A recombinant saponin and its preparation method

Abstract

This invention relates to the field of natural product separation and recombination, and particularly to a recombinant saponin from *Gleditsia sinensis* and its preparation method. The method includes: 1) crude saponin extraction: water extraction of *Gleditsia sinensis* pericarp to obtain a crude saponin solution; 2) impurity removal: loading the crude saponin aqueous solution into macroporous adsorption resin AB-8 and eluting to remove impurities; 3) saponin component separation: eluting sequentially with 25-35% ethanol solution, 40-50% ethanol solution, 55-65% ethanol solution, and 70-80% ethanol solution to obtain S30, S45, S60, and S75 saponin components, respectively; 4) saponin hydrolysis: hydrolyzing the S45 saponin component to obtain hydrolyzed saponin; 5) saponin component recombination: recombining the saponin components obtained in steps 3) and 4) with the hydrolyzed saponin to obtain recombinant saponin from *Gleditsia sinensis*. This invention enables saponin purification and improves the surface activity of *Gleditsia sinensis*.

Description

Technical Field

[0001] This invention relates to the field of natural product separation and recombination, and in particular to a recombinant saponin and its preparation method. Background Technology

[0002] The Chinese honey locust (Gleditsia sinensis), belonging to the genus Gleditsia of the legume family, is a typical ecologically and economically useful tree species, mainly distributed in Central Asia, Southeast Asia, North America, and South America. It is widely cultivated in Shanxi, Henan, Shandong, Guizhou, and Yunnan provinces of my country. The endosperm of the Chinese honey locust seed is rich in galactomannan. Due to its high carbohydrate content and low protein and fat content, Chinese honey locust polysaccharides have high economic and nutritional value. Furthermore, the structure and function of Chinese honey locust galactomannan are similar to guar gum and locust gum polysaccharides, all being non-traditional industrial polysaccharide gums. Chinese honey locust galactomannan is commonly used as a thickener, adhesive, stabilizer, and flocculant, and is widely applied in various industries such as food, daily chemicals, pharmaceuticals, oil drilling, and printing.

[0003] While utilizing the galactomannan of Gleditsia sinensis, we discovered that the pericarp of Gleditsia sinensis contains a large amount of natural saponins. The saponin structure contains hydrophilic, highly polar groups such as glycosomes, ligands, and organic acids, while the aglycone structure is relatively large, low polarity, and lipophilic. It exhibits structural characteristics similar to surfactants, and its aqueous solution, when shaken, produces a persistent, soap-like foam. It is a nonionic natural surfactant with excellent detergency. However, the production of natural saponins still faces challenges such as difficulty in removing impurities, high purification costs, instability of some saponin structures, and the need for further improvement in saponin surface activity. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a recombinant saponin and its preparation method.

[0005] In a first aspect, the present invention provides a method for preparing recombinant saponins from saponins, comprising:

[0006] 1) Extraction of crude saponin from soapberry: The pericarp of soapberry is extracted with water to obtain a crude saponin solution;

[0007] 2) Impurity removal: The crude saponin aqueous solution is loaded into macroporous adsorption resin AB-8 and impurities are removed by elution;

[0008] 3) Separation of saponin components: Elution was performed sequentially with 25-35% ethanol solution, 40-50% ethanol solution, 55-65% ethanol solution and 70-80% ethanol solution to obtain saponin components of saponin from saponin plants of S30, S45, S60 and S75 respectively;

[0009] 4) Saponin hydrolysis: The saponin component of S45 soapberry is hydrolyzed to obtain hydrolyzed saponin;

[0010] 5) Recombination of saponin components: The saponin components and hydrolyzed saponins obtained in steps 3) and 4) are recombined to obtain recombined saponin.

[0011] This invention first uses water extraction to prepare crude saponin from natural saponins. Then, the crude saponin aqueous solution is passed through macroporous adsorption resin AB-8 to remove impurities and separate the saponin components. The separated saponin S45 is hydrolyzed and optimized and recombined with other saponin components, thereby purifying the saponin and improving its surface activity.

[0012] Preferably, in step 1), deionized water is used as the extractant, and the solid-liquid ratio is preferably 1:6 to 10; more preferably, the soapberry peel is crushed and passed through a 30 to 50 mesh sieve, deionized water is used as the extractant, the solid-liquid ratio is 1:6 to 8, the mixture is stirred and extracted for 5 to 8 hours, and then filtered to obtain a crude soapberry saponin aqueous solution.

[0013] Further optimization, in step 1), crude saponin extraction: the soapberry peel is crushed and passed through a 40-mesh sieve, deionized water is used as the extraction solvent, the solid-liquid ratio is 1:7-8, the mixture is stirred and extracted for 6-7 hours, and then filtered to obtain an aqueous solution of crude soapberry saponin.

[0014] In this invention, the above-mentioned water extraction method is used to treat the pericarp of Gleditsia sinensis to extract crude saponin and prepare an aqueous solution of crude Gleditsia sinensis saponin. This method not only has low extraction cost but also high saponin extraction yield.

[0015] Preferably, in step 2), the solution is eluted sequentially with deionized water and 5-15% ethanol solution; more preferably, the crude saponin solution is loaded into macroporous adsorption resin AB-8 and eluted sequentially with 2-3 times the resin volume of deionized water and 2-3 times the resin volume of 5-15% ethanol solution to remove impurities from the saponin.

[0016] Further optimization, in step 2), impurity removal: the macroporous adsorption resin AB-8 is wet-packed, the crude saponin aqueous solution is packed into the resin separation column, and the impurities in the saponin are removed by elution with 2 times the resin volume of deionized water and 2 times the resin volume of 10% ethanol solution.

[0017] In this invention, research has shown that using a specific macroporous adsorption resin, AB-8, is beneficial for saponin purification. Elution is performed sequentially with a preferred amount of deionized water and an ethanol solution of the specified concentration. The impurities in the saponin are removed to the greatest extent possible by using the macroporous adsorption resin with deionized water and a 10% ethanol aqueous solution as eluents.

[0018] Preferably, in step 3), elution is performed using 30-35% ethanol solution, 45-50% ethanol solution, 60-65% ethanol solution, or 75-80% ethanol solution; preferably, the 30-35% ethanol solution is 3-8 times the resin volume, the 45-50% ethanol solution is 8-12 times the resin volume, the 60-65% ethanol solution is 3-8 times the resin volume, and the 60-65% ethanol solution is 2-3 times the resin volume.

[0019] In this invention, after the crude saponin extraction and impurity removal of saponins described above, optimizing the eluent and elution parameters facilitates the acquisition of saponin components with better purity and performance. Furthermore, the macroporous adsorption resin, eluted sequentially with ethanol aqueous solutions of different concentrations, allows for direct separation of the next batch of saponins without regeneration. Moreover, using 30%, 45%, 60%, and 75% ethanol aqueous solutions as eluents on the macroporous adsorption resin can yield specific purified saponins with different structures and properties, which is beneficial for improving the surface activity and product purity of the recombinant saponins.

[0020] Further preferred, in step 3), the saponin components are separated by eluting with 5 times the resin volume of 30% ethanol solution, 10 times the resin volume of 45% ethanol solution, 5 times the resin volume of 60% ethanol solution, and 2 times the resin volume of 75% ethanol solution, respectively, to obtain saponin components S30, S45, S60 and S75.

[0021] Preferably, in step 4), the S45 saponin component is prepared into an aqueous solution with a concentration of 15-25%, the pH is adjusted to 1.5-2.5, and then hydrolyzed at a temperature of 75-85°C.

[0022] Further preferred, in step 4), saponin hydrolysis: the saponin component of S45 is prepared into an aqueous solution with a concentration of 20%, the pH is adjusted to 2.0 with hydrochloric acid, heated to 80°C, kept warm for 2 hours for hydrolysis, neutralized, concentrated and dried to obtain hydrolyzed saponin.

[0023] This invention employs a preferred method for saponin hydrolysis. Partial acid hydrolysis of S45 saponin can reduce the surface tension and critical micelle concentration of saponin, thereby enhancing its synergistic effect with other saponin components. Under preferred hydrolysis conditions (30% hydrolysis rate of structural sugars in saponin), this invention further improves the surface activity of saponin and enhances the stability of saponin components and saponin recombinant complexes.

[0024] Preferably, in step 5), the saponin components are recombined: the saponin components S30, S45, hydrolyzed saponin, S60, and S75 are recombined in proportion.

[0025] This invention improves the surface activity of recombinant saponins by fully leveraging synergistic effects without altering the basic structure of saponins. The complexes of specific saponin components in this invention exhibit superior surface activity.

[0026] Further preferred, step 5) includes adjusting the ratio of the saponin component and the hydrolyzed saponin to make the critical micelle concentration and surface tension of the recombinant saponin adjustable.

[0027] Secondly, the recombinant saponin provided by the present invention is obtained by the preparation method of the recombinant saponin.

[0028] Preferably, the recombinant saponin is a complex of one or more of the following: S30 saponin component, S45 saponin component, hydrolyzed saponin, S60 saponin component, and S75 saponin component; preferably, the mass ratio of S30 saponin component, hydrolyzed saponin, S60 saponin component, and S75 saponin component is 1-2:15-20:15-20:1.5-2.

[0029] Further preferably, the surface tension of the recombinant saponin is 30-35 mN / m, and the critical micelle concentration is 0.08-0.12 g / L.

[0030] The beneficial effects of this invention are at least as follows: the recombinant saponins provided by this invention have higher surface activity and product purity. 1) Crude saponins are extracted using water extraction, resulting in low extraction cost and high saponin extraction yield. 2) Impurities in the saponins are removed to the greatest extent by using macroporous adsorption resin with deionized water and 10% ethanol aqueous solution as eluents. 3) Purified saponins with different structures and properties can be obtained by using macroporous adsorption resin with 30%, 45%, 60%, and 75% ethanol aqueous solutions as eluents. 4) Partial acid hydrolysis of S45 saponins can reduce the surface tension and critical micelle concentration of saponins, improve the surface activity of saponins, and enhance their synergistic effect with other saponin components. 5) Sequential elution with macroporous adsorption resins of different concentrations of ethanol aqueous solutions allows for direct separation of the next batch of saponins without regeneration. 6) The surface activity of the complex of different saponin components is greater than that of a single saponin component. 7) The surface activity of the recombinant saponins is improved by fully utilizing the synergistic effect without changing the basic structure of the saponins. 8) Partial hydrolysis can improve the stability of saponin components and saponin recombinant complexes. Attached Figure Description

[0031] To more clearly illustrate the embodiments of the present invention and the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 This is a process flow diagram provided for Embodiment 1 of the present invention.

[0033] Figure 2 The saponin graded products obtained by separating different concentrations of ethanol eluent provided in Example 1 of the present invention are saponin graded products. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of the embodiments of the invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0035] Unless otherwise stated, all raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods. Where specific techniques or conditions are not specified in the examples, they are performed using conventional methods or in accordance with techniques or conditions described in the literature in this field, or according to the product instructions. Reagents and instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0036] The present invention will be further described below with reference to embodiments.

[0037] Example 1

[0038] This embodiment provides a recombinant saponin, the preparation process of which is as follows: Figure 1 As shown. The preparation method is as follows: The dried soapberry pericarp is crushed and passed through a 40-mesh sieve. Deionized water is used as the extraction solvent, the solid-liquid ratio is 1:8, and the mixture is stirred and extracted for 6 hours. The crude soapberry saponin aqueous solution is obtained by filtration. A certain amount of macroporous adsorption resin AB-8 is weighed and wet-packed into a column. The crude soapberry saponin aqueous solution is packed into the resin separation column and eluted sequentially with 2 times the volume (resin volume, the same below) of deionized water and 10% ethanol solution to remove impurities in the saponin. The soapberry saponin components S30, S45, S60 and S75 are obtained by elution sequentially with 30% ethanol solution (5 times the volume), 45% ethanol solution (10 times the volume), 60% ethanol solution (5 times the volume), and 75% ethanol solution (2 times the volume) to concentrate and dry them respectively. Figure 2The separated S45 saponin was prepared into a 20% aqueous solution, adjusted to pH 2.0 with hydrochloric acid, heated to 80℃, and hydrolyzed for 2 hours. After neutralization, the solution was concentrated and dried to obtain hydrolyzed saponin S45 (the hydrolysis rate of structural sugars in the saponin was 30%). Saponin was reconstituted at a mass ratio of S30:hydrolyzed S45:S60:S75 = 2:20:20:1.5. The critical micelle concentration of the reconstituted saponin was measured to be 0.08 g / L, and the surface tension was 30.7 mN / m.

[0039] Example 2

[0040] The dried soapberry pericarps were pulverized and passed through a 40-mesh sieve. Using deionized water as the extraction solvent, at a solid-liquid ratio of 1:8, extraction was carried out with stirring for 6 hours. The mixture was then filtered to obtain a crude soapberry saponin aqueous solution. A certain amount of macroporous adsorption resin AB-8 was weighed and wet-packed into a column. The crude soapberry saponin aqueous solution was packed into the resin separation column, and eluted sequentially with 2 volumes of deionized water and 10% ethanol solution to remove impurities from the saponins. Elution was then carried out sequentially with 30% ethanol solution (5 volumes), 45% ethanol solution (10 volumes), 60% ethanol solution (5 volumes), and 75% ethanol solution (2 volumes) to obtain soapberry saponin components S30, S45, S60, and S75, which were then concentrated and dried. The separated S45 saponin was prepared into a 20% aqueous solution, adjusted to pH 2.0 with hydrochloric acid, heated to 80℃, and hydrolyzed for 2 hours. After neutralization, the solution was concentrated and dried to obtain saponin hydrolysis S45. Saponins were recombined at a mass ratio of S30:hydrolyzed S45:S60:S75 = 2:15:20:1.5. The critical micelle concentration of the recombinant saponins was measured to be 0.08 g / L, and the surface tension was 33.6 mN / m.

[0041] Example 3

[0042] The dried soapberry pericarps were pulverized and passed through a 40-mesh sieve. Using deionized water as the extraction solvent, at a solid-liquid ratio of 1:8, extraction was carried out with stirring for 6 hours. The mixture was then filtered to obtain a crude soapberry saponin aqueous solution. A certain amount of macroporous adsorption resin AB-8 was weighed and wet-packed into a column. The crude soapberry saponin aqueous solution was packed into the resin separation column, and eluted sequentially with 2 volumes of deionized water and 10% ethanol solution to remove impurities from the saponins. Elution was then carried out sequentially with 30% ethanol solution (5 volumes), 45% ethanol solution (10 volumes), 60% ethanol solution (5 volumes), and 75% ethanol solution (2 volumes) to obtain soapberry saponin components S30, S45, S60, and S75, which were then concentrated and dried. The separated S45 saponin was prepared into a 20% aqueous solution, adjusted to pH 2.0 with hydrochloric acid, heated to 80℃, and hydrolyzed for 2 hours. After neutralization, the solution was concentrated and dried to obtain saponin hydrolysis S45. The critical micelle concentration of saponin hydrolysis S45 was measured to be 0.2 g / L, and the surface tension was 42.5 mN / m.

[0043] Example 4

[0044] The dried soapberry pericarp was pulverized and passed through a 40-mesh sieve. Deionized water was used as the extraction solvent, and the solid-liquid ratio was 1:8. Extraction was carried out with stirring for 6 hours, followed by filtration to obtain a crude soapberry saponin aqueous solution. A certain amount of macroporous adsorption resin AB-8 was weighed and wet-packed into a column. The crude soapberry saponin aqueous solution was packed into the resin separation column, and eluted sequentially with 2 volumes of deionized water and 10% ethanol solution to remove impurities from the saponin. Elution was then performed sequentially with 30% ethanol solution (5 volumes), 45% ethanol solution (10 volumes), 60% ethanol solution (5 volumes), and 75% ethanol solution (2 volumes) to obtain soapberry saponin components S30, S45, S60, and S75, which were then concentrated and dried. Reconstituted saponins were then carried out at a mass ratio of S30:S45:S60:S75 = 2:31:20:1.5. The critical micelle concentration of the reconstituted saponins was measured to be 0.15 g / L, and the surface tension was 37.2 mN / m.

[0045] Comparative Example 1

[0046] The dried soapberry pericarps were pulverized and passed through a 40-mesh sieve. Deionized water was used as the extraction solvent, with a solid-liquid ratio of 1:8, and the mixture was stirred and extracted for 6 hours. The resulting crude soapberry saponin aqueous solution was obtained by filtration. A certain amount of macroporous adsorption resin AB-8 was weighed and wet-packed into a column. The crude soapberry saponin aqueous solution was then packed into the resin separation column, and eluted sequentially with 2 volumes of deionized water and 10% ethanol solution to remove impurities from the saponin. Elution was then performed sequentially with 30% ethanol solution (5 volumes), 45% ethanol solution (10 volumes), 60% ethanol solution (5 volumes), and 75% ethanol solution (2 volumes) to obtain soapberry saponin components S30, S45, S60, and S75, which were then concentrated and dried. The critical micelle concentration and surface tension of soapberry saponin components S30, S45, S60, and S75 were determined, and the results are shown in the table below.

[0047] Table 1. Surface tension and critical micelle concentration of different saponin components and complexes from saponins.

[0048]

[0049] Comparative Example 2

[0050] The dried soapberry pericarp was crushed and passed through a 40-mesh sieve. Deionized water was used as the extraction solvent, and the solid-liquid ratio was 1:8. The mixture was stirred and extracted for 6 hours. After filtration, a crude soapberry saponin aqueous solution was obtained. The critical micelle concentration of crude soapberry saponin was determined to be 0.25 g / L, and the surface tension was 42.5 mN / m.

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing recombinant saponin, characterized in that, include: 1) Extraction of crude saponin from soapberry: The pericarp of soapberry is extracted with water to obtain a crude saponin solution; 2) Impurity removal: The crude saponin solution is loaded into macroporous adsorption resin AB-8 and impurities are removed by elution; 3) Separation of saponin components: Elution was performed sequentially with 25-35% ethanol solution, 40-50% ethanol solution, 55-65% ethanol solution and 70-80% ethanol solution to obtain saponin components of saponin from saponin plants of S30, S45, S60 and S75 respectively; 4) Saponin hydrolysis: The saponin component of S45 soapberry is hydrolyzed to obtain hydrolyzed saponin; 5) Recombination of saponin components: The saponin components and hydrolyzed saponins obtained in steps 3) and 4) are recombined to obtain recombined saponin; the saponin components are selected from one or more of the following: S30 saponin components, S45 saponin components, S60 saponin components and S75 saponin components.

2. The method for preparing recombinant saponin according to claim 1, characterized in that, In step 1), deionized water is used as the extractant, and the solid-liquid ratio is 1:6~10.

3. The method for preparing recombinant saponin according to claim 2, characterized in that, In step 1), the soapberry peel is crushed and passed through a 30-50 mesh sieve. Deionized water is used as the extraction solvent, the solid-liquid ratio is 1:6-8, the mixture is stirred and extracted, filtered, and crude soapberry saponin aqueous solution is obtained.

4. The method for preparing recombinant saponin according to claim 1, characterized in that, In step 2), the solution is eluted sequentially with deionized water and 5-15% ethanol solution.

5. The method for preparing recombinant saponin according to claim 4, characterized in that, In step 2), the crude saponin solution is loaded into macroporous adsorption resin AB-8 and eluted sequentially with 2-3 times the resin volume of deionized water and 2-3 times the resin volume of 5-15% ethanol solution.

6. The method for preparing recombinant saponin according to claim 1, characterized in that, In step 3), elution is performed using 30-35% ethanol solution, 45-50% ethanol solution, 60-65% ethanol solution, and 75-80% ethanol solution.

7. The method for preparing recombinant saponin according to claim 6, characterized in that, In step 3), the 30-35% ethanol solution is 3-8 times the resin volume, the 45-50% ethanol solution is 8-12 times the resin volume, the 60-65% ethanol solution is 3-8 times the resin volume, and the 60-65% ethanol solution is 2-3 times the resin volume.

8. The method for preparing recombinant saponin according to claim 1, characterized in that, In step 4), the saponin component of S45 saponin is prepared into an aqueous solution with a concentration of 15-25%, the pH is adjusted to 1.5-2.5, and then hydrolyzed at a temperature of 75-85℃.

9. The method for preparing recombinant saponin according to any one of claims 1-8, characterized in that, Step 5) includes adjusting the ratio of the saponin component and the hydrolyzed saponin to make the critical micelle concentration and surface tension of the recombinant saponin adjustable.

10. A recombinant saponin, characterized in that, It is obtained by the preparation method of recombinant saponin according to any one of claims 1-9.

11. The recombinant saponin according to claim 10, characterized in that, The recombinant saponin is a hydrolyzed saponin and a complex selected from one or more of the following saponin components: S30, S45, S60, and S75.

12. The recombinant saponin according to claim 11, characterized in that, The mass ratio of S30 saponin component, hydrolyzed saponin, S60 saponin component, and S75 saponin component is 1~2:15~20:15~20:1.5~2.

13. The recombinant saponin according to any one of claims 10-12, characterized in that, The recombinant saponin has a surface tension of 30~35 mN / m and a critical micelle concentration of 0.08~0.12 g / L.

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