CN115181192B - Extraction method of lotus flower stem polysaccharide - Google Patents
Extraction method of lotus flower stem polysaccharide Download PDFInfo
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Abstract
The invention provides an extraction method of lotus flower stem polysaccharide, and belongs to the technical field of polysaccharide extraction. The extraction method of the lotus flower stem polysaccharide provided by the invention comprises the following steps: (1) Drying the lotus stems, and pulverizing to obtain lotus stem powder; (2) Mixing the lotus flower stem powder with water, and adding pectase for enzymolysis to obtain an enzymolysis solution; (3) Flash extraction is carried out on the enzymolysis liquid, and solid-liquid separation is carried out to obtain supernatant; (4) Concentrating the supernatant, adding absolute ethyl alcohol for precipitation, and drying the precipitation to obtain crude polysaccharide; (5) And (3) purifying the crude polysaccharide to obtain the lotus stem polysaccharide. The extraction method of the lotus flower stem polysaccharide can greatly improve the extraction quantity of the lotus flower stem polysaccharide, 92% of protein can be removed in the purification process, and the polysaccharide retention rate is 78%. The obtained purified polysaccharide has remarkable antioxidant activity.
Description
Technical Field
The invention relates to the technical field of plant polysaccharide extraction, in particular to an extraction method of lotus stem polysaccharide.
Background
The lotus flower has nine major color systems of golden, yellow, purple, blue, red, tea, green, red, white and the like, so the lotus flower is called as nine-product lotus flower. The lotus flower flowers bloom in four seasons, integrates the advantages of lotus flowers and lotus flowers in various places, and has the functions of ornamental, beauty, medical treatment, edibility and the like. In addition, the extracts of the flowers, leaves and stems of the lotus flower have certain biological activity, and modern researches show that the flower of the lotus flower contains rich nutrient substances such as anthocyanin, polysaccharide, flavone, various amino acids required by human bodies and the like, and mainly has the effects of reducing blood fat, scavenging free radicals, resisting oxidation, preventing cancers and the like.
The lotus flower stems are used as rhizome parts of lotus flowers, are easy to be used as plant wastes, and are rarely studied. The research on the lotus stem polysaccharide not only reasonably utilizes plant resources, but also is hopeful to be applied to production practice as an antioxidant in the fields of cosmetics and the like.
The existing extraction method of plant polysaccharide comprises hot water extraction, enzymolysis, ultrasonic extraction, microwave-assisted extraction, supercritical fluid extraction, and multiple methods. The flash extraction method can realize high-efficiency extraction within tens of seconds, and is environment-friendly and pollution-free. Since plant cells generally contain cell walls, enzymatic hydrolysis releases active substances by breaking the cell walls, thereby greatly improving extraction efficiency. The polysaccharide deproteinizing method comprises a Sevage method, a trifluorotrichloroethane method, a trichloroacetic acid method, a TCA method and the like, the TCA method is used for removing the protein of the lotus stem, the removal rate is high, an organic solvent is not used, and the method is a safer and environment-friendly deproteinizing method.
Disclosure of Invention
The invention aims to provide the extraction method of the lotus seed stem polysaccharide with high extraction rate, high purity and high antioxidant activity.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an extraction method of lotus flower stem polysaccharide, which comprises the following steps:
(1) Drying the lotus stems, and pulverizing to obtain lotus stem powder;
(2) Mixing the lotus flower stem powder with water, and adding pectase for enzymolysis to obtain an enzymolysis solution;
(3) Flash extraction is carried out on the enzymolysis, and solid-liquid separation is carried out to obtain supernatant;
(4) Concentrating the supernatant, adding absolute ethyl alcohol for precipitation, and drying the precipitation to obtain crude polysaccharide;
(5) And (3) purifying the crude polysaccharide to obtain the lotus stem polysaccharide.
Preferably, the crushed materials are sieved by a 60-80-mesh sieve.
Preferably, the mass volume ratio of the lotus flower stem powder to the water is 1g: 40-50 mL.
Preferably, the addition amount of the pectase is 2.5-3.5% of the mass of the lotus seed stem powder.
Preferably, the enzymolysis temperature is 35-45 ℃, the pH is 2-5, and the time is 100-140 min.
Preferably, the voltage of the flash extraction is 95-105V and the time is 55-65 s.
Preferably, the purification comprises the steps of deproteinization of trichloroacetic acid and dialysis.
Preferably, the dialysis has a molecular weight cut-off of 1000-3500 Da.
The extraction method of the lotus flower stem polysaccharide can greatly improve the extraction quantity of the lotus flower stem polysaccharide, 92% of protein can be removed in the purification process, and the polysaccharide retention rate is 78%. The obtained purified polysaccharide has remarkable antioxidant activity.
The in vitro antioxidation measurement is carried out on the lotus flower stem polysaccharide provided by the invention, the IC50 for eliminating DPPH free radical is 0.361mg/mL, and the IC50 for eliminating ABTS free radical is 0.271mg/mL, which shows that the lotus flower stem polysaccharide has better effect for eliminating DPPH and ABTS free radical. Therefore, the lotus flower stem polysaccharide can be used as a natural plant antioxidant. Due to the potential toxicity of the synthetic antioxidant, the natural antioxidant is safe and environment-friendly, and provides a reference basis for the application of the lotus stem polysaccharide in foods, medicines, health products and cosmetics.
Drawings
FIG. 1 is a graph showing the effect of enzyme species on extraction of polysaccharide from lotus stems in example 1;
FIG. 2 is a graph showing the effect of the extraction pattern of example 2 on the extraction amount of polysaccharide from the stems of the lotus flower;
FIG. 3 is a graph showing the effect of enzymolysis time on extraction amount of polysaccharide from lotus stems in example 3;
FIG. 4 shows the effect of the enzymolysis temperature on the extraction amount of polysaccharide from the lotus stems in example 4;
FIG. 5 is a graph showing the effect of enzyme addition on extraction of polysaccharide from lotus stems in example 5;
FIG. 6 is a graph showing the effect of feed liquid comparison on extraction amount of polysaccharide from lotus stems in example 6;
FIG. 7 is a graph showing the effect of flash extraction time on extraction amount of lotus stem polysaccharide in example 7;
FIG. 8 is a graph showing the effect of pH of enzymolysis on extraction amount of polysaccharide from lotus stems in example 8;
FIG. 9 is a graph showing the effect of TCA method on protein removal and polysaccharide retention from lotus stems in example 9;
FIG. 10 shows DPPH scavenging ability of purified lotus flower stalk polysaccharide in example 9:
FIG. 11 is the ABTS clearance ability of purified lotus flower stem polysaccharide in example 9.
Detailed Description
The invention provides an extraction method of lotus flower stem polysaccharide, which comprises the following steps:
(1) Drying the lotus stems, and pulverizing to obtain lotus stem powder;
(2) Mixing the lotus flower stem powder with water, and adding pectase for enzymolysis to obtain an enzymolysis solution;
(3) Flash extraction is carried out on the enzymolysis, and solid-liquid separation is carried out to obtain supernatant;
(4) Concentrating the supernatant, adding absolute ethyl alcohol for precipitation, and drying the precipitation to obtain crude polysaccharide;
(5) And (3) purifying the crude polysaccharide to obtain the lotus stem polysaccharide.
The invention firstly dries and then pulverizes the freshly picked lotus flower stems to obtain lotus flower stem powder for standby.
In the present invention, the drying is preferably vacuum drying.
In the present invention, the conditions for vacuum drying are preferably a drying temperature of-20℃and a pressure of 3pa for 48 hours.
In the present invention, the pulverized material is preferably sieved through a 60 to 80 mesh sieve, more preferably a 70 mesh sieve.
Mixing the lotus flower stem powder with water, and adding pectase for enzymolysis to obtain an enzymolysis liquid.
In the invention, the mass volume ratio of the lotus flower stem powder mixed with water is preferably 1g:40 to 50mL, more preferably 1g:45ml.
In the present invention, the amount of the pectase to be added is preferably 2.5 to 3.5% by mass, more preferably 3% by mass, of the lotus seed stem powder.
In the present invention, the temperature of the enzymolysis is preferably 35 to 45 ℃, and more preferably 40 ℃.
In the present invention, the pH of the enzymatic hydrolysis is preferably 2 to 5, more preferably 5.
In the present invention, the time for the enzymolysis is preferably 100 to 140 minutes, more preferably 120 minutes.
And performing flash extraction and solid-liquid separation on the enzymolysis to obtain supernatant.
In the present invention, the voltage of the flash extraction is preferably 95 to 105V, and more preferably 100V.
In the present invention, the flash extraction time is preferably 55 to 65s, more preferably 60s.
In the present invention, the solid-liquid separation is preferably centrifugal separation.
In the present invention, the rotational speed of the centrifugal separation is preferably 8000 to 10000rpm, more preferably 9000rpm.
In the present invention, the time for the centrifugal separation is preferably 10 to 15 minutes, more preferably 12 minutes.
Concentrating the supernatant, adding absolute ethyl alcohol for precipitation, and drying the precipitation to obtain crude polysaccharide.
In the present invention, the volume of the supernatant after concentration is preferably 1/3 to 1/5, more preferably 1/4 of the volume before concentration.
In the present invention, the concentration is preferably performed by rotary evaporation, and the temperature of the rotary evaporation is preferably 20 to 70 ℃, and more preferably 50 ℃.
In the present invention, the amount of the anhydrous ethanol to be added is preferably 3 to 5 times, more preferably 4 times, the volume of the supernatant after concentration.
In the present invention, the temperature of the absolute ethanol precipitation is preferably 3 to 5 ℃, and more preferably 4 ℃.
In the present invention, the time for the precipitation of the absolute ethanol is preferably 10 to 14 hours, more preferably 12 hours.
In the present invention, the drying is preferably performed in an oven.
In the present invention, the drying temperature is preferably 40 to 60 ℃, and more preferably 50 ℃.
In the present invention, the drying is preferably to a constant weight.
And (3) purifying the crude polysaccharide to obtain the lotus stem polysaccharide.
In the present invention, the purification is preferably a step of deproteinizing trichloroacetic acid and dialyzing.
In the present invention, the method for deproteinizing trichloroacetic acid is preferably as follows: preparing 40-50 mg/mL of crude polysaccharide aqueous solution, mixing with 10-15% TCA solution in equal volume, centrifuging at room temperature (25 ℃) for 1-2 h and taking supernatant at 8000-10000 rmp. And (3) steaming the supernatant to 1/3-1/5 of the volume of the stock solution, adding 3-5 times of absolute ethyl alcohol of the concentrated solution, precipitating with alcohol for 12 hours at the temperature of 4 ℃ in a refrigerator, precipitating crude polysaccharide obtained by alcohol precipitation in a baking oven at the temperature of 40-60 ℃ and drying to constant weight. Finally, preparing a crude polysaccharide solution with the concentration of 50-150 mg/mL by distilled water, dialyzing in a 1000-3500 Da dialysis bag for 48-72 h at room temperature (25 ℃), and freeze-drying in vacuum (the drying temperature is-20 ℃ and the pressure is 3pa for 48 h) to obtain polysaccharide powder.
Further preferred is as follows: 45mg/mL of the crude polysaccharide aqueous solution was prepared and mixed with 12% TCA solution in equal volume, and the supernatant was centrifuged at 8000rmp after 1.5h at room temperature (25 ℃ C.). And (3) performing rotary evaporation (50 ℃) on the supernatant to 1/4 of the volume of the stock solution, adding absolute ethanol with the volume being 4 times that of the concentrated solution, performing alcohol precipitation for 12 hours at the temperature of 4 ℃ in a refrigerator, and drying the crude polysaccharide precipitate obtained by alcohol precipitation in an oven at the temperature of 50 ℃ to constant weight. Finally, distilled water was used to prepare a crude polysaccharide solution at a concentration of 100 mg/mL. Dialyzing with 2000Da dialysis bag at room temperature (25deg.C) for 60 hr, and vacuum freeze-drying (drying temperature-20deg.C, pressure 3pa, and time 48 hr) to obtain polysaccharide powder.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Fresh lotus flower stems were picked up and dried in vacuo (drying temperature: 20 ℃ below zero, pressure: 3pa, time: 48 hours), then crushed and sieved through a 80-mesh sieve, and the undersize product was taken to obtain lotus flower stem powder, which was used in the following examples.
Example 1
Screening the extracted enzyme: alpha-amylase, pectinase, papain, cellulase and neutral protease.
6 parts of 1g of lotus flower stem powder are weighed, 30mL of distilled water is respectively added, then 2% of enzyme by mass of the lotus flower stem powder is respectively added, and a blank control without enzyme is used. And carrying out enzymolysis for 1h under the conditions of optimal temperature and pH of the five enzymes.
The optimal temperature of the alpha-amylase is 65 ℃ and the pH value is 6; the optimal temperature of pectase is 40 ℃, the pH is 4.5, the optimal temperature of papain is 40, and the pH is 6.5; the optimal temperature of the cellulase is 50 ℃ and the pH value is 4.8; the neutral protease has an optimum temperature of 45℃and a pH of 7.5.
After the enzymolysis is finished, the extraction amount (mg/g) of the polysaccharide of the lotus stems is used as an index to determine the polysaccharide content of the lotus stems in each group of enzymolysis liquid.
The content of the polysaccharide in the lotus flower stems is determined by a phenol-sulfuric acid method. The measurement method comprises the steps of preparing 0.2mg/mL glucose standard solution, respectively taking 0mL, 0.1mL, 0.2mL, 0.3mL, 0.4mL, 0.5mL and 0.6mL in test tubes, supplementing distilled water to 1.0mL, adding 1.0mL distilled water into a blank control group, respectively adding 0.5mL 6% phenol solution and 2.5mL concentrated sulfuric acid, standing for 20min, heating in a boiling water bath for 15min, and standing for 1h. And finally, measuring absorbance at the wavelength of 490nm by using an ultraviolet-visible spectrophotometer, performing linear regression analysis by using software, drawing a standard curve by taking the concentration of glucose as x and the absorbance as y, and obtaining a regression equation y=0.0303x+0.0021.
Calculating polysaccharide extraction amount:
in the formula, R: yield of lotus flower stem polysaccharide (mg/g); c: calculating polysaccharide concentration (mg/mL) of the obtained reaction sample from the standard curve; v: volume of extract (mL); n is dilution multiple; w is the mass (g) of the lotus flower stem powder.
The results are shown in FIG. 1. As can be seen from fig. 1, the pectase group can significantly increase the extraction amount of the polysaccharide from the lotus stems, compared with other groups, so that pectase is selected for enzymolysis.
Example 2
Screening of extraction modes
The influence of leaching, ultrasound, flash type, enzymolysis, flash type compound ultrasound, flash type compound enzymolysis (pectase) and ultrasonic compound enzymolysis (pectase) on the extraction amount of the polysaccharide of the lotus flower stems is examined. The specific extraction conditions are as follows:
leaching: accurately weighing 3g of lotus flower stem powder, adding 90mL of distilled water, and leaching at room temperature (25 ℃) for 1h;
b, ultrasonic treatment: accurately weighing 3g of lotus flower stem powder, adding 90mL of distilled water, and performing 400W ultrasonic treatment for 30min;
c flash type: accurately weighing 3g of lotus flower stem powder, adding 90mL of distilled water and extracting for 60s in 100V flash;
d, enzymolysis: accurately weighing 3g of lotus flower stem powder, adding 90mL of distilled water, and performing enzymolysis for 1h at 50 ℃ with 2% pectase;
eultrasound+flash: accurately weighing 3g of lotus flower stem powder, adding 90mL of distilled water, performing 400W ultrasonic treatment for 30min, and performing 100V flash extraction for 60s;
f enzymolysis + flash: accurately weighing 3g of lotus flower stem powder, adding 90mL of distilled water, performing enzymolysis for 1h at 50 ℃ with 2% pectase, and then performing 100V flash extraction for 60s;
g enzymolysis and ultrasonic treatment: accurately weighing 3g of lotus flower stem powder, adding 90mL of distilled water, performing enzymolysis for 1h at 50 ℃ with 2% pectase, and performing 400W ultrasonic treatment for 30min.
After the extraction, the content of the polysaccharide of the lotus flower stems in each group of enzymolysis liquid was measured by using the extraction amount (mg/g) of the polysaccharide of the lotus flower stems as an index (the measuring method is the same as that of example 1). The results are shown in FIG. 2.
As can be seen from FIG. 2, the flash type complex enzymolysis method has the highest polysaccharide content, which indicates that the flash type complex enzymolysis method adopted by the invention has higher extraction rate of the polysaccharide of the lotus stems of the perfume, compared with the single extraction method or the complex extraction method. Meanwhile, the flash extraction has the characteristics of shorter extraction time and higher efficiency.
Example 3
Accurately weighing 3g of lotus flower stem powder and pectase with mass of 2% and adding into a 250mL beaker, and then mixing the materials according to a feed liquid ratio of 1:30 (g: mL) adding buffer solution with ph=4 into a 250mL beaker, respectively carrying out water bath for 40, 60, 80, 100, 120, 140 and 160min in a water bath kettle with the temperature of 50 ℃, and inactivating enzyme in the water bath kettle with the temperature of 100 ℃ for 10min after enzymolysis is finished. And (3) performing flash extraction after enzyme deactivation, wherein the flash extraction voltage is 100V, and the flash extraction time is 60s. After the completion of the extraction, the supernatant was centrifuged to calculate the polysaccharide extraction amount (the method is the same as in example 1). The results are shown in FIG. 3.
As can be seen from FIG. 3, the extraction amount of the polysaccharide from the lotus flower stems is in a rapid rising trend along with the prolongation of the enzymolysis time, and finally reaches equilibrium within 100-140 min, and the extraction amount is basically unchanged after the maximum extraction amount is reached. Therefore, the preferred enzymolysis time is 120min.
Example 4
Accurately weighing 3g of lotus flower stem powder and pectase with mass of 2% into a 250mL beaker, and mixing the materials according to a feed liquid ratio of 1:30 (g: mL) adding buffer solution with ph=4 into 250mL beakers, respectively carrying out water bath for 60min at 20, 30, 40, 50, 60, 70 and 80 ℃ and carrying out enzyme deactivation for 10min at 100 ℃ after enzymolysis. And (3) performing flash extraction after enzyme deactivation, wherein the flash extraction voltage is 100V, and the flash extraction time is 60s. Centrifuging after extraction, collecting supernatant, and calculating polysaccharide extraction amount. The results are shown in FIG. 4.
As can be seen from fig. 4, the polysaccharide content showed a tendency to increase with increasing enzymolysis temperature, but after the enzymolysis temperature exceeded 40 ℃, the polysaccharide extraction amount began to decrease, so the optimum extraction temperature of the polysaccharide from lotus stems was 40 ℃.
Example 5
Accurately weighing 3g of lotus flower stem powder, and adding pectase with mass of 0.5, 1, 1.5, 2, 2.5, 3 and 3.5% into a 250mL beaker respectively according to a feed liquid ratio of 1:30 (g: mL) the enzyme was quenched in a water bath at 50 ℃ for 10min after completion of the enzymatic hydrolysis by adding a buffer solution at ph=4 to a 250mL beaker and water-bathing in a water bath at 50 ℃. And (3) performing flash extraction after enzyme deactivation, wherein the flash extraction voltage is 100V, and the flash extraction time is 60s. Centrifuging after extraction, collecting supernatant, and calculating polysaccharide extraction amount. The results are shown in FIG. 5.
As can be seen from fig. 5, the polysaccharide extraction amount tended to increase with increasing pectinase addition amount. When the adding amount of pectase reaches 2.5% -3.5%, the balance is reached, and the extraction amount is basically unchanged after the maximum extraction amount is reached. Therefore, the preferable addition amount of pectase is 3%.
Example 6
Accurately weighing 3g of lotus flower stem powder and pectase with mass percent of 2% into a 250mL beaker, and respectively mixing the materials according to the feed liquid ratio of 1:25 and 1: 30. 1: 35. 1: 40. 1: 45. 1: 50. 1:55 (g: mL) the enzyme was quenched in a water bath at 50 ℃ for 10min after completion of the enzymatic hydrolysis by adding a buffer solution at ph=4 to a 250mL beaker and water-bathing in a water bath at 50 ℃. And (3) performing flash extraction after enzyme deactivation, wherein the flash extraction voltage is 100V, and the flash extraction time is 30, 45, 60, 75, 90, 105 and 120s respectively. And centrifuging after the extraction is finished, taking supernatant to react, and calculating the extraction quantity of polysaccharide. The results are shown in FIG. 6.
As can be seen from FIG. 6, the extraction amount of the lotus flower stem polysaccharide is in a rapid rising trend along with the increase of the buffer solution volume, and the equilibrium is reached when the feed-liquid ratio is 1 g:40-50 ml. Therefore, the preferred feed ratio is 1g:45ml.
Example 7
Accurately weighing 3g of lotus flower stem powder and pectase with mass of 2% into a 250mL beaker, and mixing the materials according to a feed liquid ratio of 1:30 (g: mL) the enzyme was quenched in a water bath at 50 ℃ for 10min after completion of the enzymatic hydrolysis by adding a buffer solution at ph=4 to a 250mL beaker and water-bathing in a water bath at 50 ℃. And (3) performing flash extraction after enzyme deactivation, wherein the flash extraction voltage is 100V, and the flash extraction time is 30, 45, 60, 75, 90, 105 and 120s respectively. Centrifuging after extraction, collecting supernatant, and calculating polysaccharide extraction amount. The results are shown in FIG. 7.
As can be seen from fig. 7, as the flash extraction time is prolonged, the polysaccharide content tends to increase and then decrease, with 60s being the turning point. Therefore, the optimal flash extraction time is 60s.
Example 8
Accurately weighing 3g of lotus flower stem powder and pectase with mass of 2% into a 250mL beaker, and mixing the materials according to a feed liquid ratio of 1:30 (g: mL) the enzyme was quenched in a water bath at 50℃for 60min and a water bath at 100℃for 10min after the completion of the enzyme hydrolysis, respectively, by adding buffers at pH=2, 3, 4, 5, 6, 7, 8 in a 250mL beaker. And (3) performing flash extraction after enzyme deactivation, wherein the flash extraction voltage is 100V, and the flash extraction time is 60s. Centrifuging after extraction, collecting supernatant, and calculating polysaccharide extraction amount. The results are shown in FIG. 8.
As can be seen from fig. 8, even when the pH is in the range of 2 to 5, a satisfactory polysaccharide extraction amount can be obtained, but when the pH exceeds 5, the polysaccharide extraction amount abruptly decreases. Therefore, the preferable enzymolysis pH is 2-5.
Example 9
Extracting the lotus flower stem polysaccharide according to the optimized conditions of the examples 1-8.
Accurately weighing 3g of lotus flower stem powder and 3% pectase by mass, adding into a 250ml beaker, and mixing the materials according to a feed liquid ratio of 1g:45mL of buffer at ph=5 was added to a 250mL beaker. Water bath is carried out for 120min in a water bath kettle at 40 ℃, and enzyme deactivation is carried out for 10min in the water bath kettle at 100 ℃ after enzymolysis is finished. And (3) performing flash extraction after enzyme deactivation, wherein the flash extraction voltage is 100V, and the flash extraction time is 60s. Centrifuging (10000 rpm,12 min) after extraction is completed to obtain supernatant. Concentrating to 1/4 of the volume of the stock solution by rotary evaporation (50 ℃) and then adding absolute ethyl alcohol with the volume being 4 times that of the concentrated solution, carrying out alcohol precipitation for 12 hours at the refrigerator with the temperature being 4 ℃, and drying the precipitate obtained by alcohol precipitation to constant weight at the temperature of 50 ℃ in an oven to obtain crude polysaccharide.
Crude polysaccharide is prepared into 40mg/mL crude polysaccharide solution, the crude polysaccharide solution and 10% TCA solution are mixed in equal volume to deproteinize, and after stirring for 1.5h at room temperature (25 ℃), the supernatant is centrifugally taken at 8000rpm, and the polysaccharide content in the supernatant is measured. Polysaccharide retention was calculated.
Polysaccharide retention Y 1 (％)＝(A/A 0 )×100％
Wherein: a is that 0 Represents the mass (. Mu.g) of polysaccharide in the crude polysaccharide solution prior to deproteinization; a represents the mass (. Mu.g) of polysaccharide in the crude polysaccharide solution after deproteinization.
Preparing 100 mug/mL bovine serum albumin standard solution, respectively taking 0.2mL, 0.4mL, 0.6mL, 0.8mL and 1.0mL into test tubes, supplementing 1.0mL with distilled water, adding 1.0mL of distilled water into blank control group, respectively adding 5mL of coomassie brilliant blue solution, standing at room temperature for reaction for 5min, and then completing colorimetric determination at 595nm wavelength. Performing linear regression analysis by software, and drawing a standard curve with protein concentration as x and absorbance as y to obtain regression equation y=0.023x+0.0105, R 2 ＝0.9947。
Calculation of protein removal rate: protein removal rate Y 2 (％)＝(B/B 0 )×100％
Wherein: b (B) 0 Represents the mass of protein in the crude polysaccharide solution before deproteinization (μg); b represents the mass (. Mu.g) of protein in the crude polysaccharide solution after deproteinization.
Comprehensive index was calculated, comprehensive index = polysaccharide retention x 50% + protein removal x 50%.
The results are shown in FIG. 9. As can be seen from FIG. 9, the TCA method is used for purifying the crude polysaccharide from the lotus stems, the protein removal rate reaches 92%, the polysaccharide retention rate reaches 78%, the comprehensive index reaches 85%, and the method has the effect of removing the protein more ideally and improving the purification rate of the crude polysaccharide.
And (3) spin-steaming (50 ℃) the deproteinized supernatant to 1/4 of the volume of the stock solution, adding absolute ethanol with the volume being 4 times that of the concentrated solution, carrying out alcohol precipitation for 12 hours at the temperature of 4 ℃ in a refrigerator, and drying the crude polysaccharide obtained by alcohol precipitation in an oven at the temperature of 50 ℃ to constant weight. Finally, a crude polysaccharide solution of 100mg/ml was prepared. Dialyzing with 2000Da dialysis bag at room temperature (25deg.C) for 60 hr, and vacuum freeze-drying (drying temperature is-20deg.C, pressure is 3pa, and time is 48 hr) to obtain purified polysaccharide powder.
Determination of DPPH free radical
The purified lotus flower stem polysaccharide is respectively prepared into aqueous solutions with different concentrations, namely solutions with the concentration of 0.25mg/mL, 0.5mg/mL, 1mg/mL, 1.5mg/mL, 2mg/mL and 2.5mg/mL, 2mL of each concentration solution is respectively taken and mixed with 2mL of DPPH solution with the concentration of 0.1mmol/L, sample solutions with different concentrations are obtained, the sample solutions are sealed by preservative films, and the sample solutions react in darkness for 30min at 25 ℃. Determination of the absorption at 517nmThe luminosity value is denoted as A. Mixing 2mL of absolute ethyl alcohol and 2mL of distilled water as a blank group to be zeroed; taking a solution obtained by adding 2mL of absolute ethyl alcohol into 2mL of sample solution for reaction for 30min as a blank control group, wherein the measured absorbance value at 517nm is A i The blank group is used for removing absorbance existing in the lotus flower stem polysaccharide sample; the absorbance at 517nm was recorded as A using 2mL of 0.1mmol/LDPPH solution plus 2mL of absolute ethanol as a model control 0 . And drawing a cleaning effect graph by taking concentration as an abscissa and cleaning rate as an ordinate, and taking cleaning of Vc to DPPH as a comparison. The results are shown in FIG. 10. The solvent used for preparing the DPPH solution is absolute ethyl alcohol.
The clearance rate calculation formula is:
clearance of dpph= [1- (a) i -A))/A 0 ]×100％
A is the absorbance measured by adding 2mL of DPPH to 2mL of sample solution;
A i adding 2mL of absolute ethyl alcohol to 2mL of sample solution to measure the absorbance;
A 0 the absorbance was measured by adding 2mL of absolute ethanol to 2mL of DPPH solution.
Determination of abts free radical
Mixing an ABTS solution with the concentration of 7mmol/L and a potassium persulfate solution with the concentration of 2.45mmol/L according to the volume ratio of 1:1 to prepare a cationic working solution of the ABTS free radical, and shielding from light for 12 hours, so as to excite the ABTS free radical. Diluting the ABTS free radical with distilled water until the absorbance is 0.7+ -0.02 to obtain the ABTS working solution.
Preparing the purified lotus flower stem polysaccharide into solutions with different concentrations, namely solutions with the concentrations of 0.1mg/mL, 0.3mg/mL, 0.5mg/mL, 0.7mg/mL, 0.9mg/mL, 1.1mg/mL and 1.3mg/mL, respectively taking 2mL of each concentration solution, respectively adding 2 mLABSS working solution, uniformly mixing, reacting for 10min at the temperature of 37 ℃ in the dark, and measuring the absorbance value A at 734 nm; respectively taking 2mL of each concentration solution, respectively adding 2mL of distilled water, reacting for 10min at 37 ℃ in dark place, and measuring the absorbance value A at 734nm i The method comprises the steps of carrying out a first treatment on the surface of the Then respectively using 2 mLABSS working solution and 2mL deionized water to make reaction, and measuring light absorption value A at 734nm 0 As a reference. The scavenging effect of Vc on ABTS is used as a control. The results are shown in FIG. 11.
The calculation formula is as follows:
ABTS clearance = [1- (a) i -A))/A 0 ]×100％
A is the absorbance value of 2mL of ABTS working solution added into 2mL of sample solution;
A i adding 2mL of distilled water to 2mL of sample solution to obtain a light absorption value;
A 0 absorbance was measured with 2mL distilled water added to 2mL ABTS solution.
As can be seen from FIGS. 10 and 11, the IC50 s of DPPH and ABTS for the purified polysaccharide powder were 0.361mg/mL and 0.271mg/mL, respectively.
From the above examples, the invention provides the lotus flower stem polysaccharide and the preparation method thereof, and the extraction method of the lotus flower stem polysaccharide can improve the extraction amount of the lotus flower stem polysaccharide, 92% of protein can be removed in the purification process, and the polysaccharide retention rate is 78%. The obtained purified polysaccharide has remarkable antioxidant activity.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (4)
1. The extraction method of the lotus stem polysaccharide is characterized by comprising the following steps:
(1) Drying the lotus stems, and pulverizing to obtain lotus stem powder;
(2) Mixing the lotus flower stem powder with water according to the weight ratio of 1g:45mL of the mixture is mixed, pectase accounting for 3% of the mass of the lotus flower stem powder is added for enzymolysis for 120min at 40 ℃ under the condition of pH=5, and enzymolysis liquid is obtained;
(3) Flash extraction is carried out on the enzymolysis, the flash extraction is carried out for 60s under the voltage of 100V, and the solid-liquid separation is carried out, so as to obtain supernatant;
(4) Concentrating the supernatant, adding absolute ethyl alcohol for precipitation, and drying the precipitation to obtain crude polysaccharide;
(5) Purifying the crude polysaccharide to obtain lotus stem polysaccharide;
the purification is trichloroacetic acid deproteinization, which is carried out by mixing crude polysaccharide solution with concentration of 40mg/mL with 10% TCA solution in equal volume, and stirring at 25deg.C for 1.5h.
2. The extraction method according to claim 1, wherein the crushed material is sieved with a 60-80 mesh sieve.
3. The extraction method of claim 2, wherein the purifying further comprises a step of dialysis.
4. The extraction method according to claim 3, wherein the dialysis has a molecular weight cut-off of 1000-3500 Da.
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