Preparation method of rare saponin nano microemulsion loaded with red ginseng extract and American ginseng extract

Abstract

The invention provides a preparation method of a nano microemulsion loaded with rare saponin in red ginseng extract and American ginseng extract, which comprises the following specific steps: extracting the rare saponin in the red ginseng extract and American ginseng extract, preparing a nano liposome loaded with the rare saponin, modifying with modified sesbania gum and preparing the nano microemulsion loaded with the rare saponin. Hydrophilic and hydrophobic rare ginsenoside are simultaneously embedded in the nano-liposome, in addition, the nano-liposome is modified by adopting modified sesbania gum, and the nano-liposome is endowed with an outer membrane structure by adopting the sesbania gum modified by genipin and feruloyl oligosaccharide, so that the stability of the nano-liposome can be improved, the biological activity of the rare ginsenoside is ensured, and the bioavailability of the rare ginsenoside is improved. In addition, the rare ginsenoside can be released in an organism in a targeted mode, genipin and feruloyl in the modified sesbania gum interact with phospholipid molecules in the nano-liposome in the intestinal environment, decomposition of the nano-liposome is further accelerated, rapid release of the rare ginsenoside is achieved, and a new method is provided for research and utilization of the rare ginsenoside.

Description

Technical Field

[0001] The invention belongs to the technical field of food processing, and in particular relates to a preparation method of a nano-microemulsion loaded with rare saponin in red ginseng extract and American ginseng extract. Background Art

[0002] Rare ginsenosides are secondary products of metabolic transformation of prototype ginsenosides (such as Rb1, Rb2, Rc, Rd, etc.) extracted from Araliaceae plants such as American ginseng and ginseng, mainly including Rg3, Rh2, Rg5, Rk1, Rk2, Rh3, Rk3 and other components. These rare ginsenosides have shown broad application prospects in the fields of medicine and health products due to their unique molecular structure and biological activity. They have multiple pharmacological effects such as anti-tumor, improvement of myocardial ischemia, hypoglycemia, anti-endometriosis, regulation of immune function, anti-inflammatory, anti-allergic, and anti-fatigue. In particular, ginsenosides Rh2 and Rg3 have been proven to have significant anti-tumor activity. At present, due to the disadvantages of low content, poor stability, strong molecular polarity, poor water solubility, low conversion rate, and slow oral absorption, the bioavailability of rare ginsenosides is greatly reduced, thereby limiting the application of rare ginsenosides. Studies have shown that saponins can be used as natural emulsifiers, and nano-preparations can be one of the ways to improve the utilization of rare ginsenosides. In the prior art, research on loading rare ginsenosides mainly focuses on nanoemulsions. Most forms are prepared by dissolving rare ginsenosides in an oil phase and mixing them with a water phase containing a certain protein through high-speed shearing. The proteins used include whey protein isolate, soy protein isolate, lactoferrin, chickpea protein isolate, etc. However, these proteins have certain limitations. For example, whey protein isolate has poor long-term storage stability, which will affect the effect of rare ginsenosides; soy protein isolate is easy to affect the stability of the emulsion due to the interaction between protein molecules, and commercial soy protein isolate is easy to be partially denatured during processing, thereby affecting the effect of the emulsion; the emulsification performance of lactoferrin is easily affected by factors such as pH and ions, resulting in limited functional properties. Its structure is a globular glycoprotein with fewer surface hydrophobic groups and stronger spatial repulsion, which affects the loading rate and emulsion stability of rare ginsenosides; chickpea protein isolate has poor emulsification stability under neutral conditions, and its antioxidant properties are low, and the effect of inhibiting fat oxidation is not ideal. Artificial emulsifiers are added to the existing preparation of nanoemulsions loaded with rare ginsenosides to promote emulsification and reduce the oil-water interfacial tension, but synthetic emulsifiers (such as Span, Tween series) themselves have certain toxicity, and the dosage allowed for application in food is very low, and has been gradually replaced by natural emulsifiers, and single protein nano delivery systems are often unstable and easily decomposed by pepsin, which will lead to the inability to release in the intestines that absorb nutrients. In addition to nanoemulsions, nano preparations loaded with rare ginsenosides also have nano microspheres, nano hydrogels and solid nano particles, etc., but the research is still shallow, so it is urgent to provide a preparation method of nano microemulsion loaded with rare ginsenosides, combined with nano liposomes and nano emulsion preparation, which can help broaden the application of rare ginsenosides, improve the efficiency of the body in utilizing rare ginsenosides, and lay the foundation for the high value processing of rare ginsenosides. Summary of the invention

[0003] Technical problem to be solved: In view of the above technical problems, the purpose of the present invention is to provide a method for preparing a nano-microemulsion loaded with rare saponins in red ginseng extract and American ginseng extract, the specific steps of which include extraction of rare saponins in red ginseng extract and American ginseng extract, preparation of nano-liposomes loaded with rare saponins, modification of modified sesbania gum and preparation of rare saponin nano-microemulsion, using nano-liposomes to simultaneously embed hydrophilic and hydrophobic rare ginsenosides, and further using modified sesbania gum to modify the nano-liposomes, and the sesbania gum modified with genipin and feruloyl oligosaccharides gives the nano-liposomes an outer membrane structure, which can improve the stability of the nano-liposomes and ensure the biological activity of the rare saponins. In addition, the rare saponins can be targetedly released in the body, and the genipin and feruloyl groups in the modified sesbania gum interact with the phospholipid molecules in the nano-liposomes in the intestinal environment, further accelerating the decomposition of the nano-liposomes, and realizing the rapid release of the rare saponins, providing a new method for the research and utilization of rare ginsenosides.

[0004] Technical solution: A method for preparing a nano-microemulsion loaded with rare saponins in red ginseng extract and American ginseng extract, comprising the following steps: S1. dissolving L-α-glycerophosphorylcholine and stigmasterol in anhydrous ethanol to prepare a liposome solution; S2. adding the liposome solution to the rare saponin solution and magnetically stirring at 55-60°C for 2-4h to form nanoliposome colostrum; S3. high pressure homogenization and microfiltration of the nanoliposome colostrum to obtain nanoliposomes loaded with rare saponin; S4. Add the nanoliposomes loaded with rare saponins into the modified sesbania gum solution and stir at 250-400 rpm for 1-2 hours, and shear and disperse to obtain a nano-microemulsion loaded with rare saponins in red ginseng extract and American ginseng extract. Furthermore, the method for extracting rare saponins from the red ginseng extract and the American ginseng extract comprises the following steps: Step 1. The red ginseng extract is vacuum dried for 1-2 hours, crushed and sieved to obtain red ginseng extract powder; Step 2. The red ginseng extract powder and the American ginseng extract are uniformly mixed in a mass ratio of (1-3):(1-2), and water is added in a solid-liquid ratio of 1:(5-7) to prepare a crude suspension; Step 3. Inoculate 2-8% of Bacillus subtilis culture liquid into the crude suspension, and ferment at 30-37°C for 72-120h to obtain rare saponin fermentation liquid; Step 4. The rare saponin fermentation broth is subjected to microwave treatment at 700-900W for 25-30min, and then centrifuged at 3000-4000rpm for 10-15min. The supernatant is ultrafiltered to obtain the rare saponin solution in red ginseng extract and American ginseng extract. Furthermore, in step S1, the mass ratio of L-α-glycerophosphorylcholine to stigmasterol is (2-6):(1-2). Furthermore, in step S2, the ratio of the liposome solution to the rare saponin solution is (5-15): 1 Furthermore, the conditions for high pressure homogenization in step S3 are a pressure of 400-600 MPa and a homogenization time of 10-20 min. Furthermore, the method for preparing the modified sesbania gum solution in step S4 comprises the following steps: Step I. adding 0.5-2% sesbania gum to a 10-20% by mass genipin solution, stirring at 35-40° C. and 150-200 rpm for 1-2 hours to obtain a genipin-modified sesbania gum solution; Step II. The genipin-modified sesbania gum solution and the feruloyl oligosaccharide solution are uniformly mixed in a volume ratio of (1.5-3):1, and stirred at 30-35°C and 150-200 rpm for 0.5-1 h to obtain a modified sesbania gum solution. Furthermore, in step S4, the ratio of liposome nano-colostrum to modified sesbania gum solution is (1-5):1. Furthermore, the shearing, dispersing and homogenizing conditions in step S4 are a shearing rate of 10000-15000 rpm and a shearing time of 3-5 min. The nano-microemulsion loaded with rare saponins in red ginseng extract and American ginseng extract prepared by any of the above methods. Application of the nano-microemulsion loaded with rare saponins in red ginseng extract and American ginseng extract prepared by any of the methods described above in the preparation of functional emulsions. Beneficial effects: 1. The rare ginsenosides in the red ginseng extract and the American ginseng extract of the present invention include water-soluble and water-insoluble ones. The nanoliposomes prepared from L-α-glycerophosphorylcholine and stigmasterol can embed the water-soluble rare ginsenosides in the hydrophilic region inside the liposomes, and the water-insoluble rare ginsenosides are embedded in the phospholipid bilayer of the nanoliposomes, which can embed the rare ginsenosides in the red ginseng extract and the American ginseng extract to the greatest extent, thereby improving the embedding efficiency of the rare saponins in the nano-microemulsion. 2. The nano-microemulsion loaded with rare saponins in red ginseng extract and American ginseng extract prepared by the present invention uses nano-liposomes encapsulating rare saponins as the inner core material, and the outer layer is coated with modified sesbania gum. On the one hand, the polymer chains of sesbania gum are intertwined and entangled to form a dense gel network structure, and a thin film structure is attached to the outer layer of the nano-liposomes to protect the rare saponins in the nano-liposomes from being affected by external conditions, thereby ensuring the biological activity of the rare saponins; on the other hand, genipin and feruloyl oligosaccharide modify the sesbania gum, and the network structure formed by the sesbania gum can embed genipin and feruloyl oligosaccharide, and the three form hydrogen bonds with each other, further enhancing the interaction between the molecular chains of the sesbania gum gel network, and at the same time, the feruloyl group gives the modified sesbania gum film structure good oxygen isolation, which can further ensure the biological activity of the rare saponins in red ginseng extract and American ginseng extract, thereby improving the stability of the nano-microemulsion loaded with rare saponins in red ginseng extract and American ginseng extract. 3. The nano-microemulsion loaded with rare saponins in red ginseng extract and American ginseng extract prepared by the present invention can stably exist in the stomach of the body, minimize the influence of the gastric juice environment on the rare saponins, ensure that the rare saponins can be released in the intestinal tract, and be delivered to the target site by the random body transport system to exert their effects. In addition, the enzyme system and pH in the intestine can decompose the modified sesbania gum and the nano-liposome loaded with rare saponins to release the rare saponins, and the phenolic hydroxyl groups in the feruloyl oligosaccharide can interact with the phosphate groups and carbonyl groups contained in the nano-liposomes, resulting in the colloidization of the nano-liposomes, restricting the movement of the nano-liposomes and facilitating the decomposition of the enzyme system in the intestine. At the same time, the carboxyl groups contained in genipin are ionized, and electrostatic attraction occurs with the choline structure in the nano-liposomes, accelerating the peeling of the nano-liposomes loaded with rare saponins, and promoting the efficient release of the rare saponins. 4. The method for preparing a nano-microemulsion loaded with rare saponins in red ginseng extract and American ginseng extract provided by the present invention can not only ensure the stability of rare saponins during long-term storage, but also achieve efficient release and targeted delivery, providing a new research direction for the development and utilization of rare saponins, promoting the high-value development of rare saponins, and having good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 The particle size, PDI, embedding efficiency and centrifugal stability constant of Examples 11-15 and Comparative Examples 1-4; Figure 2 is the release rate of Example 11 and Comparative Examples 1-4 in simulated gastric fluid; Figure 3 is the release rate of Example 11 and Comparative Examples 1-4 in simulated intestinal fluid; Figure 4 The particle size changes of Example 11 and Comparative Examples 1-4 during storage; Figure 5 PDI changes during storage of Example 11 and Comparative Examples 1-4; Figure 6 The centrifugal stability constant changes of Example 11 and Comparative Examples 1-4 during storage. DETAILED DESCRIPTION The present invention will be further described below in conjunction with embodiments, which are explanations of the present invention and are not limited to the following embodiments: Information on the strain used in the present invention: Bacillus subtilis CICC 24538, purchased from China Industrial Microbiological Culture Collection Management Center. Example 1 The method for extracting rare ginsenosides from red ginseng extract and American ginseng extract comprises the following steps: Step 1. The red ginseng extract is vacuum dried for 1 hour, crushed and sieved to obtain red ginseng extract powder; Step 2. Weigh 50 g of red ginseng extract powder and 50 g of American ginseng extract, mix them evenly, and add 600 mL of water to make a crude suspension; Step 3. Inoculate 56 mL of Bacillus subtilis culture liquid into the crude suspension, and ferment at 37°C for 120 h to obtain rare saponin fermentation liquid; Step 4. The rare saponin fermentation broth was microwaved at 800 W for 30 min, then centrifuged at 4000 rpm for 15 min, and the supernatant was ultrafiltered to obtain the rare saponin solution in red ginseng extract and American ginseng extract. Example 2 The method for extracting rare ginsenosides from red ginseng extract and American ginseng extract comprises the following steps: Step 1. The red ginseng extract is vacuum dried for 1 hour, crushed and sieved to obtain red ginseng extract powder; Step 2. Weigh 50g of red ginseng extract powder and 25g of American ginseng extract, mix them evenly, and add 450mL of water to make a crude suspension; Step 3. Inoculate 31.5 mL of Bacillus subtilis culture liquid into the crude suspension, and ferment at 37°C for 120 h to obtain rare saponin fermentation liquid; Step 4. The rare saponin fermentation broth was microwaved at 800 W for 30 min, then centrifuged at 4000 rpm for 15 min, and the supernatant was ultrafiltered to obtain the rare saponin solution in red ginseng extract and American ginseng extract. Example 3 The method for extracting rare ginsenosides from red ginseng extract and American ginseng extract comprises the following steps: Step 1. The red ginseng extract is vacuum dried for 1 hour, crushed and sieved to obtain red ginseng extract powder; Step 2. Weigh 50 g of red ginseng extract powder and 50 g of American ginseng extract, mix them evenly, and add 600 mL of water to make a crude suspension; Step 3. Inoculate 28 mL of Bacillus subtilis culture liquid into the crude suspension, and ferment at 37°C for 120 h to obtain rare saponin fermentation liquid; Step 4. The rare saponin fermentation broth was microwaved at 800 W for 30 min, then centrifuged at 4000 rpm for 15 min, and the supernatant was ultrafiltered to obtain the rare saponin solution in red ginseng extract and American ginseng extract. Example 4 The method for extracting rare ginsenosides from red ginseng extract and American ginseng extract comprises the following steps: Step 1. The red ginseng extract is vacuum dried for 1 hour, crushed and sieved to obtain red ginseng extract powder; Step 2. Weigh 50 g of red ginseng extract powder and 50 g of American ginseng extract, mix them evenly, and add 600 mL of water to make a crude suspension; Step 3. Inoculate 56 mL of Bacillus subtilis culture liquid into the crude suspension, and ferment at 37°C for 96 hours to obtain rare saponin fermentation liquid; Step 4. The rare saponin fermentation broth was microwaved at 800 W for 30 min, then centrifuged at 4000 rpm for 15 min, and the supernatant was ultrafiltered to obtain the rare saponin solution in red ginseng extract and American ginseng extract. Performance Testing (1) Yield of rare saponins Refer to T / CNHFA001-2021 "Determination of the content of rare ginsenosides in ginseng" to determine the yield of rare ginsenosides in Examples 1-4. Table 1 Rare saponin yields of Examples 1-4 Rare saponin yield (%) Example 1 8.61 Example 2 9.13 Example 3 7.87 Example 4 7.02 As can be seen from Table 1, the yield of rare ginsenosides extracted from red ginseng extract and American ginseng extract in Example 2 is higher than that in Example 1, Example 3 and Example 4, indicating that the ratio of red ginseng extract to American ginseng extract, the inoculation amount of strains and the fermentation time will have a certain effect on the yield of rare ginsenosides. The rare saponin solutions in the following examples are all extracted using the method of Example 2. Example 5 The method for preparing nanoliposomes loaded with rare ginsenosides in red ginseng extract and American ginseng extract comprises the following steps: S1. Weigh 80 g L-α-glycerophosphorylcholine and 20 g stigmasterol, dissolve them in anhydrous ethanol to prepare a liposome solution; S2. Add 100 mL of liposome solution to 10 mL of rare saponin solution and stir magnetically at 60 °C for 3 h to form nanoliposome colostrum; S3. Nanoliposome colostrum was homogenized at 500 MPa for 15 min and microfiltered to obtain nanoliposomes loaded with rare saponins. Example 6 The method for preparing nanoliposomes loaded with rare ginsenosides in red ginseng extract and American ginseng extract comprises the following steps: S1. Weigh 90 g L-α-glycerophosphorylcholine and 30 g stigmasterol, dissolve them in anhydrous ethanol to prepare a liposome solution; S2. Add 100 mL of liposome solution to 10 mL of rare saponin solution and stir magnetically at 60 °C for 3 h to form nanoliposome colostrum; S3. Nanoliposome colostrum was homogenized at 500 MPa for 15 min and microfiltered to obtain nanoliposomes loaded with rare saponins. Example 7 The method for preparing nanoliposomes loaded with rare ginsenosides in red ginseng extract and American ginseng extract comprises the following steps: S1. Weigh 80 g L-α-glycerophosphorylcholine and 40 g stigmasterol, dissolve them in anhydrous ethanol to prepare a liposome solution; S2. Add 100 mL of liposome solution to 10 mL of rare saponin solution and stir magnetically at 60 °C for 3 h to form nanoliposome colostrum; S3. Nanoliposome colostrum was homogenized at 500 MPa for 15 min and microfiltered to obtain nanoliposomes loaded with rare saponins. Example 8 The method for preparing nanoliposomes loaded with rare ginsenosides in red ginseng extract and American ginseng extract comprises the following steps: S1. Weigh 80 g L-α-glycerophosphorylcholine and 20 g stigmasterol, dissolve them in anhydrous ethanol to prepare a liposome solution; S2. Add 50 mL of liposome solution to 10 mL of rare saponin solution and stir magnetically at 60 °C for 3 h to form nanoliposome colostrum; S3. Nanoliposome colostrum was homogenized at 500 MPa for 15 min and microfiltered to obtain nanoliposomes loaded with rare saponins. Example 9 The method for preparing nanoliposomes loaded with rare ginsenosides in red ginseng extract and American ginseng extract comprises the following steps: S1. Weigh 80 g L-α-glycerophosphorylcholine and 20 g stigmasterol, dissolve them in anhydrous ethanol to prepare a liposome solution; S2. Add 150 mL of liposome solution to 10 mL of rare saponin solution and stir magnetically at 60 °C for 3 h to form nanoliposome colostrum; S3. Nanoliposome colostrum was homogenized at 500 MPa for 15 min and microfiltered to obtain nanoliposomes loaded with rare saponins. Example 10 The method for preparing nanoliposomes loaded with rare ginsenosides in red ginseng extract and American ginseng extract comprises the following steps: S1. Weigh 80 g L-α-glycerophosphorylcholine and 20 g stigmasterol, dissolve them in anhydrous ethanol to prepare a liposome solution; S2. Add 100 mL of liposome solution to 10 mL of rare saponin solution and stir magnetically at 60 °C for 3 h to form nanoliposome colostrum; S3. Nanoliposome colostrum was homogenized at 500 MPa for 10 min and microfiltered to obtain nanoliposomes loaded with rare saponins. Performance Testing (1) Particle size, polydispersity index (PDI) The nanoliposome solution loaded with rare saponin prepared in Examples 5-10 was diluted 100 times with ultrapure water, and the particle size and polydispersity index (PDI) were measured using a particle size analyzer. (2) Encapsulation efficiency The high-speed centrifugation-filtration method was used to determine the embedding efficiency of the rare saponin-loaded nanoliposomes prepared in Examples 5-10. The rare saponin-loaded nanoliposomes prepared in Examples 5-10 were centrifuged at 35,000 g for 30 min at 4°C. The middle layer aqueous phase was taken and passed through a 0.45 μm filter membrane to remove impurities. The rare saponin content was determined according to T / CNHFA001-2021 "Determination of the Content of Rare Ginseng Saponins", and the embedding efficiency of the nanoliposomes was further calculated. Table 2 Nanoliposome particle size, PDI and embedding efficiency of Examples 5-10 Particle size (nm) PDI Embedding efficiency (%) Example 5 133 0.16 92.15 Example 6 146 0.21 88.64 Example 7 152 0.25 86.37 Example 8 157 0.26 85.73 Example 9 148 0.23 87.49 Example 10 175 0.31 83.85 As shown in Table 2, the particle size and PDI of the nanoliposomes loaded with rare saponins prepared in Example 5 are the smallest, while the encapsulation efficiency is the highest, indicating that compared with Examples 6-10, the nanoliposomes prepared in Example 5 can better encapsulate rare saponins, and its particle size distribution is more uniform. Embodiment 11 The preparation method of the nano-microemulsion loaded with rare saponin in red ginseng extract and American ginseng extract comprises the following steps: S1. Weigh 80 g L-α-glycerophosphorylcholine and 20 g stigmasterol, dissolve them in anhydrous ethanol to prepare a liposome solution; S2. Add 100 mL of liposome solution to 10 mL of rare saponin solution and stir magnetically at 60 °C for 3 h to form nanoliposome colostrum; S3. The nanoliposome colostrum was homogenized at 500 MPa for 15 min and then microfiltered to obtain nanoliposomes loaded with rare saponins; S4. 60 mL of the rare saponin-loaded nanoliposomes were added to 20 mL of modified sesbania gum solution and stirred at 400 rpm for 2 h, and then sheared and dispersed at 12000 rpm for 3 min to obtain a rare saponin nanoemulsion loaded with red ginseng extract and American ginseng extract; The preparation method of the modified sesbania gum is as follows: Step I. Weigh 2 g of sesbania gum and 15 g of genipin, dissolve them in 83 mL of water, and then stir at 40° C. and 200 rpm for 2 h to obtain a genipin-modified sesbania gum solution; Step II. Mix 50 mL of genipin-modified sesbania gum solution and 25 mL of feruloyl oligosaccharide solution, and stir at 35° C. and 150 rpm for 1 h to obtain a modified sesbania gum solution. Example 12 The preparation method of the nano-microemulsion loaded with rare saponin in red ginseng extract and American ginseng extract comprises the following steps: S1. Weigh 80 g L-α-glycerophosphorylcholine and 20 g stigmasterol, dissolve them in anhydrous ethanol to prepare a liposome solution; S2. Add 100 mL of liposome solution to 10 mL of rare saponin solution and stir magnetically at 60 °C for 3 h to form nanoliposome colostrum; S3. The nanoliposome colostrum was homogenized at 500 MPa for 15 min and then microfiltered to obtain nanoliposomes loaded with rare saponins; S4. 40 mL of the nanoliposomes loaded with rare saponins was added to 40 mL of modified sesbania gum solution and stirred at 400 rpm for 2 h, and then sheared and dispersed at 12000 rpm for 3 min to obtain a nano-microemulsion loaded with rare saponins in red ginseng extract and American ginseng extract; The preparation method of the modified sesbania gum is as follows: Step I. Weigh 2 g of sesbania gum and 15 g of genipin, dissolve them in 83 mL of water, and then stir at 40° C. and 200 rpm for 2 h to obtain a genipin-modified sesbania gum solution; Step II. Mix 50 mL of genipin-modified sesbania gum solution and 25 mL of feruloyl oligosaccharide solution, and stir at 35° C. and 150 rpm for 1 h to obtain a modified sesbania gum solution. Embodiment 13 The preparation method of the nano-microemulsion loaded with rare saponin in red ginseng extract and American ginseng extract comprises the following steps: S1. Weigh 80 g L-α-glycerophosphorylcholine and 20 g stigmasterol, dissolve them in anhydrous ethanol to prepare a liposome solution; S2. Add 100 mL of liposome solution to 10 mL of rare saponin solution and stir magnetically at 60 °C for 3 h to form nanoliposome colostrum; S3. The nanoliposome colostrum was homogenized at 500 MPa for 15 min and then microfiltered to obtain nanoliposomes loaded with rare saponins; S4. 50 mL of the rare saponin-loaded nanoliposomes were added to 10 mL of modified sesbania gum solution and stirred at 400 rpm for 2 h, and then sheared and dispersed at 12000 rpm for 3 min to obtain a rare saponin nanoemulsion loaded with red ginseng extract and American ginseng extract; The preparation method of the modified sesbania gum is as follows: Step I. Weigh 2 g of sesbania gum and 15 g of genipin, dissolve them in 83 mL of water, and then stir at 40° C. and 200 rpm for 2 h to obtain a genipin-modified sesbania gum solution; Step II. Mix 50 mL of genipin-modified sesbania gum solution and 25 mL of feruloyl oligosaccharide solution, and stir at 35° C. and 150 rpm for 1 h to obtain a modified sesbania gum solution. Embodiment 14 The preparation method of the nano-microemulsion loaded with rare saponin in red ginseng extract and American ginseng extract comprises the following steps: S1. Weigh 80 g L-α-glycerophosphorylcholine and 20 g stigmasterol, dissolve them in anhydrous ethanol to prepare a liposome solution; S2. Add 100 mL of liposome solution to 10 mL of rare saponin solution and stir magnetically at 60 °C for 3 h to form nanoliposome colostrum; S3. The nanoliposome colostrum was homogenized at 500 MPa for 15 min and then microfiltered to obtain nanoliposomes loaded with rare saponins; S4. 60 mL of the rare saponin-loaded nanoliposomes were added to 20 mL of modified sesbania gum solution and stirred at 400 rpm for 2 h, and then sheared and dispersed at 12000 rpm for 3 min to obtain a rare saponin nanoemulsion loaded with red ginseng extract and American ginseng extract; The preparation method of the modified sesbania gum is as follows: Step I. Weigh 2 g of sesbania gum and 10 g of genipin, dissolve them in 88 mL of water, and then stir at 40° C. and 200 rpm for 2 h to obtain a genipin-modified sesbania gum solution; Step II. Mix 50 mL of genipin-modified sesbania gum solution and 25 mL of feruloyl oligosaccharide solution, and stir at 35° C. and 150 rpm for 1 h to obtain a modified sesbania gum solution. Embodiment 15 The preparation method of the nano-microemulsion loaded with rare saponin in red ginseng extract and American ginseng extract comprises the following steps: S1. Weigh 80 g L-α-glycerophosphorylcholine and 20 g stigmasterol, dissolve them in anhydrous ethanol to prepare a liposome solution; S2. Add 100 mL of liposome solution to 10 mL of rare saponin solution and stir magnetically at 60 °C for 3 h to form nanoliposome colostrum; S3. The nanoliposome colostrum was homogenized at 500 MPa for 15 min and then microfiltered to obtain nanoliposomes loaded with rare saponins; S4. 60 mL of the rare saponin-loaded nanoliposomes were added to 20 mL of modified sesbania gum solution and stirred at 400 rpm for 2 h, and then sheared and dispersed at 12000 rpm for 3 min to obtain a rare saponin nanoemulsion loaded with red ginseng extract and American ginseng extract; The preparation method of the modified sesbania gum is as follows: Step I. Weigh 2 g of sesbania gum and 15 g of genipin, dissolve them in 83 mL of water, and then stir at 40° C. and 200 rpm for 2 h to obtain a genipin-modified sesbania gum solution; Step II. Mix 60 mL of genipin-modified sesbania gum solution and 20 mL of feruloyl oligosaccharide solution, and stir at 35° C. and 150 rpm for 1 h to obtain a modified sesbania gum solution. Comparative Example 1 The difference between this comparative example and Example 11 is that the modified sesbania gum is replaced by sesbania gum. The preparation method of the nano-microemulsion loaded with rare saponin in red ginseng extract and American ginseng extract comprises the following steps: S1. Weigh 80 g L-α-glycerophosphorylcholine and 20 g stigmasterol, dissolve them in anhydrous ethanol to prepare a liposome solution; S2. Add 100 mL of liposome solution to 10 mL of rare saponin solution and stir magnetically at 60 °C for 3 h to form nanoliposome colostrum; S3. The nanoliposome colostrum was homogenized at 500 MPa for 15 min and then microfiltered to obtain nanoliposomes loaded with rare saponins; S4. Add 60 mL of nanoliposomes loaded with rare saponins into 20 mL of sesbania gum solution and stir at 400 rpm for 2 h, then shear and disperse and homogenize at 12,000 rpm for 3 min to obtain a nano-microemulsion loaded with rare saponins in red ginseng extract and American ginseng extract. Comparative Example 2 The difference between this comparative example and Example 11 is that the modified sesbania gum is modified only with genipin. The preparation method of the nano-microemulsion loaded with rare saponin in red ginseng extract and American ginseng extract comprises the following steps: S1. Weigh 80 g L-α-glycerophosphorylcholine and 20 g stigmasterol, dissolve them in anhydrous ethanol to prepare a liposome solution; S2. Add 100 mL of liposome solution to 10 mL of rare saponin solution and stir magnetically at 60 °C for 3 h to form nanoliposome colostrum; S3. The nanoliposome colostrum was homogenized at 500 MPa for 15 min and then microfiltered to obtain nanoliposomes loaded with rare saponins; S4. 60 mL of the rare saponin-loaded nanoliposomes were added to 20 mL of modified sesbania gum solution and stirred at 400 rpm for 2 h, and then sheared and dispersed at 12000 rpm for 3 min to obtain a rare saponin nanoemulsion loaded with red ginseng extract and American ginseng extract; The preparation method of the modified sesbania gum is as follows: 2 g of sesbania gum and 15 g of genipin are weighed, dissolved in 83 mL of water, and then stirred at 40° C. and 200 rpm for 2 hours to obtain the modified sesbania gum. Comparative Example 3 The difference between this comparative example and Example 11 is that the modified sesbania gum is modified only with feruloyl oligosaccharide. The preparation method of the nano-microemulsion loaded with rare saponin in red ginseng extract and American ginseng extract comprises the following steps: S1. Weigh 80 g L-α-glycerophosphorylcholine and 20 g stigmasterol, dissolve them in anhydrous ethanol to prepare a liposome solution; S2. Add 100 mL of liposome solution to 10 mL of rare saponin solution and stir magnetically at 60 °C for 3 h to form nanoliposome colostrum; S3. The nanoliposome colostrum was homogenized at 500 MPa for 15 min and then microfiltered to obtain nanoliposomes loaded with rare saponins; S4. 60 mL of the rare saponin-loaded nanoliposomes were added to 20 mL of modified sesbania gum solution and stirred at 400 rpm for 2 h, and then sheared and dispersed at 12000 rpm for 3 min to obtain a rare saponin nanoemulsion loaded with red ginseng extract and American ginseng extract; The preparation method of the modified sesbania gum is as follows: Step I. Weigh 2 g of sesbania gum and dissolve it in 98 mL of water, then stir at 40° C. and 200 rpm for 2 h to obtain a sesbania gum solution; Step II. Mix 50 mL of sesbania gum solution and 25 mL of feruloyl oligosaccharide solution evenly, and stir at 35° C. and 150 rpm for 1 h to obtain a modified sesbania gum solution. Comparative Example 4 The difference between this comparative example and Example 11 is that nanoliposomes are not used to embed the rare saponins in the red ginseng extract and the American ginseng extract. The preparation method of the nano-microemulsion loaded with rare saponin in red ginseng extract and American ginseng extract comprises the following steps: S1. 10 mL of rare saponin solution was added to 20 mL of modified sesbania gum solution and stirred at 400 rpm for 2 h, and then sheared and dispersed at 12000 rpm for 3 min to obtain a nano-microemulsion loaded with rare saponin in red ginseng extract and American ginseng extract; The preparation method of the modified sesbania gum is as follows: Step I. Weigh 2 g of sesbania gum and dissolve it in 98 mL of water, then stir at 40° C. and 200 rpm for 2 h to obtain a sesbania gum solution; Step II. Mix 50 mL of sesbania gum solution and 25 mL of feruloyl oligosaccharide solution evenly, and stir at 35° C. and 150 rpm for 1 h to obtain a modified sesbania gum solution. Performance Testing (1) Particle size and polydispersity index (PDI) The nano-microemulsion loaded with rare saponins in red ginseng extract and American ginseng extract prepared in Examples 11-15 and Comparative Examples 1-4 was diluted 300 times with ultrapure water, and the particle size and polydispersity index (PDI) were measured using a particle size analyzer. (2) Encapsulation efficiency The embedding efficiency of the rare saponin nano-microemulsion loaded with red ginseng extract and American ginseng extract prepared in Examples 11-15 and Comparative Examples 1-4 was determined by high-speed centrifugation-filtration method. (3) Centrifugal stability constant The rare saponin nano-microemulsion loaded with red ginseng extract and American ginseng extract prepared in Examples 11-15 and Comparative Examples 1-4 was diluted 100 times with ultrapure water, and the absorbance A0 was measured at 490 nm using an ultraviolet spectrophotometer. The diluted nano-microemulsion was centrifuged at 4°C and 4000 rpm for 20 min, and the lower layer of liquid was diluted 100 times with ultrapure water. After mixing, the absorbance A1 was measured at 490 nm using an ultraviolet-visible spectrophotometer, so the centrifugal stability constant = A1 / A0×100%. Depend on Figure 1 It can be seen that among Examples 11-15, the particle size and PDI of Example 11 are the smallest, while the embedding efficiency and the centrifugal stability constant are the largest, indicating that the proportion of modified sesbania gum and the degree of modification of sesbania gum will affect the performance of the rare saponin nano-microemulsion loaded with red ginseng extract and American ginseng extract; in addition, the particle size and PDI of Example 11 are lower than those of Comparative Examples 1-4, while the embedding efficiency and the centrifugal stability constant are higher than those of Comparative Examples 1-4, indicating that the nanoliposomes can efficiently embed the rare ginsenosides from red ginseng extract and American ginseng extract, and the sesbania gum modified with genipin and feruloyl oligosaccharide can be coated on the outer layer of the nanoliposomes, interact with the structure of the nanoliposomes, enhance the embedding effect of the rare ginsenosides, ensure the embedding efficiency of the rare saponins in the nano-microemulsion, and the interaction between the modified sesbania gum and the nanoliposomes can further improve the stability of the rare saponins in the nano-microemulsion. (4) Release rate of rare saponins in simulated gastrointestinal digestion test Simulated gastric juice (pH 1.2) and simulated intestinal juice (pH 6.8) were prepared according to the method in Part IV of the 2015 edition of the Pharmacopoeia of the People's Republic of China

[16] . 10 mL of the nano-microemulsion prepared in Example 11 and Comparative Examples 1-4 were added with 40 mL of simulated gastric juice and simulated intestinal juice, respectively. The mixture was incubated in a constant temperature oscillating water bath at 37°C and 100 r / min for 4 h. A certain amount of simulated digestive fluid was taken out every 30 min, allowed to stand for a period of time, and then centrifuged to determine the content of rare saponins. The release rate of rare saponins in the in vitro simulated gastrointestinal digestion test was calculated. Depend on Figure 2 It can be seen that in the simulated gastric juice, the release rate of rare saponins in the nano-microemulsion prepared in Comparative Example 1 is the largest. In Comparative Example 1, sesbania gum is not modified with genipin and feruloyl oligosaccharide. Sesbania gum can be decomposed by enzymes in the extreme pH of gastric juice, thereby destroying the membrane structure of the outer layer of the nanoliposomes, causing the nanoliposomes to be further decomposed and release rare saponins as the digestion time increases, causing the biological activity of the rare saponins to be inactivated in gastric juice; Comparative Examples 2 and 3 are both modified with a single component of genipin or feruloyl oligosaccharide, and the release rate of rare saponins is The rate was significantly reduced, indicating that modification with genipin or feruloyl oligosaccharide can improve the performance of sesbania gum, so that it can reduce the pH of gastric juice and the influence of enzymes in gastric juice to a certain extent; Comparative Example 4 did not use nanoliposomes to first embed rare saponins, and its release rate of rare saponins was significantly higher than that of Example 11, indicating that modified sesbania gum has a certain protective effect on rare saponins, and nanoliposomes can be used in combination with modified sesbania gum. The unique interaction between the two greatly reduces the influence of gastric juice on the nano-microemulsion, thereby further ensuring the biological activity of rare saponins. Depend on Figure 3It can be seen that in the simulated intestinal fluid, the release rate of the rare saponins in Example 11 is significantly higher than that in Comparative Examples 1-4, indicating that the nano-microemulsion loaded with rare saponins can be decomposed in the simulated intestinal fluid, and the sesbania gum modified with genipin and feruloyl oligosaccharide is decomposed, wherein the phenolic hydroxyl groups in the feruloyl oligosaccharide can interact with the phosphate groups and carbonyl groups contained in the nanoliposomes, causing the nanoliposomes to become gelled, restricting the movement of the nanoliposomes and facilitating the enzymatic decomposition in the simulated intestinal fluid. At the same time, the carboxyl groups contained in genipin are ionized, and electrostatic mutual attraction occurs with the choline structure in the nanoliposomes, accelerating the peeling of the nanoliposomes loaded with rare saponins, and promoting the efficient release of rare saponins. (4) Storage stability The nano-microemulsions prepared in Example 11 and Comparative Examples 1-4 were stored at 25° C. for 60 days, and the particle size, PDI and centrifugal stability constant of the nano-microemulsions were measured every 10 days. Depend on Figure 4 , Figure 5 and Figure 6 It can be seen that with the extension of storage time, the particle size and PDI of Example 11 and Comparative Examples 1-4 both increase, while the centrifugal stability constant decreases. The stability of the nano-microemulsion prepared in Example 11 during storage is better than that of Comparative Examples 1-4, indicating that the preparation of nano-microemulsions by embedding rare saponins in modified sesbania gum-modified nanoliposomes can improve the stability of rare saponins during storage and ensure the biological activity of rare saponins. In addition, the genipin and feruloyl oligosaccharides in the modified sesbania gum have antioxidant and antibacterial effects, which can prevent the lipid components in the nano-microemulsion from being oxidized during storage and minimize the influence of microorganisms in the storage environment. At the same time, the nanoliposomes also have a protective effect on rare saponins, further improving the storage stability of the nano-microemulsion. The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the art can make many possible changes and modifications to the technical solution of the present invention by using the above disclosed methods and technical contents without departing from the spirit and technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the scope of protection of the technical solution of the present invention.

Claims

1. A method for preparing a nano-microemulsion loaded with rare saponins in red ginseng extract and American ginseng extract, characterized in that: The following steps are involved: S1. dissolving L-α-glycerophosphorylcholine and stigmasterol in anhydrous ethanol to prepare a liposome solution; S2. adding the liposome solution to the rare saponin solution and stirring magnetically at 55-60°C for 2-4 hours to form nanoliposome colostrum; S3. The nanoliposome colostrum is subjected to high pressure homogenization and microfiltration to obtain nanoliposomes loaded with rare saponins; S4. Add the nanoliposomes loaded with rare saponins into the modified sesbania gum solution and stir at 250-400 rpm for 1-2 hours, and shear and disperse to obtain a nano-microemulsion loaded with rare saponins in red ginseng extract and American ginseng extract.

2. The method for preparing the nano-microemulsion loaded with rare saponins in red ginseng extract and American ginseng extract according to claim 1, characterized in that: The method for extracting rare saponins from the red ginseng extract and the American ginseng extract comprises the following steps: Step 1. The red ginseng extract is vacuum dried for 1-2 hours, crushed and sieved to obtain red ginseng extract powder; Step 2. The red ginseng extract powder and the American ginseng extract are uniformly mixed in a mass ratio of (1-3):(1-2), and water is added in a solid-liquid ratio of 1:(5-7) to prepare a crude suspension; Step 3. Inoculate 2-8% of Bacillus subtilis culture liquid into the crude suspension, and ferment at 30-37°C for 72-120h to obtain rare saponin fermentation liquid; Step 4. The rare saponin fermentation broth is subjected to microwave treatment at 700-900W for 25-30min, and then centrifuged at 3000-4000rpm for 10-15min. The supernatant is ultrafiltered to obtain the rare saponin solution in red ginseng extract and American ginseng extract.

3. The method for preparing the nano-microemulsion loaded with rare saponins in red ginseng extract and American ginseng extract according to claim 1, characterized in that: In the step S1, the mass ratio of L-α-glycerophosphorylcholine to stigmasterol is (2-6):(1-2).

4. The method for preparing the nano-microemulsion loaded with rare saponins in red ginseng extract and American ginseng extract according to claim 1, characterized in that: The ratio of the liposome solution to the rare saponin solution in step S2 is (5-15):

1.

5. The method for preparing the nano-microemulsion loaded with rare saponins in red ginseng extract and American ginseng extract according to claim 1, characterized in that: The conditions for high pressure homogenization in step S3 are a pressure of 400-600 MPa and a homogenization time of 10-20 min.

6. The method for preparing the nano-microemulsion loaded with rare saponins in red ginseng extract and American ginseng extract according to claim 1, characterized in that: The method for preparing the modified sesbania gum solution in step S4 comprises the following steps: Step I. adding 0.5-2% sesbania gum to a 10-20% by mass genipin solution, stirring at 35-40° C. and 150-200 rpm for 1-2 hours to obtain a genipin-modified sesbania gum solution; Step II. The genipin-modified sesbania gum solution and the feruloyl oligosaccharide solution are uniformly mixed in a volume ratio of (1.5-3):1, and stirred at 30-35°C and 150-200 rpm for 0.5-1 h to obtain a modified sesbania gum solution.

7. The method for preparing the nano-microemulsion loaded with rare saponins in red ginseng extract and American ginseng extract according to claim 1, characterized in that: In step S4, the ratio of liposome nano-colostrum to modified sesbania gum solution is (1-5):

1.

8. The method for preparing the nano-microemulsion loaded with rare saponins in red ginseng extract and American ginseng extract according to claim 1, characterized in that: The conditions for shearing, dispersing and homogenizing in step S4 are a shearing rate of 10000-15000 rpm and a shearing time of 3-5 min.

9. The nano-microemulsion loaded with rare saponins in red ginseng extract and American ginseng extract prepared according to the method of any one of claims 1 to 8.

10. Use of the nano-microemulsion loaded with rare saponins in red ginseng extract and American ginseng extract prepared according to the method of claim 9 in preparing functional emulsion.

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