurate 5ml of absorption extracts solution, adds hydrochloric acid solution, and the concentration of hydrochloric acid is 0.2mol/L in the mixed liquor, at 30 ℃ of lower hydrolysis 15-60min, transfers pH to neutral after the cooling; Polysaccharide extraction liquid after the acid hydrolysis is put in the polyamide chromatography post, with 60 ℃ of hot water elutions, collected filtrate, be settled to 100ml, as need testing solution.
2. the preparation of reference substance solution: with embodiment 1.
3. the drafting of typical curve: with embodiment 1.
4. the mensuration of need testing solution is calculated tea polysaccharide content in Pu'er tea or the Pu'er tea sample.
Three. the result
Make typical curve with absorbance (Y) and quality (X), regression equation is: Y=8.0339X-0.0332
In the formula: Y is the absorbance of reference substance solution; X is the quality (mg) of D-anhydrous dextrose.
Absorbance A tea polysaccharide content (%)
Puerh tea leaves 0.124 1.96
Embodiment 4: tea polysaccharide assay in the Pu'er tea
One. instrument and reagent: with embodiment 1.
Two. method:
1. the preparation of need testing solution
Puerh tea leaves need testing solution preparation method: the Pu'er tea sample ligand is made detection liquid may further comprise the steps: get Pu'er tea 2g, put in the 250ml flask, add respectively 30ml boiling water lixiviate 2 times, each 20min, merging filtrate; Add ethanol and be adjusted to ethanol content 90%; Filtering, is 90% ethanol washing filter residue 4 times with hot concentration, 40ml/ time, volatilize solvent, and filter paper is put in the flask together with filter residue, adds 80ml water, adds hot reflux 1.5h, filters while hot, and with 100 ℃ of hot washes, merging filtrate is settled to 100ml; The accurate 5ml of absorption extracts solution, adds hydrochloric acid solution, and the concentration of hydrochloric acid is 1.0mol/L in the mixed liquor, at 70 ℃ of lower hydrolysis 15-60min, transfers pH to neutral after the cooling; Polysaccharide extraction liquid after the acid hydrolysis is put in the polyamide chromatography post, with 100 ℃ of hot water elutions, collected filtrate, be settled to 100ml, as need testing solution.
2. the preparation of reference substance solution: with embodiment 1.
3. the drafting of typical curve: with embodiment 1.
4. the mensuration of need testing solution is calculated tea polysaccharide content in Pu'er tea or the Pu'er tea sample.
Three. the result
Make typical curve with absorbance (Y) and quality (X), regression equation is: Y=8.0339X-0.0332
In the formula: Y is the absorbance of reference substance solution; X is the quality (mg) of D-anhydrous dextrose.
Absorbance A tea polysaccharide content (%)
Puerh tea leaves 0.055 1.10
Embodiment 5: tea polysaccharide assay in the Pu'er tea
One. instrument and reagent: with embodiment 1.
Two. method:
1. the preparation of need testing solution
Pu'er tea need testing solution preparation method: get Pu'er tea 1g, put in the 250ml flask, add 60% ethanol 20ml, 80 ℃ of water-bath backflow 15min filter while hot, with hot 60% ethanol washing 2 times, 20-40ml/ time, volatilize solvent, filter paper is put in the flask together with filter residue, add 20ml water, add hot reflux 0.5h, filter while hot, use hot wash, merging filtrate is settled to 100ml; The accurate 5ml of absorption extracts solution, adds hydrochloric acid solution, and the concentration of hydrochloric acid is 0.2mol/L in the mixed liquor, at 30 ℃ of lower hydrolysis 15-60min, transfers pH to neutral after the cooling; Polysaccharide extraction liquid after the acid hydrolysis is put in the polyamide chromatography post, with 60 ℃ of hot water elutions, collected filtrate, be settled to 100ml, as need testing solution.
2. the preparation of reference substance solution: with embodiment 1.
3. the drafting of typical curve: with embodiment 1.
4. the mensuration of need testing solution is calculated tea polysaccharide content in Pu'er tea or the Pu'er tea sample.
Three. the result
Make typical curve with absorbance (Y) and quality (X), regression equation is: Y=8.0339X-0.0332
In the formula: Y is the absorbance of reference substance solution; X is the quality (mg) of D-anhydrous dextrose.
Absorbance A tea polysaccharide content (%)
Pu'er tea 0.249 7.01
Embodiment 6: the mensuration of tea polysaccharide content in the Pu'er tea
One. instrument and reagent: with embodiment 1.
Two. method:
1. the preparation of need testing solution
Pu'er tea need testing solution preparation method: get Pu'er tea 1g, put in the 250ml flask, add 90% ethanol 80ml, 95 ℃ of water-bath backflow 60min filter while hot, with hot 90% ethanol washing 4 times, 20-40ml/ time, volatilize solvent, filter paper is put in the flask together with filter residue, add 80ml water, add hot reflux 1.5h, filter while hot, use hot wash, merging filtrate is settled to 100ml; The accurate 5ml of absorption extracts solution, adds hydrochloric acid solution, and the concentration of hydrochloric acid is 1.0mol/L in the mixed liquor, at 70 ℃ of lower hydrolysis 15-60min, transfers pH to neutral after the cooling; Polysaccharide extraction liquid after the acid hydrolysis is put in the polyamide chromatography post, with 100 ℃ of hot water elutions, collected filtrate, be settled to 100ml, as need testing solution.
2. the preparation of reference substance solution: with embodiment 1.
3. the drafting of typical curve: with embodiment 1.
4. the mensuration of need testing solution is calculated tea polysaccharide content in Pu'er tea or the Pu'er tea sample.
Three. the result
Make typical curve with absorbance (Y) and quality (X), regression equation is: Y=8.0339X-0.0332
In the formula: Y is the absorbance of reference substance solution; X is the quality (mg) of D-anhydrous dextrose.
Absorbance A tea polysaccharide content (%)
Pu'er tea 0.196 5.71.
Claims (10)
1. the detection method of tea polysaccharide in a Pu'er tea or the Pu'er tea is characterized in that, may further comprise the steps:
Step 1, preparation glucose standard solution is in contrast;
Step 2 is separated Pu'er tea or Pu'er tea sample through alcohol precipitation, acid hydrolysis and polyamide column, be mixed with detection liquid;
Step 3 is with the absorbance of determined by ultraviolet spectrophotometry standard reference material solution and detection liquid, drawing standard curve;
Step 4 is calculated tea polysaccharide content in Pu'er tea or the Pu'er tea sample.
2. according to claim 1 detection method is characterized in that, wherein, the glucose standard solution in the step 1 is the D-anhydrous dextrose.
3. according to claim 1 detection method is characterized in that, wherein, the preparation glucose standard solution in the step 1 may further comprise the steps: get D-anhydrous dextrose reference substance, be dissolved in water and dilute and make the solution that contains 0.2mg among every 1ml, in contrast product solution.
4. according to claim 1 detection method is characterized in that, wherein, in the step 2 the Pu'er tea sample ligand is made detection liquid, may further comprise the steps: get Pu'er tea 2g, put in the 250ml flask, add respectively 10-30ml boiling water lixiviate 2-4 time, each 5-20min, merging filtrate; Add ethanol and be adjusted to ethanol content 60-90%; Filtering, is 60-90% ethanol washing filter residue 2-4 time with hot concentration, 20-40ml/ time, volatilizes solvent, filter paper is put in the flask together with filter residue, adds 20-80ml water, adds hot reflux 0.5-1.5h, filters while hot, with 60-100 ℃ of hot wash, merging filtrate is settled to 100ml; The accurate 5ml of absorption extracts solution, adds hydrochloric acid solution, and the concentration of hydrochloric acid is 0.2-1.0mol/L in the mixed liquor, at 30-70 ℃ of lower hydrolysis 15-60min, transfers pH to neutral after the cooling; Polysaccharide extraction liquid after the acid hydrolysis is put in the polyamide chromatography post, with 60-100 ℃ of hot water elution, collected filtrate, be settled to 100ml, as need testing solution;
Or get Pu'er tea 1g, and put in the 250ml flask, add 60-90% ethanol 20-80ml, 80-95 ℃ of water-bath backflow 15-60min filters while hot, with hot 60-90% ethanol washing 2-4 time, 20-40ml/ time, volatilize solvent, filter paper is put in the flask together with filter residue, add 20-80ml water, add hot reflux 0.5-1.5h, filter while hot, use hot wash, merging filtrate is settled to 100ml; The accurate 5ml of absorption extracts solution, adds hydrochloric acid solution, and the concentration of hydrochloric acid is 0.2-1.0mol/L in the mixed liquor, at 30-70 ℃ of lower hydrolysis 15-60min, transfers pH to neutral after the cooling; Polysaccharide extraction liquid after the acid hydrolysis is put in the polyamide chromatography post, with 60-100 ℃ of hot water elution, collected filtrate, be settled to 100ml, as need testing solution.
5. according to claim 4 detection method is characterized in that, wherein, in the step 2 the Pu'er tea sample ligand is made detection liquid, may further comprise the steps: get Pu'er tea 2g, put in the 250ml flask, add respectively 20ml boiling water lixiviate 3 times, each 10min, merging filtrate; Add ethanol and be adjusted to ethanol content 80%; Filtering, is 80% ethanol washing filter residue 3 times with hot concentration, 30ml/ time, volatilize solvent, and filter paper is put in the flask together with filter residue, adds 50ml water, adds hot reflux 1h, filters while hot, and with 60-100 ℃ of hot wash, merging filtrate is settled to 100ml; The accurate 5ml of absorption extracts solution, adds hydrochloric acid solution, and the concentration of hydrochloric acid is 0.5mol/L in the mixed liquor, at 40 ℃ of lower hydrolysis 30min, transfers pH to neutral after the cooling; Polysaccharide extraction liquid after the acid hydrolysis is put polyamide chromatography post (among the 15cm * 2cm), with 70-90 ℃ of hot water elution, to be collected filtrate, is settled to 100ml, as need testing solution;
Or get Pu'er tea 1g, put in the 250ml flask preferred 80% ethanol 50ml, 95 ℃ of water-bath backflow 30min filter while hot, with hot 80% ethanol washing 3 times, 30ml/ time, volatilize solvent, filter paper is put in the flask together with filter residue, add 50ml water, add hot reflux 1h, filter while hot, use hot wash, merging filtrate is settled to 100ml; The accurate 5ml of absorption extracts solution, adds hydrochloric acid solution, and the concentration of hydrochloric acid is 0.5mol/L in the mixed liquor, at 40 ℃ of lower hydrolysis 30min, transfers pH to neutral after the cooling; Polysaccharide extraction liquid after the acid hydrolysis is put polyamide chromatography post (among the 15cm * 2cm), with 70-90 ℃ of hot water elution, to be collected filtrate, is settled to 100ml, as need testing solution.
6. according to claim 1 detection method, it is characterized in that, wherein, may further comprise the steps with determined by ultraviolet spectrophotometry in the step 3, with the glucose standard solution with detect liquid and add respectively 5% phenol solution 1.0ml, the jolting mixing adds 5.0ml sulfuric acid, with the rapid jolting mixing of miniature whirlpool mixed instrument, placed 30 minutes under the room temperature, take the 1st pipe as blank, according to an appendix VB of Chinese Pharmacopoeia version in 2005 UV-VIS spectrophotometry, measure absorbance at 487nm wavelength place.
7. according to claim 1 detection method, it is characterized in that, wherein, the drafting of step 3 Plays curve may further comprise the steps: precision is drawn reference substance solution 0.0 respectively, 0.1,0.2,0.3,0.4,0.5ml put respectively in the 10ml tool plug test tube, add respectively two pure water to 1.0ml, the phenol solution 1.0ml of each accurate adding 5%, the jolting mixing, add 5.0ml sulfuric acid, with the rapid jolting mixing of miniature whirlpool mixed instrument, placed 30 minutes under the room temperature, take the 1st pipe as blank, according to an appendix VB of Chinese Pharmacopoeia version in 2005 UV-VIS spectrophotometry, measure absorbance at 487nm wavelength place, make typical curve with absorbance and quality.
8. according to claim 1 detection method is characterized in that, wherein, described Pu'er tea is Pu'er tea and the goods thereof that can buy on any market, such as the tea cake, and piece tea, liquid tea beverage, instant tea.
9. according to claim 1 detection method, it is characterized in that, wherein, described Pu'er tea is that any Pu'er tea extraction by prior art or new technology separated the Pu'er tea that obtains, as prepare the raw material of instant tea, and the raw material of preparation liquid tea beverage, preparation contains the food of Pu'er tea composition, medicine, the raw material of health products.
10. according to claim 1 detection method is characterized in that, measures complete in 1 hour with determined by ultraviolet spectrophotometry.
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| CN103257116A (en) * | 2013-03-29 | 2013-08-21 | 江汉大学 | Method for determining green tea polyphenol palmitate content |
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| CN103728263A (en) * | 2013-12-31 | 2014-04-16 | 无限极（中国）有限公司 | Method for quantitatively detecting polysaccharide of semen cassiae |
| CN103792202A (en) * | 2014-02-25 | 2014-05-14 | 重庆烟草工业有限责任公司 | Method for measuring amount of tea dust added to composite filter stick |
| CN104568534A (en) * | 2013-10-28 | 2015-04-29 | 云南天士力帝泊洱生物茶集团有限公司 | Determination method for taste quality and aroma quality of Pu-Er ripe tea |
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| CN107014761A (en) * | 2017-02-28 | 2017-08-04 | 梧州市雅正农业科技有限公司 | The detection method of tea polysaccharide in a kind of tea extraction of six fort |
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| CN103353502A (en) * | 2013-07-22 | 2013-10-16 | 安徽农业大学 | Pu-Er raw tea fingerprint identification method based on GC/MS technology |
| CN104568534A (en) * | 2013-10-28 | 2015-04-29 | 云南天士力帝泊洱生物茶集团有限公司 | Determination method for taste quality and aroma quality of Pu-Er ripe tea |
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| CN103674865A (en) * | 2013-11-25 | 2014-03-26 | 北京工业大学 | Method of using phenol-sulfuric acid to measure polysaccharides |
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| CN103792202A (en) * | 2014-02-25 | 2014-05-14 | 重庆烟草工业有限责任公司 | Method for measuring amount of tea dust added to composite filter stick |
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| CN108680526A (en) * | 2018-03-27 | 2018-10-19 | 天津科技大学 | Use the method for tea polysaccharide in acid precipitation pigment detection Pu'er tea and its extract |
| CN111189865A (en) * | 2020-01-08 | 2020-05-22 | 南京航空航天大学 | Method for distinguishing different producing areas of Pu' er tea in small area |
| CN111189865B (en) * | 2020-01-08 | 2021-07-27 | 南京航空航天大学 | Method for distinguishing different producing areas of Pu' er tea in small area |
| CN116268138A (en) * | 2023-04-26 | 2023-06-23 | 四川轻化工大学 | Method for improving tea polysaccharide content in Tibetan tea |
| CN116268138B (en) * | 2023-04-26 | 2024-05-03 | 四川轻化工大学 | Method for improving tea polysaccharide content in Tibetan tea |
| CN116425899A (en) * | 2023-05-17 | 2023-07-14 | 华侨大学 | Pu' er tea polysaccharide and preparation method and application thereof |
| CN117433865A (en) * | 2023-12-21 | 2024-01-23 | 云南省计量测试技术研究院 | Heavy metal-containing puer raw tea matrix standard substance and preparation method thereof |
| CN117433865B (en) * | 2023-12-21 | 2024-04-12 | 云南省计量测试技术研究院 | Heavy metal-containing puer raw tea matrix standard substance and preparation method thereof |
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