LU501444B1 - Extraction method for polysaccharide in plants - Google Patents
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- LU501444B1 LU501444B1 LU501444A LU501444A LU501444B1 LU 501444 B1 LU501444 B1 LU 501444B1 LU 501444 A LU501444 A LU 501444A LU 501444 A LU501444 A LU 501444A LU 501444 B1 LU501444 B1 LU 501444B1
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- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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Abstract
The disclosure belongs to the technical field of biochemical engineering, and particularly relates to an extraction method for polysaccharide in plants. The extraction method comprises the following steps: enzymatically hydrolyzing plant raw materials to obtain enzymatic hydrolysate; sequentially mixing the enzymatic hydrolysate with an inorganic salt aqueous solution and an organic solvent, and performing two-phase salting-out extraction on the obtained mixed liquid to obtain alcohol-soluble polysaccharide and water-soluble polysaccharide. The alcohol-soluble polysaccharide can be synchronously separated and obtained while the water-soluble polysaccharide is efficiently extracted. Meanwhile, the method has moderate operating conditions, is easy and convenient to operate, low in equipment requirement and high in applicability, and facilitates large-scale industrial production.
Description
BL-5421
LU501444
EXTRACTION METHOD FOR POLYSACCHARIDE IN PLANTS
[01] The disclosure belongs to the technical field of biochemical engineering, and particularly relates to an extraction method for polysaccharide in plants.
[02] Plant polysaccharide is one of most widely sourced polysaccharide in nature, but structural features and bioactivity of different kinds of plant polysaccharide differ greatly, and development of the polysaccharide becomes a hot field in the health care product industry along with rapid development of biology, chemistry and other subjects.
[03] In the prior art, the polysaccharide is usually extracted in an ultrasonic manner or a microwave manner or an enzyme-assisted manner or else. However, the plant polysaccharide extracted in the prior art is low in acquisition rate, and only water-soluble polysaccharide is extracted, while alcohol-soluble polysaccharide is not extracted yet. It is shown through recent correlational studies that the alcohol-soluble polysaccharide also has important bioactivity, for example, can improve activity of certain immune cells or restrain growth of cell tumor cells.
Thus, providing an extraction method capable of simultaneously extracting the alcohol-soluble polysaccharide and the water-soluble polysaccharide and meanwhile achieving high yield of the extracted polysaccharide has great practical significance.
[04] By this, the disclosure provides an extraction method for polysaccharide in plants. The extraction method provided by the disclosure can simultaneously extract alcohol-soluble polysaccharide and water-soluble polysaccharide in the plants.
[05] In order to achieve the above objective, the disclosure provides the following technical solution:
[06] The disclosure provides an extraction method for polysaccharide in plants, comprising the following steps:
[07] enzymatically hydrolyzing plant raw materials to obtain enzymatic hydrolysate; and
[08] sequentially mixing the enzymatic hydrolysate with an inorganic salt aqueous solution and an organic solvent, and performing two-phase salting-out extraction on the obtained mixed liquid to obtain alcohol-soluble polysaccharide and water-soluble polysaccharide; wherein
[09] inorganic salt in the inorganic salt aqueous solution comprises one or more of sulfate, phosphate, citrate and chloride; and
[10] the organic solvent comprises one or more of ethyl alcohol, iso-glycerol, acetone, acetonitrile and tert-butyl alcohol.
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[11] Preferably, enzymes for the enzymatic hydrolysis comprise one or more of cellulase, hemicellulase, pectinase, trypsin, papain, Beta-glucanase and amylase.
[12] Preferably, the mass of the enzymes for the enzymatic hydrolysis is 0.6%-1.8% of the mass of the plant raw materials.
[13] Preferably, the temperature of the enzymatic hydrolysis is 40-60 degrees Celsius, and the time of the enzymatic hydrolysis is 30-150 min.
[14] Preferably, the mass percent of the inorganic salt in the mixed liquid is 20%-24%.
[15] Preferably, the mass percent of the organic solvent in the mixed liquid is 19%-23%.
[16] Preferably, the temperature of the two-phase salting-out extraction is 25-40 degrees
Celsius, and the time of the two-phase salting-out extraction is 65-80 min.
[17] Preferably, an upper phase organic solvent extracting solution and a lower phase inorganic salt aqueous extract are obtained through the two-phase salting-out extraction, and the method further comprises after-treating the upper phase organic solvent extracting solution and the lower phase inorganic salt aqueous extract respectively after the two-phase salting-out extraction; and
[18] the after-treatment comprises sequentially performing dialysis, deproteinization, decoloration and purification.
[19] Preferably, the purification comprises sequentially performing DEAE-52 cellulose column chromatography separation and Sephadex G-100 gel column chromatography separation.
[20] Preferably, the dialysis is performed in a dialysis bag with the molecular weight cut-off of 3500 Da, and the time of the dialysis is 48-72 h.
[21] The disclosure provides the extraction method for the polysaccharide in the plants, comprising the following steps: enzymatically hydrolyzing the plant raw materials to obtain the enzymatic hydrolysate; sequentially mixing the enzymatic hydrolysate with the inorganic salt aqueous solution and the organic solvent, and performing the two-phase salting-out extraction on the obtained mixed liquid to obtain the alcohol-soluble polysaccharide and the water-soluble polysaccharide; wherein the inorganic salt aqueous solution and the organic solvent form reagents for the two-phase salting-out extraction; the inorganic salt in the inorganic salt aqueous solution comprises one or more of sulfate, phosphate, citrate and chloride; and the organic solvent comprises one or more of ethyl alcohol, iso-glycerol, acetone, acetonitrile and tert-butyl alcohol. In the disclosure, firstly, the plant raw materials are enzymatically hydrolyzed to obtain the enzymatic hydrolysate, and in this process, enzymatic hydrolysis can preliminarily damage cell tissue structures of the plant raw materials and release the polysaccharide in endochylema; then the inorganic salt is added to further damage the cell tissue structures of the plants on the basis of enzymatic hydrolysis and further release the polysaccharide; and finally, the organic solvent is added and uniformly mixed for polysaccharide extraction, the alcohol-soluble polysaccharide is enriched in an upper phase organic phase, the water-soluble polysaccharide is enriched in a lower phase saline phase, and the alcohol-soluble polysaccharide and the water-
BL-5421 . . _, LU501444 soluble polysaccharide can be synchronously separated while the water-soluble polysaccharide is efficiently extracted. Meanwhile, the method has moderate operating conditions, is easy and convenient to operate, low in equipment requirement and high in applicability, and facilitates large-scale industrial production.
[22] FIG. 1 is a flow diagram of an extraction method of an embodiment of the disclosure.
[23] The disclosure provides an extraction method for polysaccharide in plants, comprising the following steps:
[24] enzymatically hydrolyzing plant raw materials to obtain enzymatic hydrolysate; and
[25] sequentially mixing the enzymatic hydrolysate with an inorganic salt aqueous solution and an organic solvent, and performing two-phase salting-out extraction on the obtained mixed liquid to obtain alcohol-soluble polysaccharide and water-soluble polysaccharide; wherein
[26] inorganic salt in the inorganic salt aqueous solution comprises one or more of sulfate, phosphate, citrate and chloride; and
[27] The organic solvent comprises one or more of ethyl alcohol, iso-glycerol, acetone, acetonitrile and tert-butyl alcohol.
[28] Inthe disclosure, the raw materials used in the disclosure are all commercially available products preferably unless otherwise specified.
[29] The plant raw materials are enzymatically hydrolyzed to obtain the enzymatic hydrolysate.
[30] Inthe disclosure, the particle size of the plant raw materials is 40-200 meshes preferably, and is 100-160 meshes more preferably. In the disclosure, the plant materials comprise one or more of liquorice roots, stevia rebaudiana and tagetes erecta preferably. In the disclosure, the plant raw materials are prepared preferably through a method comprising the following steps: the plant raw materials are sequentially dried in the shade naturally and smashed to obtain the plant raw materials. In the disclosure, parts of the plant raw materials are roots, stems, leaves, flowers or corresponding dregs preferably. The smashing operation is not specifically limited in the disclosure, and it is only needed that the particle size of the plant raw materials is 40-200 meshes.
[31] In the disclosure, enzymes for the enzymatic hydrolysis comprise one or more of cellulase, hemicellulase, pectinase, trypsin, papain, Beta-glucanase and amylase preferably, and are mixed enzymes of the cellulase and the Beta-glucanase more preferably, and the mass ratio of the cellulase to the Beta-glucanase in the mixed enzymes of the cellulase and the Beta- glucanase is 2 : 1 preferably. In the disclosure, the enzyme activity of the cellulase is 500 U/g preferably, and the enzyme activity of the Beta-glucanase is 250 U/g. In the disclosure, the mass of the enzymes is 0.6%-1.8% of the mass of the plant raw materials preferably, and is 1.5%
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[32] In the disclosure, the enzymes for the enzymatic hydrolysis are used in the form of an enzyme solution; a solvent in the enzyme solution comprises buffer preferably; the buffer 1s citric acid-disodium hydrogen phosphate preferably; and the pH value of the buffer 1s 3.5-6.5 preferably, and is 4.0 more preferably. In the disclosure, the buffer is configured to guarantee the optimal activity of the enzymes.
[33] Inthe disclosure, the temperature of the enzymatic hydrolysis is 40-60 degrees Celsius preferably; and the time of the enzymatic hydrolysis is 30-150 min preferably, and is 120 min most preferably. In the embodiment of the disclosure, the enzymatic hydrolysis is performed in a water bath kettle preferably.
[34] After being obtained, the enzymatic hydrolysate is sequentially mixed with the inorganic salt aqueous solution and the organic solvent, and the two-phase salting-out extraction is performed on the obtained mixed liquid to obtain the alcohol-soluble polysaccharide and the water-soluble polysaccharide.
[35] In the disclosure, the liquid-material ratio of the mixed liquid to the plant raw materials is (35-55) mL : 1 g preferably.
[36] The reagents for the two-phase salting-out extraction are a mixture of the inorganic salt aqueous solution and the organic solvent; and the organic salt in the inorganic salt aqueous solution comprises one or more of sulfate, phosphate, citrate and chloride preferably, and is sulfate and phosphate more preferably. In the disclosure, the sulfate is ammonium sulfate preferably. In the disclosure, the phosphate is dipotassium phosphate and/or disodium hydrogen phosphate preferably. In the disclosure, the citrate is ammonium citrate and/or sodium citrate preferably; and in the disclosure, the chloride is sodium chloride preferably. In the disclosure, the mass percent of the inorganic salt in the mixed liquid is 20%-24%.
[37] In the disclosure, the organic solvent comprises one or more of ethyl alcohol, iso- glycerol, acetone, acetonitrile and tert-butyl alcohol preferably, and is absolute ethyl alcohol more preferably. In the disclosure, the mass percent of the organic solvent in the mixed liquid is 19%-23%.
[38] In the disclosure, the mixing time of the enzymatic hydrolysate and the inorganic salt aqueous solution is 8-10 min preferably, and the mixing time of the enzymatic hydrolysate and the organic solvent is 8-10 min preferably. The mixing specific manner is not specifically limited in the disclosure, and the materials are uniformly mixed through operations well known by those skilled in the art.
[39] The manner of the two-phase salting-out extraction of the disclosure comprises sequentially performing standing and centrifuging to obtain an upper phase organic solvent extracting solution and a lower phase inorganic salt aqueous extract.
[40] In the disclosure, the time of the standing is 60-70 min preferably, and is 60 min more preferably; the rotating speed of the centrifuging is 1000-8000 rmp preferably, and is 3000- 8000 rmp more preferably; and the time is 5-10 min preferably. In the disclosure, the 40 temperature of the two-phase salting-out extraction is 25-40 degrees Celsius preferably, and is 25-35 degrees Celsius more preferably. In the disclosure, the time of the two-phase salting-out
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[41] The disclosure preferably further comprises after-treating the upper phase organic solvent extracting solution and the lower phase inorganic salt aqueous extract respectively after the two-phase salting-out extraction. 5 [42] In the disclosure, the after-treatment preferably comprises sequentially performing dialysis, deproteinization and centrifugation on the upper phase organic solvent extracting solution and the lower phase inorganic salt aqueous extract respectively to obtain supernate; then decoloring the supernate to obtain an organic phase polysaccharide solution and a water phase polysaccharide solution respectively; and sequentially performing vacuum concentration, alcohol precipitation and vacuum freeze drying on the obtained organic phase polysaccharide solution and water phase polysaccharide solution to obtain organic phase crude polysaccharide and water phase crude polysaccharide. Then the obtained organic phase crude polysaccharide and water phase crude polysaccharide are purified respectively to obtain the alcohol-soluble polysaccharide and water-soluble polysaccharide.
[43] Inthe disclosure, the dialysis is performed in a dialysis bag with the molecular weight cut-off of 3500 Da preferably, and the time of the dialysis is 48-72 h preferably, and is 72 h more preferably.
[44] In the disclosure, a reagent for the deproteinization is a Sevag solution preferably, and in the disclosure, the centrifuging rotating speed of the deproteinization is 4000-6000 r/min preferably, and the centrifuging time is 5-10 min preferably.
[45] Inthe disclosure, the decoloration is performed in an AB-8 macroporous resin column preferably, and in the disclosure, the use quantity ratio of the supernate to AB-8 macroporous resin is 25-50 mL : 2-5 g. In the disclosure, the temperature of the decoloration is 30-50 degrees
Celsius preferably. In the disclosure, the decoloration is performed in a shaker preferably, and the rotating speed of the shaker is 100-140 rpm.
[46] In the disclosure, the temperature of the vacuum concentration is 40-60 degrees Celsius preferably, and is 50 degrees Celsius more preferably. In the disclosure, the dialyzed extracting solution is vacuum-concentrated to 1/3-1/4 of the volume of the extracting solution preferably, and is 1/4 more preferably.
[47] Inthe disclosure, during the alcohol precipitation, the volume percent of absolute ethyl alcohol in an alcohol precipitation system is 80%. In the disclosure, the temperature of the alcohol precipitation is 4 degrees Celsius preferably, and the precipitation time is 12-24 h preferably.
[48] In the disclosure, the temperature of the vacuum freeze drying is -50 to -65 degrees
Celsius preferably, and is -55 degrees Celsius more preferably.
[49] In the disclosure, the purification comprises separating the organic phase crude polysaccharide and the water phase crude polysaccharide through a DEAE-52 cellulose chromatographic column preferably to obtain a polysaccharide head product, separating the obtained polysaccharide head product through a Sephadex G-100 gel chromatographic column, 40 then detecting the optical density value of eluant at an ultraviolet 490 nm position through a phenol-sulfuric acid method in a tube insulating manner, drawing an elution curve, combining
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[50] In the disclosure, the eluant during separation of the Sephadex G-100 gel chromatographic column is deionized water.
[51] FIG. 1 is an extraction flow of an extraction method of the disclosure.
[52] It can be seen from FIG. 1 that the extraction flow of the disclosure is as follows: the raw materials are smashed firstly, and then enzymatically hydrolyzed to obtain enzymatic hydrolysate; and then the enzymatic hydrolysate is subjected to two-phase salting-out extraction to obtain the alcohol-soluble polysaccharide and the water-soluble polysaccharide.
[53] The extraction method for the polysaccharide in the plants provided by the disclosure is illustrated in detail with reference to embodiments below, but they shall not be understood as limiting the protection scope of the disclosure.
Embodiment 1
[54] Liquorice root dregs dried in the shade are smashed and screened at 120 meshes to obtain liquorice root dreg powder.
[55] 0.20 g of liquorice root dreg powder is added into cellulase accounting for 1.50% of the mass of the liquorice root dreg powder and 2.50 g of citric-acid-disodium hydrogen phosphate buffer with the pH value of 4.50, and after the materials are uniformly mixed, the mixture is enzymatically hydrolyzed in a water bath kettle at 40 degrees Celsius for 90 min to obtain enzymatic hydrolysate; and
[56] 2.30 g of ammonium sulfate and 3.20 g of water are mixed with the obtained enzymatic hydrolysate for 10 min, then 2.0 g of absolute ethyl alcohol is added and mixed for 10 min, a two-phase salting-out system is formed, the obtained two-phase salting-out system stands at room temperature for 60 min and then is centrifuged for 5 min, and an upper phase organic solvent extracting solution and a lower phase inorganic salt extract are obtained respectively.
[57] The upper phase organic solvent extracting solution and the lower phase inorganic salt aqueous extract are sequentially dialyzed, deproteinized by adding a Sevag solution and centrifuged respectively to obtain supernate; and then the supernate is decolored in an AB-8 macroporous resin column to obtain an organic phase polysaccharide solution and a water phase polysaccharide solution respectively. Then, the obtained organic phase polysaccharide solution and water phase polysaccharide solution are separated through DEAE-52 cellulose column chromatography to obtain a polysaccharide head product, then the obtained polysaccharide head product is dissolved in distilled water to be centrifuged to obtain supernate, and the obtained supernate is eluted with deionized water through Sephadex G-100 gel column chromatography to obtain eluant. Then, the optical density value of the eluant at an ultraviolet 490 nm position is detected through a phenol-sulfuric acid method in a tube insulating manner, an elution curve is drawn, polysaccharide components with single peaks in the elution curve are combined, and vacuum concentration, dialysis for 72 h and freeze drying are performed on 40 the polysaccharide components to obtain alcohol-soluble polysaccharide and water-soluble polysaccharide.
BL-5421 . . . ,, . LU501444
[58] Through analysis and calculation, the yield of the alcohol-soluble polysaccharide is 0.79%, and the yield of the water-soluble polysaccharide is 2.12%.
[59] Inthe disclosure, the yield of the polysaccharide is measured through a phenol-sulfuric acid colorimetric method.
[60] 1 mL of extracting solution diluted by a certain times is taken and put in a 10 mL test tube, 1 mL of 5% phenol solution and 5 mL of 98% of sulfuric acid are added, the materials stand for 10 min and are uniformly mixed, the mixture is subjected to an ice bath for 1 min and then subjected to a water bath at 30 degrees Celsius for 20 min, and the absorbance is measured at the 490 nm position. The concentration of the polysaccharide in the upper phase organic solvent phase and the concentration of the polysaccharide in the lower phase inorganic salt aqueous phase extract are calculated respectively according to a standard curve and substituted into a formula (1), and the extraction rates of the alcohol-soluble polysaccharide and the water- soluble polysaccharide are calculated.
Polysaccharide extraction rate (%) wx soos 3% FT LH}
FHM ww
[61] Inthe formula: m is the mass of the liquorice root dregs, g;
[62] C is the concentration of the polysaccharide in the upper phase organic solvent phase or the lower phase inorganic salt aqueous phase extract, mg/mL;
[63] V isthe volume of the upper phase organic solvent phase or the lower phase inorganic salt aqueous phase extract, mL; and
[64] Dis a dilution times.
Embodiment 2
[65] The embodiment 2 only differs from the embodiment 1 in that the cellulase is replaced with Beta-glucanase, and the pH value of the buffer is changed into 6.00 from 4.50.
[66] Through analysis and calculation, the yield of the alcohol-soluble polysaccharide in the embodiment is 0.85%, and the yield of the water-soluble polysaccharide is 1.90%.
Embodiment 3
[67] The embodiment 3 only differs from the embodiment 1 in that the cellulase is replaced with complex enzymes of the cellulase and the Beta-glucanase,
[68] wherein the mass ratio of the cellulase to the Beta-glucanase is 2 : 1. The pH value of the buffer is changed into 4.00 from 4.50.
[69] Through analysis and calculation, the yield of the alcohol-soluble polysaccharide in the embodiment is 0.86%, and the yield of the water-soluble polysaccharide is 2.39%.
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[70] It can be seen from the embodiments 1-3 that when the cellulase, the Beta-glucanase and the complex enzymes of the cellulase and the Beta-glucanase are adopted for enzymatic hydrolysis respectively,
[71] the yield ofthe polysaccharide finally obtained through the complex enzymes is highest.
Embodiment 4
[72] In order to test the effects of different enzymes on polysaccharide extraction, 5 tests are performed in the embodiment 4 in total.
[73] Test4.1
[74] The test 4.1 only differs from the embodiment 1 in that the pH value of the citric acid- disodium hydrogen phosphate buffer is adjusted into 4.00 from 4.5; and the temperature of enzymatic hydrolysis is adjusted into 50 degrees Celsius from 40 degrees Celsius.
[75] The test 4.2 only differs from the test 4.1 in that the cellulase is replaced with the Beta- glucanase.
[76] The test 4.3 only differs from the test 4.1 in that the cellulase is replaced with the complex enzymes of the cellulase and the Beta-glucanase at the mass ratio of 1 : 1.
[77] The test 4.4 only differs from the test 4.1 in that the cellulase is replaced with the complex enzymes of the cellulase and the Beta-glucanase at the mass ratio of 1 : 2.
[78] The test 4.5 only differs from the test 4.1 in that the cellulase is replaced with the complex enzymes of the cellulase and the Beta-glucanase at the mass ratio of 2 : 1.
[79] Table | Experimental conditions and analysis and detection results of the 5 tests in the embodiment 4
Table 1 Experimental conditions and detection results of the embodiment 4
Tn [wer [wan | eis | es]
Beta- Cellulase : Cellulase : Cellulase :
Enzyme Cellulase Beta- Beta- Beta- glucanase glucanase glucanase glucanase
PTT ma a]
Alcohol-soluble | 0.91% 0.86% 0.78% 0.74% 0.93%
Detection polysaccharide result
Water-soluble | 243% | 226% 1.73% 1.53% 2.54% polysaccharide
[80] It can be seen from Table 1 that the yield of the alcohol-soluble polysaccharide and the yield of the water-soluble polysaccharide are highest when the mass ratio of the cellulase to the
Beta-glucanase in the complex enzymes is 2 : 1.
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Embodiment 5
[81] In order to test the effects of different enzyme quantities on polysaccharide extraction, tests are performed in the embodiment 5 in total.
[82] The test 5.1 only differs from the embodiment 3 in that the temperature of enzymatic 5 hydrolysis is adjusted into 50 degrees Celsius from 40 degrees Celsius; and the mass percent of the enzyme quantity in the liquorice root dreg powder is 0.6%.
[83] Thetest 5.2 only differs from the test 5.1 in that the mass percent of the enzyme quantity in the liquorice root dreg powder is 0.9%.
[84] Thetest 5.3 only differs from the test 5.1 in that the mass percent of the enzyme quantity in the liquorice root dreg powder is 1.2%.
[85] The test 5.4 only differs from the test 5.1 in that the mass percent of the enzyme quantity in the liquorice root dreg powder is 1.5%.
[86] Thetest 5.5 only differs from the test 5.1 in that the mass percent of the enzyme quantity in the liquorice root dreg powder is 1.8%.
[87] Table 2 is the experimental conditions and analysis and detection results of the 5 tests in the embodiment 5
Table 2 Experimental conditions and detection results of the embodiment 5
Test [ws [we | ess | tess
Alcohol-soluble | 0.75% | 0.80% 0.81% 0.85% 0.85%
Detection | Polysaccharide result _
Water-soluble | 177% 1.92% 2.16% 2.24% 2.24% polysaccharide
[88] It can be seen from Table 2 that the yield of the polysaccharide is highest when the use quantity of the complex enzymes is 1.50%.
Embodiment 6
[89] In order to test the effects of different enzymatic hydrolysis times on polysaccharide extraction, 5 tests are performed in the embodiment 6 in total.
[90] The test 6.1 only differs from the embodiment 3 in that the temperature of enzymatic hydrolysis is adjusted into 50 degrees Celsius from 40 degrees Celsius; and the enzymatic hydrolysis time is
[91] 30 min. Through analysis and calculation, the yield of the alcohol-soluble polysaccharide is 0.63%; and the yield of the water-soluble polysaccharide is 2.0%.
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[92] The test 6.2 only differs from the test 6.1 in that the enzymatic hydrolysis time is 60 min.
[93] | The test 6.3 only differs from the test 6.1 in that the enzymatic hydrolysis time is 90min.
[94] The test 6.4 only differs from the test 6.1 in that the enzymatic hydrolysis time is 120min.
[95] The test 6.5 only differs from the test 6.1 in that the enzymatic hydrolysis time is 150min.
[96] Table 3 is the experimental conditions and analysis and detection results of the 5 tests in the embodiment 6
Table 3 Experimental conditions and analysis and calculation results of the embodiment 6
Teer [wer | wen | tees | teres
Alcohol-soluble | 0.63% 0.64% 0.71% 0.77% 0.76%
Detection | Polysaccharide result _
Water-soluble | 20% | 2.08% 2.13% 2.30% 2.26% polysaccharide
[97] It can be seen from Table 3 that the yield of the polysaccharide is highest when the enzymatic hydrolysis time is 120 min.
Embodiment 7
[98] In order to test the effects of different enzymatic hydrolysis temperatures on polysaccharide extraction, 5 tests are performed in the embodiment 7 in total.
[99] The test 7.1 only differs from the test 6.4 in the embodiment 6 in that the enzymatic hydrolysis temperature is set at 40 degrees Celsius.
[100] The test 7.2 only differs from the test 7.1 in that the enzymatic hydrolysis temperature is set at 45 degrees Celsius.
[101] The test 7.3 only differs from the test 7.1 in that the enzymatic hydrolysis temperature is set at 50 degrees Celsius.
[102] The test 7.4 only differs from the test 7.1 in that the enzymatic hydrolysis temperature is set at 55 degrees Celsius.
[103] The test 7.5 only differs from the test 7.1 in that the enzymatic hydrolysis temperature is setat 60 degrees Celsius. Table 4 Experimental conditions and analysis and calculation results of the 5 tests in the embodiment 7
Table 4 Experimental conditions and analysis and calculation results of the embodiment 7
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Tw [eva | ten | mars | tes temperature
Detection | Aleehol-soluble | 077% 0.80% 0.82% 0.81% 0.77% polysaccharide
Result | Water-soluble | 2.03% 2.13% 2.39% 2.34% 2.00% polysaccharide
[104] It can be seen from Table 4 that the yield of the polysaccharide is highest when the enzymatic hydrolysis temperature is 50 degrees Celsius.
Embodiment 8
[105] In order to test the effects of different buffer pH values on polysaccharide extraction, 5 tests are performed in the embodiment 8 in total.
[106] The test 8.1 only differs from the test 7.1 in the embodiment 7 in that the pH value of the buffer is set at 3.50;
[107] the test 8.2 only differs from the test 8.1 in that the pH value of the buffer 1s set at 4.00;
[108] the test 8.3 only differs from the test 8.1 in that the pH value of the buffer is set at 4.50;
[109] the test 8.4 only differs from the test 8.1 in that the pH value of the buffer is set at 5.00;
[110] the test 8.5 only differs from the test 8.1 in that the pH value of the buffer is set at 5.50.
Table 5 Experimental conditions and analysis and calculation results of the 5 tests in the embodiment 8
Table 5 Experimental conditions and analysis and calculation results of the embodiment 8
Tent [resta | wn | tens | Terns
Alcohol-soluble | 0.79% | 083% 0.78% 0.80% 0.81%
Detection | Polysaccharide result _
Water-soluble | 2.03% | 2.18% 2.12% 2.11% 2.10% polysaccharide
[111] It can be seen from Table 5 that the yield of the obtained alcohol-soluble polysaccharide and the yield of the obtained water-soluble polysaccharide are highest when the pH value of the buffer is 4.00.
Embodiment 9
[112] In order to test the effects of the mass of ammonium sulfate on polysaccharide extraction, 2 tests are performed in the embodiment 9 in total.
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[113] The test 9.1 only differs from the test 4.5 in the embodiment 4 in that the mass of the ammonium sulfate is 2.00 g, and through analysis and calculation, the yield of the alcohol- soluble polysaccharide is 0.61%, and the yield of the water-soluble polysaccharide is 2.60%.
[114] The test 9.2 only differs from the test 4.5 in the embodiment 4 in that the mass of the ammonium sulfate is 2.40g, and through analysis and calculation, the yield of the alcohol- soluble polysaccharide is 0.77%, and the yield of the water-soluble polysaccharide is 1.49%.
[115] It can be seen from the embodiment 9 that when the mass percent of the inorganic salt in the mixed liquid is 20%-24%, the method has a good extraction effect on the alcohol-soluble polysaccharide and the water-soluble polysaccharide.
Embodiment 10
[116] In order to test the effects of the mass of absolute ethyl alcohol on polysaccharide extraction, 2 tests are performed in the embodiment 10 in total.
[117] The test 10.1 only differs from the test 4.5 in the embodiment 4 in that the mass of the absolute ethyl alcohol is 1.90 g, and through analysis and calculation, the yield of the alcohol- soluble polysaccharide is 0.74%, and the yield of the water-soluble polysaccharide is 2.14%.
[118] The test 10.2 only differs from the test 4.5 in the embodiment 4 in that the mass of the absolute ethyl alcohol is 2.30 g, and through analysis and calculation, the yield of the alcohol- soluble polysaccharide is 0.70%, and the yield of the water-soluble polysaccharide is 1.80%.
[119] It can be seen from the embodiment 10 that when the mass percent of the inorganic solvent in the mixed liquid is 19%-23%, the method has a good extraction effect on the alcohol- soluble polysaccharide and the water-soluble polysaccharide.
Embodiment 11
[120] In order to test the effects of the mass of raw materials on polysaccharide extraction, 2 tests are performed in the embodiment 11 in total.
[121] The test 11.1 only differs from the test 4.5 in the embodiment 4 in that the mass of the liquorice root dreg powder is 0.18 g, and through analysis and calculation, the yield of the alcohol-soluble polysaccharide is 0.95%, and the yield of the water-soluble polysaccharide is 2.09%.
[122] The test 11.2 only differs from the test 4.5 in the embodiment 4 in that the mass of the liquorice root dreg powder is 0.29g, and through analysis and calculation, the yield of the alcohol-soluble polysaccharide is 1.00%, and the yield of the water-soluble polysaccharide is 1.84%.
[123] It can be seen from the embodiment 11 that when the mass percent of the liquorice root dreg powder is 0.18-0.29 g, the method has a good extraction effect on the alcohol-soluble polysaccharide and the water-soluble polysaccharide.
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Embodiment 12
[124] Liquorice roots are cut into small sections to be kept out of the sun, ventilated and dried in the shade, smashed and screened at 120 meshes to obtain the liquorice root powder;
[125] 0.22 g of liquorice root powder is added into cellulase accounting for 1.40% of the mass ofthe liquorice root powder and 2.50 g of citric-acid-disodium hydrogen phosphate buffer with the pH value of 5.00, and after the materials are uniformly mixed, the mixture is enzymatically hydrolyzed in a water bath kettle at 40 degrees Celsius for 60 min to obtain enzymatic hydrolysate; and
[126] 2.40 g of ammonium sulfate and 3.10 g of water are added into the obtained enzymatic hydrolysate to be mixed for 10 min, then 2.00 g of absolute ethyl alcohol is added and mixed for 10 min, a two-phase salting-out system is formed, the obtained two-phase salting-out system stands at room temperature for 60 min and then is centrifuged for 5 min, and an upper phase organic solvent extracting solution and a lower phase inorganic salt extract are obtained respectively.
[127] Follow-up treatment of the upper phase organic solvent extracting solution and the lower phase inorganic salt extract is the same as after-treatment steps of the upper phase organic solvent extracting solution and the lower phase inorganic salt extract in the embodiment 1, and repetitions are omitted.
[128] Through analysis and calculation, the yield of the alcohol-soluble polysaccharide in the embodiment is 1.58%, and the yield of the water-soluble polysaccharide is 4.24%.
Embodiment 13
[129] Stevia rebaudiana leaves are kept out of the sun, ventilated and dried in the shade, smashed and screened at 160 meshes to obtain stevia rebaudiana powder;
[130] 0.20 g of stevia rebaudiana powder is added into cellulase accounting for 1.50% of the mass of the stevia rebaudiana powder and 2.5 g of citric-acid-disodium hydrogen phosphate buffer with the pH value of 4.00, and after the materials are uniformly mixed, the mixture is enzymatically hydrolyzed in a water bath kettle at 50 degrees Celsius for 120 min to obtain enzymatic hydrolysate; and
[131] 1.70 g of dipotassium phosphate and 3.10 g of water are added into the obtained enzymatic hydrolysate to be mixed for 10 min, then 2.70 g of absolute ethyl alcohol is added and mixed for 10 min, a two-phase salting-out system is formed, the obtained two-phase salting- out system stands at room temperature for 60 min and then is centrifuged for 5 min, and an upper phase organic solvent extracting solution and a lower phase inorganic salt extract are obtained respectively.
[132] Follow-up treatment of the upper phase organic solvent extracting solution and the lower phase inorganic salt extract is the same as after-treatment steps of the upper phase organic solvent extracting solution and the lower phase inorganic salt extract in the embodiment 1, and repetitions are omitted.
BL-5421 . . . 5 LU501444
[133] Through analysis and calculation, the yield of the alcohol-soluble polysaccharide in the embodiment is 18.91%, and the yield of the water-soluble polysaccharide is 4.61%.
Embodiment 14
[134] Tagetes erecta flowers are kept out of the sun, ventilated and dried in the shade, smashed and screened at 120 meshes to obtain tagetes erecta powder;
[135] 0.22 g oftagetes erecta powder is added into cellulase accounting for 1.50% of the mass of the tagetes erecta powder and 4.00 g of citric-acid-disodium hydrogen phosphate buffer with the pH value of 4.00, and after the materials are uniformly mixed, the mixture is enzymatically hydrolyzed in a water bath kettle at 45 degrees Celsius for 90 min to obtain enzymatic hydrolysate; and
[136] 1.80 g of ammonium sulfate and 1.40 g of water are added into the obtained enzymatic hydrolysate to be mixed for the first time for 10 min, then 2.80 g of absolute ethyl alcohol is added and mixed for 10 min, a two-phase salting-out system is formed, the obtained two-phase salting-out system stands at room temperature for 60 min and then is centrifuged for 5 min, and an upper phase organic solvent extracting solution and a lower phase inorganic salt extract are obtained respectively.
[137] Follow-up treatment of the upper phase organic solvent extracting solution and the lower phase inorganic salt extract is the same as after-treatment steps of the upper phase organic solvent extracting solution and the lower phase inorganic salt extract in the embodiment 1, and repetitions are omitted.
[138] Through analysis and calculation, the yield of the alcohol-soluble polysaccharide in the embodiment is 4.70%, and the yield of the water-soluble polysaccharide is 11.62%.
[139] The above descriptions are only preferred embodiments of the prevent disclosure, and it should be noted that a person of ordinary skill in the art can further make several improvements and modifications without departing from the principle of the disclosure, and those improvements and modifications should be included in the protection scope of the present disclosure,
Claims (10)
1. An extraction method for polysaccharide in plants, comprising the following steps: enzymatically hydrolyzing plant raw materials to obtain enzymatic hydrolysate; and sequentially mixing the enzymatic hydrolysate with an inorganic salt aqueous solution and an organic solvent, and performing two-phase salting-out extraction on the obtained mixed liquid to obtain alcohol-soluble polysaccharide and water-soluble polysaccharide; wherein inorganic salt in the inorganic salt aqueous solution comprises one or more of sulfate, phosphate, citrate and chloride; and the organic solvent comprises one or more of ethyl alcohol, iso-glycerol, acetone, acetonitrile and tert-butyl alcohol.
2. The extraction method according to claim 1, characterized in that enzymes for the enzymatic hydrolysis comprise one or more of cellulase, hemicellulase, pectinase, trypsin, papain, Beta-glucanase and amylase.
3. The extraction method according to claim 1 or 2, characterized in that the mass of the enzymes for the enzymatic hydrolysis is 0.6%-1.8% of the mass of the plant raw materials.
4. The extraction method according to claim 1 or 2, characterized in that the temperature of the enzymatic hydrolysis is 40-60 degrees Celsius, and the time of the enzymatic hydrolysis is 30-150 min.
5. The extraction method according to claim 1, characterized in that the mass percent of the inorganic salt in the mixed liquid is 20%-24%.
6. The extraction method according to claim 1, characterized in that the mass percent of the organic solvent in the mixed liquid is 19%-23%.
7. The extraction method according to claim 1 or 5 or 6, characterized in that the temperature of the two-phase salting-out extraction is 25-40 degrees Celsius, and the time of the two-phase salting-out extraction is 65-80 min.
8. The extraction method according to claim 1, characterized in that an upper phase organic solvent extracting solution and a lower phase inorganic salt aqueous extract are obtained through the two-phase salting-out extraction, and the method further comprises after-treating the upper phase organic solvent extracting solution and the lower phase inorganic salt aqueous extract respectively after the two-phase salting-out extraction; and the after-treatment comprises sequentially performing dialysis, deproteinization, decoloration and purification.
9. The extraction method according to claim 1 or 8, characterized in that the purification comprises sequentially performing DEAE-52 cellulose column chromatography separation and
BL-5421 LU501444 Sephadex G-100 gel column chromatography separation.
10. The extraction method according to claim 1 or 8, characterized in that the dialysis is performed in a dialysis bag with the molecular weight cut-off of 3500 Da, and the time of the dialysis is 48-72 h.
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