A preparation method of alcohol-soluble kudzu vine polysaccharide extract

Abstract

The invention discloses a preparation method of an alcohol-soluble kudzu vine polysaccharide extract, comprising the steps of extraction, deodorization, decolorization, enzymatic protein removal, alcohol precipitation purification, drying, etc. The preparation process of the invention is simple and green, and the obtained alcohol-soluble kudzu vine polysaccharide has good alcohol solubility, light color and no fishy smell, which is conducive to industrial production and product application.

Description

Technical Field

[0001] The invention relates to the technical field of polysaccharide extract preparation, and in particular to a method for preparing an alcohol-soluble kudzu vine polysaccharide extract. Background Art

[0002] Gexianmi, scientifically known as Nostoc pseudospherical, is a rare specialty of Hubei. It is a characteristic lower algae plant that can be used as both medicine and food. It is composed of many rosary-shaped algae filaments embedded in a gelatinous polysaccharide matrix to form a spherical or ellipsoidal protophyte. It is dark green and can reach 2 cm in diameter. According to the "Supplement to Compendium of Materia Medica" and "Yao Xing Kao", Gexianmi is edible and can also be used as medicine. In recent years, due to the application of pesticides and fertilizers, the natural ecological environment has been destroyed, and the wild resources of Gexianmi have sharply decreased. In March 2021, it was listed as a precious endangered species in the "Yangtze River Protection Law of the People's Republic of China" for key protection. Although Gexianmi was approved as a new food raw material by the National Health Commission in 2018, it is difficult to compete with mass food as a health food. Opening up new channels for the resource utilization of Gexianmi is the key to revitalizing the development of the Gexianmi industry.

[0003] Pueraria lobata is rich in polysaccharides and has unique biological activity and pharmacological effects. Research reports point out that Pueraria lobata polysaccharide has good solubility in water, but Pueraria lobata polysaccharide has poor solubility in alcohol products, resulting in its application scope being restricted. CN114181326A discloses a method for preparing Pueraria lobata polysaccharide, the main steps of which are crushing, water extraction, enzymolysis, and multiple organic solvent extractions, and finally obtaining water-soluble Pueraria lobata polysaccharide. Its preparation process requires the use of a large amount of organic solvents, and the obtained Pueraria lobata polysaccharide is a water-soluble polysaccharide. CN105622775A discloses a Pueraria lobata polysaccharide and an extraction method thereof, which mainly prepares Pueraria lobata polysaccharide through the steps of material preparation, soaking, water extraction, enzymolysis, ultrafiltration, concentration, sterilization and drying. The invention uses pure water as the extraction solvent, and the molecular weight distribution range of the obtained Pueraria lobata polysaccharide is 10,000 Daltons to 1 million Daltons, and its product molecular weight is specific, and the solubility of the product in alcoholic beverages is not described. CN110713555A discloses a method for extracting polysaccharide from kudzu vine, the main steps of which are pre-crushing, water extraction, acid decolorization, centrifugal filtration, primary alcohol precipitation, re-dissolution, secondary alcohol precipitation and drying. The alcohol solubility of the polysaccharide obtained is unknown, the fishy smell of the raw material of kudzu vine is not removed, and the acid decolorization during the preparation process will destroy the structure of the polysaccharide itself. It can be seen that the above-mentioned prior art has deficiencies in both preparation process and product properties. Summary of the invention

[0004] The object of the present invention is to provide a method for preparing an alcohol-soluble scutellaria baicalensis polysaccharide extract. The scutellaria baicalensis polysaccharide obtained by the method has good solubility in alcoholic beverages.

[0005] The technical solution adopted by the present invention is:

[0006] A method for preparing an alcohol-soluble kudzu vine polysaccharide extract comprises the following steps:

[0007] Step 1, ethanol extraction: coarsely crush the kudzu vine and pass it through a sieve to collect the sieve, then use a 20-50% ethanol solution for multiple condensation reflux extractions, and collect the kudzu vine extract after solid-liquid separation;

[0008] Step 2, deodorizing: adding dried perilla leaves to the kudzu vine extract, with the mass ratio of perilla leaves to kudzu vine being 0.05-0.1:1, stirring at low temperature for a period of time, and performing solid-liquid separation to obtain the deodorized kudzu vine extract;

[0009] Step 3, decolorization: the extract of Pueraria lobata obtained in step 2 is decolorized by a macroporous resin, and the permeate is collected;

[0010] Step 4: Enzymatic protein removal: adding 0.8-1.5% trypsin to the kudzu vine permeate obtained in step 3 to remove kudzu vine protein;

[0011] Step 5, alcohol precipitation purification: the protein-removed pueraria lobata permeate is concentrated, the alcohol content of the concentrate is adjusted with 95% ethanol for alcohol precipitation, and the precipitate is collected by centrifugation to obtain pueraria lobata polysaccharide;

[0012] Step 6, drying: drying the obtained kudzu vine polysaccharide to obtain an alcohol-soluble kudzu vine polysaccharide extract.

[0013] Preferably, in step 1, the mesh size of the sieve is 30-50 meshes.

[0014] Preferably, in step 1, the extraction temperature is 80-95° C., the number of extractions is 1-3 times, the extraction solvent multiple is 15-25 times, and the extraction time is 1.5-2.5 h.

[0015] Preferably, in step 2, the deodorization temperature is 30-45°C, the stirring speed is 150-250r / min, and the deodorization stirring time is 0.5-1.5h.

[0016] Preferably, in step 3, the macroporous resin for decolorization is ADS-17 macroporous resin or D941 macroporous resin, and the permeate flow rate is 1-3 BV / h.

[0017] Preferably, in step 4, the enzymolysis temperature is 45-60° C., and the enzymolysis time is 1-3 h.

[0018] Preferably, in the step 5, the protein-removed pueraria lobata permeate is concentrated to 1 / 45-1 / 55 of the original volume.

[0019] Preferably, in step 5, 95% ethanol is used to adjust the alcohol content of the concentrated solution to 60-80%, the centrifugal speed is 5000-7000 r / min, and the centrifugal time is 10-30 min.

[0020] Preferably, in step six, the drying method is vacuum freeze drying or low-temperature vacuum drying.

[0021] The beneficial effects produced by the present invention are:

[0022] 1. The kudzu vine polysaccharide prepared by the method of the present invention has good solubility in alcoholic beverages and the product properties are stable.

[0023] 2. The kudzu vine polysaccharide prepared by the method of the present invention is light in color and has no fishy smell of kudzu vine itself.

[0024] 3. The preparation process of the alcohol-soluble Myrica ovata polysaccharide of the present invention is simple, no toxic organic solvent is used, and the process is green and pollution-free. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the dissolution amount of samples in 38% vol base wine in different embodiments and comparative examples. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical scheme and advantages of the present invention clearer, the following examples are specifically described. It is necessary to point out that the following examples are only used to explain and illustrate the present invention and are not intended to limit the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above invention content still belong to the protection scope of the present invention.

[0027] Example 1

[0028] A method for preparing an alcohol-soluble kudzu vine polysaccharide extract, the method steps are as follows:

[0029] (1) First, 1 kg of dried kudzu vine root was coarsely crushed and passed through a 30-mesh sieve to collect the sieve residue, and then condensed and refluxed with a 15-fold alcohol strength 20% ethanol solution at 80°C for 2 times, each extraction for 1.5 hours, and the kudzu vine root extract was obtained. 50 g of dried kudzu vine root perilla leaves were added to the obtained kudzu vine root extract, and the mixture was stirred at a speed of 150 r / min for 0.5 hours at 30°C, and the kudzu vine root extract was obtained by solid-liquid separation. The obtained kudzu vine root extract was decolorized by D941 macroporous resin, and the permeate flow rate was 1 BV / h, and the permeate was collected.

[0030] (2) 8 g of trypsin was added to the permeate of the kudzu vine, and the protein in the permeate of the kudzu vine was removed by enzymolysis at a temperature of 45°C for 1 h. The permeate of the kudzu vine after protein removal was then concentrated to 1 / 45 of the original volume, and the alcohol content of the kudzu vine concentrate was adjusted to 60% with 95% ethanol. The concentrate was centrifuged at a speed of 5000 r / min for 10 min, and the precipitate was collected to obtain kudzu vine polysaccharide. Finally, the obtained kudzu vine polysaccharide was vacuum freeze-dried to obtain 201 g of alcohol-soluble kudzu vine polysaccharide extract.

[0031] Example 2

[0032] A method for preparing an alcohol-soluble kudzu vine polysaccharide extract, the method steps are as follows:

[0033] (1) First, 1 kg of dried kudzu vine root was coarsely crushed and passed through a 50-mesh sieve to collect the sieve residue, and then condensed and refluxed with a 50% ethanol solution with a 25-fold alcohol strength at 95°C for extraction, and the extraction times were 2 times, each extraction was 2.5 hours, and the kudzu vine root extract was obtained. 100 g of dried kudzu vine root perilla leaves were added to the obtained kudzu vine root extract, and the mixture was stirred at a speed of 250 r / min for 1.5 hours at 45°C, and the kudzu vine root extract after deodorization was obtained by solid-liquid separation. The obtained kudzu vine root extract was decolorized by D941 macroporous resin, and the permeate flow rate was 3 BV / h, and the permeate was collected.

[0034] (2) 1.5 g of trypsin was added to the permeate of the radix puerariae, and the protein in the permeate of the radix puerariae was removed by enzymolysis at a temperature of 60°C for 3 h. The permeate of the radix puerariae from which the protein was removed was then concentrated to 1 / 55 of the original volume, and the alcohol content of the radix puerariae concentrate was adjusted to 80% with 95% ethanol, and the concentrate was centrifuged at a centrifugal speed of 7000 r / min for 30 min to collect the precipitate to obtain radix puerariae polysaccharide. Finally, the obtained radix puerariae polysaccharide was subjected to vacuum freeze drying to obtain 176 g of alcohol-soluble radix puerariae polysaccharide extract.

[0035] Comparative Example 1

[0036] Without using the deodorization process in step 2, decolorization and subsequent steps were directly performed after using ethanol to extract the kudzu vine, and the remaining steps were the same as in Example 1 to obtain 204 g of alcohol-soluble kudzu vine polysaccharide extract.

[0037] Comparative Example 2

[0038] Without using the decolorization process in step three, enzymatic protein removal was directly performed after ethanol extraction and deodorization, and the remaining steps were the same as in Example 1 to obtain 206 g of alcohol-soluble Pueraria lobata polysaccharide extract.

[0039] Comparative Example 3

[0040] According to the method in Example 1 of invention patent CN105622775A, the polysaccharide extract of Puerariae lobata was prepared, and the steps were as follows:

[0041] (1) First, the kudzu vine is dried at 60°C for 2 hours, then crushed through an 80-mesh sieve, the sieve residue is collected, and the residue is soaked in 30 times the amount of purified water for 24 hours to obtain soaked kudzu vine. 15 times the amount of purified water is added to the soaked kudzu vine, the temperature is raised to 90°C, and then extracted for 2 hours, allowed to stand to room temperature, and then centrifuged to collect the extract.

[0042] (2) After the extract was heated to 55°C, 0.5% of papain (10 4 u / g)(w / v) enzymolysis for 2h, then the enzymolysis solution was cooled to room temperature and centrifuged to collect the feed liquid, the feed liquid was inactivated at 100°C for 10min, and then filtered to remove impurities to obtain the enzymolysis solution. The enzymolysis solution was separated once using an ultrafiltration membrane with a molecular weight of 10,000 Daltons, the retentate was collected, and the retentate was reverse osmosis using a reverse osmosis membrane (reverse osmosis feed temperature <40°C, pressure 2.0Mpa) to concentrate to 0.3 times the original volume to obtain a concentrated solution. Finally, the concentrated solution was sterilized at 80°C for 20min, and vacuum freeze-dried to obtain 212g of Pueraria polysaccharide extract.

[0043] Test Example 1

[0044] Tasting experiment: The fishy smell of the samples in each embodiment and comparative example was tested by a multi-person tasting method. The fishy smell of the samples in different embodiments and comparative examples is shown in Table 1.

[0045] Table 1 Odor of samples in different embodiments and comparative examples

[0046]

[0047] Test Example 2

[0048] Dissolution test: Take an excess of the samples in each embodiment and comparative example, place them in 100 mL of 38% base wine, shake and stir, centrifuge at 10000 r / min for 20 min, take the centrifugal supernatant to detect the content of the kudzu vine polysaccharide, and calculate the dissolved amount of the sample. The dissolved amount of the samples in different embodiments and comparative examples is as follows: Figure 1 shown.

[0049] from Figure 1It can be seen that the dissolution amount of the sample prepared by the method of the present invention in 38%vol base wine is much higher than that of Comparative Example 3, which indicates that the alcohol-soluble kudzu vine polysaccharide extract obtained by the present invention has good solubility in alcoholic solvents. In addition, the dissolution amount of the samples in Comparative Examples 1 and 2 is significantly lower than that in Example 1, which indicates that the deodorization and decolorization steps used in the present invention have a significant effect on improving the solubility of the kudzu vine extract. If the deodorization and decolorization steps are removed, the integrity of the process of the present invention will be destroyed, resulting in a decrease in the solubility of the alcohol-soluble kudzu vine extract.

[0050] Test Example 3

[0051] Chromaticity stability experiment: The chromaticity stability of the samples in each embodiment and comparative example was tested using a colorimeter. The chromaticity values ​​and chromaticity stability of the samples in different embodiments and comparative examples are shown in Table 2.

[0052] Table 2 Chromaticity values ​​of samples at different times in different embodiments and comparative examples

[0053]

[0054] As can be seen from Table 2, the chromaticity values ​​of the samples in Comparative Examples 2 and 3 are significantly higher than those in Examples 1 and 2, while the chromaticity stability is significantly lower than that in Examples 1 and 2, which indicates that the decolorization step used in the present invention can significantly reduce the chromaticity value of the sample and increase its chromaticity stability. The alcohol-soluble Pueraria lobata extract prepared by the present invention has the characteristics of light color and high chromaticity stability.

[0055] Based on the above data analysis results, the alcohol-soluble Puerariae polysaccharide extract prepared in the embodiment of the present invention has the characteristics of good alcohol solubility, light color and no fishy smell. In summary, the above embodiments are only examples of the preferred embodiments of the present invention and do not include all the scope of the invention. Those of ordinary skill in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be subject to the scope defined in the claims.

Claims

1. A method for preparing an alcohol-soluble kudzu vine polysaccharide extract, characterized in that: The following steps are involved: Step 1, ethanol extraction: coarsely crush the kudzu vine and pass it through a sieve to collect the sieve, then use a 20-50% ethanol solution for multiple condensation reflux extractions, and collect the kudzu vine extract after solid-liquid separation; Step 2, deodorization: adding dried perilla leaves to the kudzu extract, the mass ratio of perilla leaves to kudzu is 0.05-0.1:1, stirring at low temperature for a period of time, and separating the solid and liquid to obtain the deodorized kudzu extract; the deodorization temperature is 30-45 ° C, the stirring speed is 150-250 r / min, and the deodorization stirring time is 0.5-1.5 h; Step 3, decolorization: the extract of Pueraria lobata obtained in step 2 is decolorized by a macroporous resin, and the permeate is collected; Step 4: Enzymatic protein removal: 0.8-1.5% trypsin is added to the kudzu vine permeate obtained in step 3 to remove kudzu vine protein; Step 5, alcohol precipitation purification: the protein-removed pueraria lobata permeate is concentrated, the alcohol content of the concentrate is adjusted with 95% ethanol for alcohol precipitation, and the precipitate is collected by centrifugation to obtain pueraria lobata polysaccharide; Step 6, drying: drying the obtained kudzu vine polysaccharide to obtain an alcohol-soluble kudzu vine polysaccharide extract.

2. The method for preparing an alcohol-soluble Puerariae polysaccharide extract according to claim 1, characterized in that: In the step 1, the mesh size of the sieve is 30-50 meshes.

3. The method for preparing an alcohol-soluble Puerariae polysaccharide extract according to claim 1, characterized in that: In the step 1, the extraction temperature is 80-95° C., the number of extractions is 1-3 times, the extraction solvent multiple is 15-25 times, and the extraction time is 1.5-2.5 h.

4. The method for preparing an alcohol-soluble kudzu vine polysaccharide extract according to claim 1, characterized in that: In the step 3, the macroporous resin used for decolorization is ADS-17 macroporous resin or D941 macroporous resin, and the permeate flow rate is 1-3 BV / h.

5. The method for preparing an alcohol-soluble Puerariae polysaccharide extract according to claim 1, characterized in that: In the step 4, the enzymolysis temperature is 45-60°C and the enzymolysis time is 1-3 h.

6. The method for preparing an alcohol-soluble Puerariae polysaccharide extract according to claim 1, characterized in that: In the step 5, the protein-removed pueraria lobata permeate is concentrated to 1 / 45-1 / 55 of the original volume.

7. The method for preparing an alcohol-soluble Puerariae polysaccharide extract according to claim 1, characterized in that: In the step 5, 95% ethanol is used to adjust the alcohol content of the concentrated solution to 60-80%, the centrifugal speed is 5000-7000 r / min, and the centrifugal time is 10-30 min.

8. The method for preparing an alcohol-soluble Puerariae polysaccharide extract according to claim 1, characterized in that: In step six, the drying method is vacuum freeze drying or low-temperature vacuum drying.

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