Method for extracting and purifying purple sweet potato polysaccharide

Abstract

The invention discloses a method for extracting and purifying purple sweet potato polysaccharide, and belongs to the technical field of polysaccharide extraction. The method comprises the following steps: S1, pretreating purple sweet potatoes to obtain purple sweet potato powder; s2, coarse extraction of purple sweet potato powder: adding absolute ethyl alcohol, refluxing to a degreased part through coagulation, and extracting; concentrating under reduced pressure to obtain a concentrated solution; carrying out alcohol precipitation on the concentrated solution, adding distilled water to carry out deproteinization treatment, then carrying out decoloring treatment, and centrifuging to remove a decoloring agent; dialyzing and freeze-drying to obtain a purple sweet potato polysaccharide crude product; s3, purifying the purple sweet potato polysaccharide, namely redissolving the purple sweet potato polysaccharide, putting into a cellulose column, standing and adsorbing, eluting by adopting a NaCl solution, collecting eluent for developing, measuring absorbance until no elution peak exists, combining the eluent, concentrating, dialyzing, redissolving, separating, eluting, collecting the eluent, and freeze-drying to obtain a pure purple sweet potato polysaccharide product. The obtained purple sweet potato polysaccharide is high in purity and high in yield, and the purified purple sweet potato polysaccharide has remarkable antioxidant activity and has a good antioxidant effect on grease and the like; the purified purple sweet potato polysaccharide can be stored under natural light or dark conditions, and has good stability.

Description

Technical Field

[0001] The invention belongs to the technical field of polysaccharide extraction, and particularly relates to a method for extracting and purifying purple sweet potato polysaccharide. Background Art

[0002] Polysaccharides are important bioactive macromolecules in animals, plants and microorganisms, and have numerous physiological effects. In addition, some polysaccharides have been developed as drugs and health foods for treating diseases. The extraction, separation and purification of polysaccharides are the primary and necessary processes for subsequent research on polysaccharides, including the structure and functional activity of polysaccharides. Therefore, effective extraction, separation and purification of polysaccharides are particularly important for the research of polysaccharides.

[0003] At present, many technologies have been developed for the extraction of polysaccharides, mainly including hot water extraction (or water extraction and alcohol precipitation), ultrasonic and microwave assisted extraction, enzyme extraction, etc. However, these extraction technologies have their own advantages and disadvantages. We should choose the appropriate extraction technology according to the properties of polysaccharides and our own experimental conditions. In recent years, the rich nutrients in purple sweet potatoes have attracted people's attention, but there are few reports on the research of purple sweet potato polysaccharides. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a method for extracting and purifying purple sweet potato polysaccharides to solve the problem that there are few methods for extracting purple sweet potato polysaccharides.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides a method for extracting and purifying purple sweet potato polysaccharide, comprising the following steps:

[0007] S1. Prepare purple sweet potato, auxiliary reagents and instruments;

[0008] S2, peeling, washing, slicing, drying, crushing and grinding the purple sweet potato to obtain purple sweet potato powder;

[0009] S3, adding the purple sweet potato powder into anhydrous ethanol, performing condensation reflux degreasing treatment, discarding the supernatant and adding distilled water for leaching;

[0010] S4, centrifuging the extracted mixture, collecting the supernatants, combining them, and concentrating them under reduced pressure to obtain a concentrate;

[0011] S5, adding anhydrous ethanol to the concentrated solution, leaving it overnight and collecting the polysaccharide precipitate; then washing the polysaccharide precipitate with ethanol and acetone in turn and freeze-drying it, and then adding distilled water to dissolve it; and then deproteinizing it, and then adding a decolorizing agent to the dissolved solution for decolorization, stirring it and centrifuging it to remove the decolorizing agent;

[0012] S6, putting the solvent after removing the decolorizing agent in step S5 into a dialysis bag for dialysis, and obtaining a crude product of purple sweet potato polysaccharide after freeze-drying;

[0013] S7, weighing purple sweet potato polysaccharide, re-dissolving it with distilled water, filtering it with a microporous filter membrane, and then loading it into a cellulose column to allow it to stand for adsorption, and then eluting it with a NaCl solution, and collecting the eluate;

[0014] S8, after the collected eluate is developed by the phenol-sulfuric acid method, the absorbance at a wavelength of 490 nm is measured by a UV-visible spectrophotometer until there is no elution peak, and the eluate with the main absorption peak is combined;

[0015] S9. The collected eluate is concentrated, dialyzed, freeze-dried, and then reconstituted with distilled water. It is then further separated using Sephadex G-100 gel, eluted with distilled water, and the eluate is collected. The eluate is monitored and collected under the same conditions as the DEAE-52 cellulose column, and the components obtained are collected and freeze-dried to obtain pure purple sweet potato polysaccharide.

[0016] Furthermore, in step S3, the mass ratio of purple sweet potato powder to anhydrous ethanol is 0.8-1.2:6; the extraction condition is 80° C., and the extraction is performed twice, each time for 2 hours.

[0017] Furthermore, in step S4, the reduced pressure concentration amount is 0.15-0.25 times the original amount before concentration.

[0018] Furthermore, in step S5, the volume ratio of anhydrous ethanol to concentrated solution is 3.8-4.2:1-1.2, the overnight temperature is 4°C; the mass ratio of polysaccharide to distilled water is 5:2; Sevage reagent is used for deproteinization; and the decolorizing agent is diatomaceous earth.

[0019] Furthermore, in step S6, the dialysis condition is to use distilled water for 72 hours.

[0020] Furthermore, in step S7, the concentration of purple sweet potato polysaccharide after redissolution is 5 mg / mL; the pore size of the microporous filter membrane is 0.45 μm; the DEAE-52 cellulose column is φ2×30 cm; the NaCl solution is washed 6 times, each concentration is 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mol / L, and the washing flow rate is 1.2 mL / min.

[0021] Furthermore, in step S8, when measuring the absorbance, an elution curve is drawn with the number of tubes as the abscissa and A490nm as the ordinate.

[0022] Furthermore, in step S9, the elution is performed at a flow rate of 0.5 mL / min.

[0023] The beneficial effects of the present invention are:

[0024] 1. In the present invention, the protein removal rate was 93.14% by the Sevage method, which was only 0.86% different from the predicted result. The verification results showed that there was no significant difference between the experimental value and the predicted value, indicating that the response model can fully reflect the expected optimization.

[0025] 2. In the present invention, the crude polysaccharide of purple sweet potato is separated and purified by DEAE-52 cellulose column and Sephadex G-100 gel column. The results show that the extract contains almost no protein and uronic acid, and the sugar content is as high as 92.52%, indicating that the crude polysaccharide of purple sweet potato is effectively extracted.

[0026] 3. The extraction and purification method of the present invention can obtain purple sweet potato polysaccharides with high purity and high yield. The purified purple sweet potato polysaccharides have significant antioxidant activity and good antioxidant effect on oils and fats. In addition, the purified purple sweet potato polysaccharides have good stability when stored under neutral or alkaline pH, temperature below 45°C, natural light or light-proof conditions.

[0027] 4. The invention provides an efficient and simple extraction and purification method, which provides strong support for the development and utilization of purple sweet potato polysaccharides.

[0028] Other advantages, objectives and features of the present invention will be described in the following description and will be apparent to those skilled in the art to some extent, or those skilled in the art may be taught from the practice of the present invention. The objectives and other advantages of the present invention may be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to make the purpose, technical solution and beneficial effects of the invention clearer, the present invention provides the following drawings for illustration:

[0030] Figure 1 This is a finished product display diagram of the purple sweet potato pure polysaccharide of the present invention;

[0031] Figure 2 The figure is an elution curve of the crude purple sweet potato polysaccharide of the present invention on a DEAE-52 cellulose column;

[0032] Figure 3 The figure is an elution curve diagram of the crude purple sweet potato polysaccharide of the present invention on a Sephadex G-100 gel column. DETAILED DESCRIPTION

[0033] A method for extracting and purifying purple sweet potato polysaccharide:

[0034] A method for extracting and purifying purple sweet potato polysaccharide comprises the following steps:

[0035] S1. Prepare purple sweet potato, auxiliary reagents and instruments;

[0036] S2, pretreatment: peeling, washing, slicing, drying, and crushing and grinding the purple sweet potato to obtain purple sweet potato powder;

[0037] S3, crude polysaccharide extraction: 50g purple sweet potato powder was added to 300mL anhydrous ethanol, and condensed and refluxed for defatting twice; then centrifuged and discarded the supernatant, and 1000mL distilled water was added to the defatted purple sweet potato powder, and extracted twice at 80°C, each time for 2h;

[0038] S4, centrifuging the mixture of the two extractions, collecting the supernatants, combining them, and concentrating them under reduced pressure to 0.15-0.25 times the original concentration;

[0039] S5. Add 4 times the volume of anhydrous ethanol to the concentrated solution, place it at 4°C overnight, and collect the polysaccharide precipitate by alcohol precipitation; then wash the polysaccharide precipitate with ethanol and acetone twice in sequence, freeze-dry it, and then add distilled water to dissolve it (the mass ratio of polysaccharide to distilled water is 5:2); and use Sevage reagent to deproteinize twice (the volume ratio of the dissolved solution to the Sevage reagent is 2:1); then add diatomaceous earth powder with a content of 2% of the dissolved solution to decolorize it, stir it for 30 minutes, then centrifuge to remove the diatomaceous earth, and repeat the operation twice;

[0040] S6, putting the solvent after removing the decolorant in step S5 into a 3500Da dialysis bag, dialyzing with distilled water for 72 hours, and lyophilizing to obtain a crude product of purple sweet potato polysaccharide;

[0041] S7, weigh purple sweet potato polysaccharide and re-dissolve it in distilled water (concentration of 5 mg / mL), filter it through a 0.45 μm microporous filter membrane, and load it into a DEAE-52 cellulose column (φ2×30 cm). After standing to ensure that the sample is fully adsorbed, elute it with 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L NaCl solutions at a flow rate of 1.2 mL / min, and collect 4 mL of the eluate per tube;

[0042] S8, after the collected eluate is developed by the phenol-sulfuric acid method, the absorbance at a wavelength of 490 nm is measured by a UV-visible spectrophotometer (the elution curve is drawn with the number of tubes as the abscissa and A490 nm as the ordinate), until there is no elution peak, and the eluate with the main absorption peak is combined;

[0043] S9. The collected main eluate was concentrated, dialyzed, freeze-dried, and then reconstituted with distilled water. It was then further separated using a Sephadex G-100 gel column (φ1×30cm), eluted with distilled water at a flow rate of 0.5mL / min, and 2mL of the eluate was collected in a tube. The eluate was monitored and collected under the same conditions as the DEAE-52 cellulose column, and the resulting components were collected and freeze-dried to obtain pure purple sweet potato polysaccharide.

[0044] verify:

[0045] The use of DEAE-52 cellulose column to separate and purify crude purple sweet potato polysaccharides can effectively remove residual pigments in crude purple sweet potato polysaccharides. Figure 2 As shown, after separation of purple sweet potato crude polysaccharide by DEAE-52 cellulose column, 4 obvious components were obtained, which were 0.05 mol / L, 0.1 mol / L, 0.2 mol / L and 0.5 mol / L NaCl solution elution components, and they were named purple sweet potato crude polysaccharide-1, purple sweet potato crude polysaccharide-2, purple sweet potato crude polysaccharide-3 and purple sweet potato crude polysaccharide-4, respectively.

[0046] From the peak areas of the four components, it can be seen that the purple sweet potato crude polysaccharide-1 is the main component of purple sweet potato polysaccharide. The purple sweet potato crude polysaccharide-1 was collected multiple times, and after concentration, dialysis and freeze-drying, its yield was calculated to be 30.07%. The purple sweet potato crude polysaccharide-1 after enrichment and freeze-drying was purified twice using Sephadex G-100 gel column. The results are shown in FIG. Figure 3 As shown, after the purple sweet potato crude polysaccharide-1 was eluted through the Sephadex G-100 gel column, the elution curve had only one set of single peaks, and the peak shape was relatively symmetrical, indicating that this component was a pure component of purple sweet potato polysaccharide.

[0047] like Figure 1 As shown, the secondary purified product was collected several times, and after concentration and freeze-drying, a white, shiny, hard solid powder was obtained, which was named purple sweet potato pure polysaccharide-1A. According to the linear regression equation of the standard curve, the total sugar content, protein content and glucuronic acid content of purple sweet potato polysaccharide before and after purification were calculated, respectively. The results are shown in Table 1:

[0048] Table 1

[0049] Polysaccharides Yield (%) Total sugar content (%) Protein content (%) Uronic acid content (%) Purple sweet potato crude polysaccharide-4 3.32 89.68 1.28 3.78 Purple sweet potato polysaccharide-1A 30.07 92.52 0.12 0.78

[0050] As shown in Table 1, after deproteinization, decolorization, dialysis, column chromatography separation and purification, the purple sweet potato crude polysaccharide-1A almost no longer contains protein and uronic acid, and its sugar content is 92.52%, which further shows that the purity of the purple sweet potato crude polysaccharide-1A is relatively high.

[0051] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A method for extracting and purifying purple sweet potato polysaccharides, characterized in that: The following steps are included: S1. Prepare purple sweet potato, auxiliary reagents and instruments; S2, peeling, washing, slicing, drying, crushing and grinding the purple sweet potato to obtain purple sweet potato powder; S3, adding the purple sweet potato powder into anhydrous ethanol, performing condensation reflux degreasing treatment, discarding the supernatant and adding distilled water for leaching; S4, centrifuging the extracted mixture, collecting the supernatants, combining them, and concentrating them under reduced pressure to obtain a concentrate; S5, adding anhydrous ethanol to the concentrated solution, leaving it overnight and collecting the polysaccharide precipitate; then washing the polysaccharide precipitate with ethanol and acetone in turn and freeze-drying it, and then adding distilled water to dissolve it; and then deproteinizing it, and then adding a decolorizing agent to the dissolved solution for decolorization, stirring it and centrifuging it to remove the decolorizing agent; S6, putting the solvent after removing the decolorizing agent in step S5 into a dialysis bag for dialysis, and obtaining a crude product of purple sweet potato polysaccharide after freeze-drying; S7, weighing purple sweet potato polysaccharide, re-dissolving it with distilled water, filtering it with a microporous filter membrane, and then loading it into a cellulose column to allow it to stand for adsorption, and then eluting it with a NaCl solution, and collecting the eluate; S8, after the collected eluate is developed by the phenol-sulfuric acid method, the absorbance at a wavelength of 490 nm is measured by a UV-visible spectrophotometer until there is no elution peak, and the eluate with the main absorption peak is combined; S9. The collected eluate is concentrated, dialyzed, freeze-dried, and then reconstituted with distilled water. It is then further separated using Sephadex G-100 gel, eluted with distilled water, and the eluate is collected. The eluate is monitored and collected under the same conditions as the DEAE-52 cellulose column, and the components obtained are collected and freeze-dried to obtain pure purple sweet potato polysaccharide.

2. The method for extracting and purifying purple sweet potato polysaccharides according to claim 1, characterized in that: In step S3, the mass ratio of purple sweet potato powder to anhydrous ethanol is 0.8-1.2:6; the extraction condition is 80° C., and the extraction is performed twice, each time for 2 hours.

3. The method for extracting and purifying purple sweet potato polysaccharides according to claim 1, characterized in that: In step S4, the reduced pressure concentration amount is 0.15-0.25 times of the original amount before concentration.

4. The method for extracting and purifying purple sweet potato polysaccharides according to claim 1, characterized in that: In step S5, the volume ratio of anhydrous ethanol to concentrated solution is 3.8-4.2:1-1.2, the overnight temperature is 4°C; the mass ratio of polysaccharide to distilled water is 5:2; Sevage reagent is used for deproteinization; and diatomaceous earth is used as the decolorizing agent.

5. The method for extracting and purifying purple sweet potato polysaccharides according to claim 1, characterized in that: In step S6, the dialysis condition is to use distilled water for 72 hours.

6. The method for extracting and purifying purple sweet potato polysaccharides according to claim 1, characterized in that: In the step S7, the concentration of purple sweet potato polysaccharide after redissolution is 5 mg / mL; the pore size of the microporous filter membrane is 0.45 μm; The DEAE-52 cellulose column was φ2×30 cm. The NaCl solution was washed 6 times with concentrations of 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L each time. The washing flow rate was 1.2 mL / min.

7. The method for extracting and purifying purple sweet potato polysaccharides according to claim 1, characterized in that: In the step S8, when measuring the absorbance, an elution curve is drawn with the number of tubes as the horizontal axis and A490nm as the vertical axis.

8. The method for extracting and purifying purple sweet potato polysaccharides according to claim 1, characterized in that: In the step S9, the flow rate during elution is 0.5 mL / min.

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