Method for preparing high-purity sapindoside powder

Abstract

The invention discloses a method for preparing high-purity sapindoside powder, and belongs to the technical field of forest chemical industry. The method provided by the invention comprises the following steps: extracting saponin in soapberry pericarp by using a leaching solution containing a mixed enzyme, Mg < 2 + > and a phosphate buffer system, performing ultrafiltration by using an ultrafiltration membrane with the molecular weight cutoff of 6000-10000 Daltons, concentrating by using an ultrafiltration membrane with the molecular weight cutoff of 200-600 Daltons, and drying, the soapberry peel solid powder with the saponin purity reaching 90% or above can be obtained.

Description

Technical Field

[0001] The invention belongs to the technical field of forest product chemical industry, and particularly relates to a method for preparing high-purity soapberry saponin powder. Background Art

[0002] Sapindus mukorossi Gaertn. is a deciduous tree species of the Sapindaceae family. The saponin content in its peel is as high as 10.8%. It is a non-ionic surfactant with strong surface activity. Due to its good detergency and foaming properties and its non-toxicity to the human body, soapberry is an important biological detergent raw material species.

[0003] Currently, there are mainly the following methods for extracting saponins from soapberry peel: 1) aqueous two-phase extraction method, but the two-phase aqueous system of this method is not stable, the added reagents are complex, and the operation is cumbersome; 2) ethanol extraction-n-butanol extraction method, which obtains soapberry saponins and sapogenin crystals, but the required solvent dosage is large, the process is complex, and the product purity is not high; 3) water extraction or dilute alcohol extraction combined with macroporous resin, which has high solvent consumption and difficult impurity removal and concentration processes; 4) resin separation method, in which the resin is difficult to parse and the regeneration process is cumbersome. Patent document CN111423485A discloses a method for extracting saponins from soapberry peel by filtering out macromolecules using an ultrafiltration membrane with a molecular weight cutoff of 6,000 to 10,000 daltons, and then concentrating and extracting saponins using nanofiltration with a molecular weight cutoff of 200 to 600 daltons. This method uses distilled water for water immersion, avoiding the use of organic solvents such as ethanol; and only uses filter membranes with different molecular weight cutoffs for ultrafiltration and concentration, which is convenient to operate and avoids the use of resin materials that are difficult to analyze and have a cumbersome regeneration process. However, the purity of the saponins in the soapberry saponin concentrate or soapberry saponin solid obtained by the method disclosed in this document is low, only about 70%, which limits the large-scale promotion and use of this method. Patent document CN102617695A discloses a production process for soapberry saponins, the purity of the product obtained can reach 90%, but this method involves supercritical extraction, nanofiltration, spray drying and other technologies, which will bring inconvenience to industrial production. Moreover, the method still requires the use of resin materials with a cumbersome regeneration process, which is relatively costly. Summary of the Invention

[0004] In view of the problems existing in the prior art, the present invention provides a method for preparing high-purity soapberry saponin powder, which comprises the following steps:

[0005] 1) Preparation of extract: Add the mixed enzyme powder to the 2+ phosphate buffer solution to obtain an extract, wherein the mixed enzyme powder consists of cellulase, amylase and pectinase;

[0006] 2) Extraction: extracting saponins from the soapberry peel using the extract obtained in step 1), filtering, and obtaining a saponin extract;

[0007] 3) Ultrafiltration: Ultrafiltration of the saponin extract using an ultrafiltration membrane with a molecular weight cutoff of 6,000 to 10,000 Daltons to obtain an ultrafiltrate;

[0008] 4) Concentrating: using a filter membrane with a molecular weight cut-off of 200 to 600 Daltons to concentrate the ultrafiltrate to obtain a soapberry peel saponin concentrate; and

[0009] 5) drying the soapberry peel saponin concentrate to obtain soapberry peel saponin solid powder.

[0010] In some embodiments, the dosage relationship of cellulase, amylase and pectinase in the mixed enzyme powder is: cellulase: amylase: pectinase = 1: (0.5-1.0): (0.5-1.0), and the total mass volume concentration of the enzyme in the extract is 0.01-0.05 g / 100 ml.

[0011] In some embodiments, the molecular weight of the cellulase, amylase and pectinase are all greater than 10,000 Daltons, and the activity of the cellulase is ≥10,000 U / g, the activity of the pectinase is ≥30,000 U / g, and the activity of the amylase is ≥10,000 U / g.

[0012] In some embodiments, the pH value of the phosphate buffer solution in step 1) is 4.5-5.5.

[0013] In some embodiments, the phosphate buffer solution in step 1) contains 0.5 mM to 1.0 mM Mg 2+ .

[0014] In some embodiments, step 2) specifically includes the following operations:

[0015] S1) drying the soapberry peel and then crushing it into 100-150 meshes to obtain soapberry peel powder; and

[0016] S2) extracting saponins from the crushed soapberry peel powder with the extracting solution at 40° C. to 50° C. for 2 to 3 hours; wherein the mass volume ratio of the soapberry peel powder to the extracting solution is 5 to 10 g / 100 ml.

[0017] In some embodiments, the filtration in step 2) is performed using a 200-300 mesh sieve.

[0018] In some embodiments, the concentration in step 4) is to concentrate the soapberry peel saponin concentrate by 5 to 10 times.

[0019] In some embodiments, the specific operations of step 5) include:

[0020] M1) cooling the freezer containing the soapberry peel saponin concentrate to -15°C within 1 hour and pre-freezing for 1 hour;

[0021] M2) After the exotherm ends, the temperature is lowered to -30°C within 1 hour, and then to -45°C within 1 hour, and the pressure in the freezing chamber is controlled to be below 10 Pa and maintained for 1 hour to freeze;

[0022] M3) Raise the temperature of the freezing chamber to -4°C, maintain the vacuum below 10 Pa, and keep it warm for 4 hours to perform preliminary sublimation;

[0023] M4) adjusting the vacuum degree to 50 Pa and keeping at -4°C for 8 hours for post-sublimation; and

[0024] M5) After sublimation, adjust the temperature to 28-34°C and the vacuum to 25 Pa and perform desorption until completely dry.

[0025] The method for preparing high-purity soapberry saponin powder based on the above technical solution first uses a mixture of enzymes (composed of cellulase, pectinase and amylase), Mg 2+ The saponins in the soapberry peel are extracted with an extract of a phosphate buffer solution. This step can significantly improve the saponin extraction rate in the soapberry peel. The saponin extract obtained by the extraction is then ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 6,000 to 10,000 Daltons. This step can remove large molecular weight enzyme molecules in the saponin extract. The ultrafiltrate from which the enzyme molecules have been removed is then concentrated using an ultrafiltration membrane with a molecular weight cutoff of 200 to 600 Daltons to obtain a soapberry peel saponin concentrate. This step can effectively remove phosphate and Mg. 2+ The results of the embodiment show that the use of the enzyme mixture, Mg 2+ When extracting saponins from soapberry peel with the extract of phosphate buffer solution, the enzymes, Mg 2+ The method can work synergistically with the phosphate buffer system to significantly improve the extraction rate of saponins from the soapberry peel (the saponin extraction rate can be as high as over 90%). After subsequent ultrafiltration, concentration and drying, the purity of the saponins in the obtained solid powder is also as high as over 90%. In addition, the method provided by the present invention completely avoids the use of organic solvents and resin materials with cumbersome regeneration processes, and can be effectively used in the extraction of saponins from the soapberry peel. DETAILED DESCRIPTION

[0026] The present invention will be further described below with reference to specific examples. It should be understood that the specific examples are only used to further illustrate the present invention, rather than to limit the present invention.

[0027] The methods for obtaining the various materials or reagents described in the examples are merely provided as experimental means to achieve the disclosed objectives and should not be construed as limiting the sources of the materials or reagents used in the present invention. In fact, the sources of the materials or reagents used are diverse, and any legally and ethically accessible materials or reagents may be substituted for and used as indicated in the examples.

[0028] Example 1

[0029] 1) Preparation of extract: Add equal amounts of cellulase (purchased from Sinopharm Chemical Reagent Co., Ltd., activity ≥ 10,000 U / g, molecular weight greater than 10,000 Dalton), amylase (purchased from Sinopharm Chemical Reagent Co., Ltd., activity ≥ 10,000 U / g, molecular weight greater than 10,000 Dalton) and pectinase (purchased from Sinopharm Chemical Reagent Co., Ltd., activity ≥ 30,000 U / g, molecular weight greater than 10,000 Dalton) to a sieve containing Mg. 2+ Phosphate buffer solution (take 0.2 mol / L sodium dihydrogen phosphate solution and adjust the pH value to 5.0 with sodium hydroxide solution, add MgCl2 solid to make Mg 2+ The concentration of the enzyme was 0.5 mM) so that the total mass volume content of the enzyme was 0.01 g / 100 ml to prepare an extract;

[0030] 2) Extraction: Dry the soapberry peel and grind it through a 100-mesh sieve to obtain soapberry peel powder; add 500 g of the soapberry peel powder to 10 L of the extract from step 1), extract at 45° C. for 2.5 hours, and filter through a 200-mesh sieve to obtain a saponin extract;

[0031] 3) Ultrafiltration: Ultrafiltration of the saponin extract using an ultrafiltration membrane with a molecular weight cutoff of 8000 Daltons to obtain an ultrafiltrate;

[0032] 4) Concentration: Using an ultrafiltration membrane with a molecular weight cut-off of 300 Daltons, the ultrafiltrate was concentrated 8-fold to obtain a soapberry peel saponin concentrate;

[0033] 5) drying the soapberry peel saponin concentrate to obtain soapberry peel saponin solid powder; wherein the specific drying operation is:

[0034] M1) cooling the freezer containing the soapberry peel saponin concentrate to -15°C within 1 hour and pre-freezing for 1 hour;

[0035] M2) After the exotherm ends, the temperature is lowered to -30°C within 1 hour, and then to -45°C within 1 hour, and the pressure in the freezing chamber is controlled to be below 10 Pa and maintained for 1 hour to freeze;

[0036] M3) Raise the temperature of the freezing chamber to -4°C, maintain the vacuum below 10 Pa, and keep it warm for 4 hours to perform preliminary sublimation;

[0037] M4) Adjust the vacuum degree to 50 Pa and keep at -4°C for 8 hours for post-sublimation;

[0038] M5) After sublimation, adjust the temperature to 28-34°C and the vacuum to 25 Pa and perform desorption until completely dry.

[0039] Examples 2-6

[0040] Examples 2-6 were carried out according to the procedure of Example 1, except that:

[0041] The mass ratio of cellulase, amylase and pectinase in the extract prepared in Example 2 is: 1:0.5:0.5.

[0042] Mg in the phosphate buffer solution used in Example 3 2+ The concentration is 1.0 mM.

[0043] The total mass volume content of the enzyme in the extract prepared in Example 4 was 0.05 g / 100 ml.

[0044] In Example 5, in step 3), an ultrafiltration membrane with a molecular weight cutoff of 6000 Daltons is selected to ultrafilter the saponin extract to obtain an ultrafiltrate, and in step 4), an ultrafiltration membrane with a molecular weight cutoff of 600 Daltons is selected to concentrate the ultrafiltrate 5 times to obtain a soapberry peel saponin concentrate.

[0045] In Example 6, in step 3), an ultrafiltration membrane with a molecular weight cutoff of 10,000 Daltons is selected to ultrafilter the saponin extract to obtain an ultrafiltrate, and in step 4), an ultrafiltration membrane with a molecular weight cutoff of 200 Daltons is selected to concentrate the ultrafiltrate 10 times to obtain a soapberry peel saponin concentrate.

[0046] Comparative Examples 1-9

[0047] Comparative Examples 1-9 were carried out according to the procedure of Example 1, except that:

[0048] The extract prepared in Comparative Example 1 contains cellulase but does not contain amylase and pectinase.

[0049] The extract prepared in Comparative Example 2 contained equal amounts of cellulase and pectinase, but did not contain amylase.

[0050] The extract prepared in Comparative Example 3 contained equal amounts of cellulase and amylase, but no pectinase.

[0051] The extract prepared in Comparative Example 4 contained equal amounts of amylase and pectinase, but no cellulase.

[0052] The mass ratio of cellulase, amylase and pectinase in the extract prepared in Comparative Example 5 is 1:1.5:1.5.

[0053] The phosphate buffer solution used in Comparative Example 6 does not contain Mg 2+ .

[0054] The phosphate buffer solution used in Comparative Example 7 contained Mg 2+ The concentration is 2.0 mM.

[0055] The phosphate buffer solution used in Comparative Example 8 contained Mg 2+ The concentration is 0.1 mM.

[0056] The leaching solution prepared in Comparative Example 9 does not contain phosphate, so that the Mg content in the final leaching solution is 2+ The concentration is 0.5 mM.

[0057] Comparative Example 10

[0058] 1) Comparative Example 10 used the extract prepared in Example 1;

[0059] 2) drying the soapberry peel and grinding it through a 100-mesh sieve to obtain soapberry peel powder; adding 500 g of the soapberry peel powder to 10 L of the extract from step 1), extracting at 45° C. for 2.5 hours, filtering through a 200-mesh sieve, and centrifuging at 8000 rpm for 10 minutes to obtain a saponin extract;

[0060] 3) Extraction was performed twice with the volume ratio of extraction solvent (n-butanol) to saponin extract being 1:1. The extracts were combined, evaporated to dryness, and freeze-dried to obtain a solid powder of saponin from the peel of soapberry fruit.

[0061] The mass of the soapberry peel saponin solid powder obtained in Examples 1-6 and Comparative Examples 1-10 was measured, and the saponin purity thereof was detected by UV (the reference substance used was sapindoside B isolated from soapberry). The results are shown in Table 1 below. The saponin extraction rate of each example and comparative example was calculated based on the soapberry peel saponin content of 10.8%, and the results are shown in Table 1 below.

[0062] Table 1: Saponin purity and extraction rate of soapberry peel saponin solid powder obtained in Examples 1-6 and Comparative Examples 1-10

[0063]

[0064]

[0065] Based on the results of Examples 1-6 and Comparative Examples 1-10, it can be seen that when using a mixture of enzyme powder (cellulase, amylase and pectinase), an appropriate concentration of Mg 2+ When extracting saponins from soapberry peel with the extract of phosphate buffer solution, the mixed enzyme powder, Mg 2+ The phosphate buffer system can play a synergistic role (this is different from the report of Wei Fengyu et al. (Study on the process of extracting saponins by enzymatic method, Applied Chemical Industry, Vol. 39, No. 8, August 2010), which believes that in the extraction process of saponins, the effect of amylase is not obvious, and pectinase has an inhibitory effect on saponin extraction. In the method of the present invention, by using a mixture containing Mg 2+ When the phosphate buffer solution was used to prepare the extract containing the enzyme, the system containing the above three enzymes had a significantly better extraction effect, which may be attributed to the Mg content in the extract. 2+ The presence of a phosphate buffer system) allows more saponins in the soapberry peel to be extracted, and then membrane ultrafiltration and membrane concentration are used to significantly improve the extraction rate of soapberry peel saponins and the purity of the saponins in the obtained solid powder, and can effectively remove the enzymes and phosphates used in the extraction process as well as Mg 2+ Ingredients. The results of the examples show that the saponin extraction rate can be as high as over 90%, and the purity of the saponins in the obtained solid powder is also as high as over 90%. In addition, the present invention combines the enzymatic extraction method of soapberry peel saponins with membrane ultrafiltration and membrane concentration processes, which can avoid the use of organic solvents (such as ethanol, n-butanol, etc.) and avoid the use of resin materials with cumbersome regeneration processes. It is an environmentally friendly, efficient and low-cost method.

[0066] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for preparing high-purity soapberry saponin powder, characterized in that: The following steps are involved: 1) Preparation of extract: Add the mixed enzyme powder to the 2+ phosphate buffer solution to obtain an extract, wherein the mixed enzyme powder consists of cellulase, amylase and pectinase; 2) Extraction: extracting saponins from the soapberry peel using the extract obtained in step 1), filtering, and obtaining a saponin extract; 3) Ultrafiltration: Ultrafiltration of the saponin extract using an ultrafiltration membrane with a molecular weight cutoff of 6,000 to 10,000 Daltons to obtain an ultrafiltrate; 4) Concentrating: using a filter membrane with a molecular weight cut-off of 200 to 600 Daltons to concentrate the ultrafiltrate to obtain a soapberry peel saponin concentrate; and 5) drying the soapberry peel saponin concentrate to obtain soapberry peel saponin solid powder.

2. The method according to claim 1, characterized in that The dosage relationship of cellulase, amylase and pectinase in the mixed enzyme powder is: cellulase:amylase:pectinase=1:(0.5-1.0):(0.5-1.0), and the total mass volume concentration of the enzyme in the extract is 0.01-0.05g / 100ml.

3. The method according to claim 1 or 2, characterized in that The molecular weights of the cellulase, amylase and pectinase are all greater than 10,000 Daltons, and the activity of the cellulase is ≥10,000 U / g, the activity of the pectinase is ≥30,000 U / g, and the activity of the amylase is ≥10,000 U / g.

4. The method according to any one of claims 1 to 3, characterized in that The pH value of the phosphate buffer solution in step 1) is 4.5-5.

5.

5. The method according to any one of claims 1 to 4, characterized in that The phosphate buffer solution in step 1) contains 0.5mM to 1.0mM Mg 2+ .

6. The method according to any one of claims 1 to 5, characterized in that Step 2) specifically includes the following operations: S1) drying the soapberry peel and then crushing it into 100-150 meshes to obtain soapberry peel powder; and S2) extracting saponins from the crushed soapberry peel powder with the extracting solution at 40° C. to 50° C. for 2 to 3 hours; wherein the mass volume ratio of the soapberry peel powder to the extracting solution is 5 to 10 g / 100 ml.

7. The method according to any one of claims 1 to 6, characterized in that The filtration in step 2) is performed using a 200-300 mesh sieve.

8. The method according to any one of claims 1 to 7, characterized in that The concentration in step 4) is to concentrate the soapberry peel saponin concentrate by 5 to 10 times.

9. The method according to any one of claims 1 to 8, characterized in that The specific operations of step 5) include: M1) cooling the freezer containing the soapberry peel saponin concentrate to -15°C within 1 hour and pre-freezing for 1 hour; M2) After the exotherm ends, the temperature is lowered to -30°C within 1 hour, and then to -45°C within 1 hour, and the pressure in the freezing chamber is controlled to be below 10 Pa and maintained for 1 hour to freeze; M3) Raise the temperature of the freezing chamber to -4°C, maintain the vacuum below 10 Pa, and keep it warm for 4 hours to perform preliminary sublimation; M4) adjusting the vacuum degree to 50 Pa and keeping at -4°C for 8 hours for post-sublimation; and M5) After sublimation, adjust the temperature to 28-34°C and the vacuum to 25 Pa and perform desorption until completely dry.

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