Method for improving extraction efficiency of royal jelly exosome

Abstract

The invention discloses a method for improving the extraction efficiency of a royal jelly exosome, and belongs to the field of nanotechnology and functional food. The method for improving the extraction efficiency of the royal jelly exosome comprises the following steps: dissolving royal jelly in a PBS (Phosphate Buffer Solution) with the pH value of 5.0-7.4; the dissolved solution is subjected to low-speed centrifugal treatment, and the low-speed centrifugal parameters are as follows: 200-3000g, 4-10 DEG C and 10-15 minutes; and filtering the centrifuged supernate by a filter membrane, then centrifuging for 1-1.5 hours at the speed of 100,000-110,000 g at the temperature of 4-10 DEG C, then centrifuging for 60-90 minutes at the speed of 130,000-150,000 g, and collecting the precipitate, namely the royal jelly exosome. The method solves the problem of unstable extraction effect in a traditional method, the extraction process is simple and easy to implement, and the extraction efficiency of the royal jelly exosome can be effectively improved.

Description

Technical Field

[0001] The invention relates to a method for improving the extraction efficiency of royal jelly exosomes, and belongs to the field of nanotechnology and functional food. Background Art

[0002] Exosomes are a type of nano-scale vesicles that are widely present in the body fluids of various organisms and have important biological functions. In recent years, the study of exosomes as drug carriers and functional food ingredients has received widespread attention. In particular, royal jelly, due to its natural, safe and non-toxic characteristics, has shown great potential in the delivery of active substances, especially in improving the stability and bioavailability of poorly soluble bioactive ingredients. Therefore, as a natural carrier, royal jelly exosomes have shown broad application prospects in the field of functional foods and medicines.

[0003] Although the extraction method of exosomes has been widely studied, the existing technology still has many shortcomings. For example, Chinese patent CN110343664B discloses a method for extracting exosomes and exosome proteins, which specifically adds metal oxides or functional materials modified with metal oxides to the liquid to be extracted containing exosomes, thereby achieving efficient extraction of exosomes; however, the recycling of the extraction materials will affect the extraction effect and exosome function, and the potential pollution control measures during the extraction process are unclear. These factors may affect the universality, repeatability and stability of the extraction method.

[0004] Chinese patent CN113943692A discloses a method for extracting exosomes from stem cells. This method relies on commercial kits, which limits the universality of the method. Chinese patent CN114958721A discloses a method for extracting stem cell exosomes. Although it improves the extraction efficiency of exosomes, it still has problems such as exosome loss, potential residual risk of polyethylene glycol, and insufficient recovery rate of low-speed centrifugation. In addition, the impurity removal effect and universality are not clearly described, which may affect the reliability of the method in different application scenarios. Summary of the invention

[0005] In view of the defects and shortcomings of the prior art, the purpose of the present invention is to provide a method for improving the extraction efficiency of royal jelly exosomes, which aims to significantly improve the extraction efficiency of exosomes and enhance the universality, repeatability and stability of the method by optimizing buffer conditions, simplifying the operating procedures, reducing impurity contamination and ensuring the function and biological activity of exosomes; and lay a technical foundation for its application in functional foods.

[0006] In order to achieve the above purpose, the technical solutions provided are as follows:

[0007] The present invention provides a method for improving the extraction efficiency of royal jelly exosomes, the method comprising the following steps:

[0008] (1) dissolving royal jelly in PBS buffer, wherein the pH value of the PBS buffer is adjusted to 5.0-7.4;

[0009] (2) subjecting the dissolved solution in step (1) to low-speed centrifugation: the low-speed centrifugation parameters are: 200-3000 g, 4° C.-10° C., 10-15 minutes;

[0010] (3) The supernatant after low-speed centrifugation in step (2) is filtered through a membrane, and then centrifuged at 100,000 g to 110,000 g for 1 to 1.5 hours at 4° C. to 10° C., and then centrifuged at 130,000 g to 150,000 g for 60 to 90 minutes to collect the precipitate, which is the royal jelly exosomes.

[0011] In one embodiment, the volume ratio of the royal jelly to the PBS buffer in step (1) is 1:20-30.

[0012] In one embodiment, the pH value in step (1) is adjusted to 5.0-6.5.

[0013] In one embodiment, the pH value in step (1) is adjusted to 5.8-6.0.

[0014] In one embodiment, the low-speed centrifugation parameters in step (2) are: 500-2500 g, 4° C.-10° C., 10-15 minutes.

[0015] In one embodiment, the low-speed centrifugation in step (2) is performed three times; the first low-speed centrifugation: 200-500g, 4°C-10°C, centrifugation for 10-15 minutes; the second low-speed centrifugation: 1000-1500g, 4°C-10°C, centrifugation for 10-15 minutes; the third low-speed centrifugation: 2000-3000g, 4°C-10°C, centrifugation for 10-15 minutes;

[0016] In one embodiment, the low-speed centrifugation in step (2) is performed three times; the first low-speed centrifugation: 500g, 4°C, centrifugation for 15 minutes; the second low-speed centrifugation: 1500g, 4°C, centrifugation for 15 minutes; the third low-speed centrifugation: 2500g, 4°C, centrifugation for 15 minutes.

[0017] In one embodiment, the centrifugation parameters of step (3) are: centrifugation at 100,000 g for 1 hour at 4° C. to 10° C., and then centrifugation at 135,000 g for 90 minutes.

[0018] The present invention also provides application of the above method in the production and processing of royal jelly.

[0019] Beneficial effects:

[0020] The extraction effect of exosomes depends largely on the pH value of the solvent during the extraction process. Too low or too high a pH value may destroy the structure of the exosomes and affect their protein content and biological activity. The method of improving the extraction efficiency of royal jelly exosomes of the present invention has significant advantages over the prior art:

[0021] 1) By adjusting the pH value of the PBS buffer solution, the amount of exosome extraction can be significantly increased;

[0022] 2) Simple process, suitable for large-scale production: This method is simple to operate and can be easily industrialized by adjusting the pH value of PBS buffer and optimizing the centrifugation steps;

[0023] 3) High extraction efficiency and high activity of exosomes: The optimized extraction conditions and step-by-step centrifugation process ensure the extraction efficiency and biological activity of exosomes, which are suitable for subsequent applications.

[0024] 4) Potential for application in multiple fields: The extracted exosomes have broad application value in functional foods, drug research, immune regulation and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the centrifugation operation during the royal jelly exosome extraction process of Examples 1 to 8;

[0026] Figure 2 This is a data diagram of the extraction efficiency of royal jelly exosomes in Examples 1 to 8;

[0027] Figure 3 TEM morphological characterization diagram of royal jelly exosomes extracted from Examples 1 to 8. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The following specific implementation methods further describe the present invention.

[0029] The royal jelly involved in the embodiment of the present invention is taken from an organic bee farm in Benxi, Liaoning Province, and is stored at -20°C for future use.

[0030] Example 1

[0031] A method for improving the extraction efficiency of royal jelly exosomes, the method comprising the following steps:

[0032] (1) Dissolve the royal jelly sample in PBS buffer, wherein the pH value of the PBS buffer is adjusted to 2.0, and the volume ratio of royal jelly to PBS buffer is 1:20;

[0033] (2) The solution dissolved in step (1) is subjected to three low-speed centrifugation treatments:

[0034] The first low-speed centrifugation: 500g, 4°C, centrifugation for 15 minutes to remove large particles;

[0035] Second low-speed centrifugation: 1500 g, 4 °C, centrifugation for 15 min to remove medium-sized particle impurities;

[0036] The third low-speed centrifugation: 2500g, 4°C, centrifugation for 15 minutes to further remove fine particle impurities;

[0037] (3) The supernatant after the third centrifugation in step (2) was filtered through a 0.22 μm filter membrane to further remove residual particulate impurities, and then the sample was centrifuged using a P70AT ultracentrifuge. The solution was ultracentrifuged at 4°C, first at 100,000 g for 1 hour, and then at 135,000 g for 90 minutes, and the precipitate was collected, which was the royal jelly exosomes.

[0038] Example 2

[0039] A method for improving the extraction efficiency of royal jelly exosomes, the method comprising the following steps:

[0040] (1) Dissolve the royal jelly sample in PBS buffer, wherein the pH value of the PBS buffer is adjusted to 2.5, and the volume ratio of royal jelly to PBS buffer is 1:20;

[0041] (2) The solution dissolved in step (1) is subjected to three low-speed centrifugation treatments:

[0042] The first low-speed centrifugation: 500g, 4°C, centrifugation for 15 minutes to remove large particles;

[0043] Second low-speed centrifugation: 1500 g, 4 °C, centrifugation for 15 min to remove medium-sized particle impurities;

[0044] The third low-speed centrifugation: 2500g, 4°C, centrifugation for 15 minutes to further remove fine particle impurities;

[0045] (3) The supernatant after the third centrifugation in step (2) was filtered through a 0.22 μm filter membrane to further remove residual particulate impurities, and then the sample was centrifuged using a P70AT ultracentrifuge. The solution was ultracentrifuged at 4°C, first at 100,000 g for 1 hour, and then at 135,000 g for 90 minutes, and the precipitate was collected, which was the royal jelly exosomes.

[0046] Example 3

[0047] A method for improving the extraction efficiency of royal jelly exosomes, the method comprising the following steps:

[0048] (1) Dissolve the royal jelly sample in PBS buffer, wherein the pH value of the PBS buffer is adjusted to 5.0, and the volume ratio of royal jelly to PBS buffer is 1:20;

[0049] (2) The solution dissolved in step (1) is subjected to three low-speed centrifugation treatments:

[0050] The first low-speed centrifugation: 500g, 4°C, centrifugation for 15 minutes to remove large particles;

[0051] Second low-speed centrifugation: 1500 g, 4 °C, centrifugation for 15 min to remove medium-sized particle impurities;

[0052] The third low-speed centrifugation: 2500g, 4°C, centrifugation for 15 minutes to further remove fine particle impurities;

[0053] (3) The supernatant after the third centrifugation in step (2) was filtered through a 0.22 μm filter membrane to further remove residual particulate impurities, and then the sample was centrifuged using a P70AT ultracentrifuge. The solution was ultracentrifuged at 4°C, first at 100,000 g for 1 hour, and then at 135,000 g for 90 minutes, and the precipitate was collected, which was the royal jelly exosomes.

[0054] Example 4

[0055] A method for improving the extraction efficiency of royal jelly exosomes, the method comprising the following steps:

[0056] (1) Dissolve the royal jelly sample in PBS buffer, wherein the pH value of the PBS buffer is adjusted to 5.8, and the volume ratio of royal jelly to PBS buffer is 1:20;

[0057] (2) The solution dissolved in step (1) is subjected to three low-speed centrifugation treatments:

[0058] The first low-speed centrifugation: 500g, 4°C, centrifugation for 15 minutes to remove large particles;

[0059] Second low-speed centrifugation: 1500 g, 4 °C, centrifugation for 15 min to remove medium-sized particle impurities;

[0060] The third low-speed centrifugation: 2500g, 4°C, centrifugation for 15 minutes to further remove fine particle impurities;

[0061] (3) The supernatant after the third centrifugation in step (2) was filtered through a 0.22 μm filter membrane to further remove residual particulate impurities, and then the sample was centrifuged using a P70AT ultracentrifuge. The solution was ultracentrifuged at 4°C, first at 100,000 g for 1 hour, and then at 135,000 g for 90 minutes, and the precipitate was collected, which was the royal jelly exosomes.

[0062] Example 5

[0063] A method for improving the extraction efficiency of royal jelly exosomes, the method comprising the following steps:

[0064] (1) Dissolve the royal jelly sample in PBS buffer, wherein the pH value of the PBS buffer is adjusted to 6.0, and the volume ratio of royal jelly to PBS buffer is 1:20;

[0065] (2) The solution dissolved in step (1) is subjected to three low-speed centrifugation treatments:

[0066] The first low-speed centrifugation: 500g, 4°C, centrifugation for 15 minutes to remove large particles;

[0067] Second low-speed centrifugation: 1500 g, 4 °C, centrifugation for 15 min to remove medium-sized particle impurities;

[0068] The third low-speed centrifugation: 2500g, 4°C, centrifugation for 15 minutes to further remove fine particle impurities;

[0069] (3) The supernatant after the third centrifugation in step (2) was filtered through a 0.22 μm filter membrane to further remove residual particulate impurities, and then the sample was centrifuged using a P70AT ultracentrifuge. The solution was ultracentrifuged at 4°C, first at 100,000 g for 1 hour, and then at 135,000 g for 90 minutes, and the precipitate was collected, which was the royal jelly exosomes.

[0070] Example 6

[0071] A method for improving the extraction efficiency of royal jelly exosomes, the method comprising the following steps:

[0072] (1) Dissolve the royal jelly sample in PBS buffer, wherein the pH value of the PBS buffer is adjusted to 6.5, and the volume ratio of royal jelly to PBS buffer is 1:20;

[0073] (2) The solution dissolved in step (1) is subjected to three low-speed centrifugation treatments:

[0074] The first low-speed centrifugation: 500g, 4°C, centrifugation for 15 minutes to remove large particles;

[0075] Second low-speed centrifugation: 1500 g, 4 °C, centrifugation for 15 min to remove medium-sized particle impurities;

[0076] The third low-speed centrifugation: 2500g, 4°C, centrifugation for 15 minutes to further remove fine particle impurities;

[0077] (3) The supernatant after the third centrifugation in step (2) was filtered through a 0.22 μm filter membrane to further remove residual particulate impurities, and then the sample was centrifuged using a P70AT ultracentrifuge. The solution was ultracentrifuged at 4°C, first at 100,000 g for 1 hour, and then at 135,000 g for 90 minutes, and the precipitate was collected, which was the royal jelly exosomes.

[0078] Example 7

[0079] A method for improving the extraction efficiency of royal jelly exosomes, the method comprising the following steps:

[0080] (1) Dissolve a royal jelly sample in PBS buffer, wherein the pH value of the PBS buffer is adjusted to 7.0, and the volume ratio of royal jelly to PBS buffer is 1:20;

[0081] (2) The solution dissolved in step (1) is subjected to three low-speed centrifugation treatments:

[0082] The first low-speed centrifugation: 500g, 4°C, centrifugation for 15 minutes to remove large particles;

[0083] Second low-speed centrifugation: 1500 g, 4 °C, centrifugation for 15 min to remove medium-sized particle impurities;

[0084] The third low-speed centrifugation: 2500g, 4°C, centrifugation for 15 minutes to further remove fine particle impurities;

[0085] (3) The supernatant after the third centrifugation in step (2) was filtered through a 0.22 μm filter membrane to further remove residual particulate impurities, and then the sample was centrifuged using a P70AT ultracentrifuge. The solution was ultracentrifuged at 4°C, first at 100,000 g for 1 hour, and then at 135,000 g for 90 minutes, and the precipitate was collected, which was the royal jelly exosomes.

[0086] Example 8

[0087] A method for improving the extraction efficiency of royal jelly exosomes, the method comprising the following steps:

[0088] (1) Dissolve the royal jelly sample in PBS buffer, wherein the pH value of the PBS buffer is adjusted to 7.4, and the volume ratio of royal jelly to PBS buffer is 1:20;

[0089] (2) The solution dissolved in step (1) is subjected to three low-speed centrifugation treatments:

[0090] The first low-speed centrifugation: 500g, 4°C, centrifugation for 15 minutes to remove large particles;

[0091] Second low-speed centrifugation: 1500 g, 4 °C, centrifugation for 15 min to remove medium-sized particle impurities;

[0092] The third low-speed centrifugation: 2500g, 4°C, centrifugation for 15 minutes to further remove fine particle impurities;

[0093] (3) The supernatant after the third centrifugation in step (2) was filtered through a 0.22 μm filter membrane to further remove residual particulate impurities, and then the sample was centrifuged using a P70AT ultracentrifuge. The solution was ultracentrifuged at 4°C, first at 100,000 g for 1 hour, and then at 135,000 g for 90 minutes, and the precipitate was collected, which was the royal jelly exosomes.

[0094] Results Analysis

[0095] 1. The actual picture of the precipitate collected by centrifugation in Examples 1 to 8 is as follows Figure 1 shown by Figure 1 The results show that in Examples 4 and 5, that is, at pH 5.8-6.0, the extraction amount of royal jelly exosomes is the largest.

[0096] 2. The royal jelly exosomes obtained in Examples 1 to 8 were respectively dissolved in 1 mL of pre-cooled PBS buffer of the corresponding pH value to obtain a royal jelly exosome solution; the protein content in the royal jelly exosome solution was detected by the BCA method, and the specific operation was as follows: using a BCA protein assay kit, the sample was mixed with a BCA working solution (reagent A and reagent B were mixed in a ratio of 50:1), incubated at 37°C for 30 minutes, and the absorbance was measured at a wavelength of 562nm, and the protein content was calculated using a standard curve. The results are shown in Figure 2 As shown in Table 1:

[0097] Figure 2 The results showed that the extraction efficiency was highest at pH 5.8 and pH 6.0, which was significantly better than buffer solutions at other pH values. When the pH value was too low (pH 2.0, pH 2.5) or too high (pH 7.4), the extraction efficiency of royal jelly exosomes decreased significantly.

[0098] Table 1 Content of royal jelly exosome protein in PBS solutions with different pH values

[0099]

[0100] 3. The morphology of the royal jelly exosomes obtained in Examples 1 to 8 was observed by transmission electron microscopy (TEM). Figure 3 As shown:

[0101] Depend on Figure 3 The results showed that the exosome morphology of Example 5 was optimal under pH 6.0 conditions, the number of extracted exosomes was large, the particle size distribution was uniform, and the surface structure was intact.

[0102] In summary, in general, combined with the illustrated results and the examples, the optimal extraction conditions are: the pH value of the PBS buffer solution plays a crucial role in the extraction efficiency of royal jelly exosomes. When the pH is adjusted to 5.8-6.0, the royal jelly exosomes extracted under this condition have complete morphology and the highest extraction efficiency, which is suitable for large-scale application and subsequent research.

[0103] The embodiments provided above are not intended to limit the scope of the present invention, and the steps described are not intended to limit the execution order thereof. Those skilled in the art may make obvious improvements to the present invention in combination with existing common knowledge, which also fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for improving the extraction efficiency of royal jelly exosomes, characterized in that: The method comprises the following steps: (1) dissolving royal jelly in PBS buffer, wherein the pH value of the PBS buffer is adjusted to 5.0-7.4; (2) subjecting the solution dissolved in step (1) to low-speed centrifugation: the low-speed centrifugation parameters are: 200-3000 g, 4° C.-10° C., 10-15 minutes; (3) The supernatant after low-speed centrifugation in step (2) is filtered through a membrane, and then centrifuged at 100,000 g to 110,000 g for 1 to 1.5 hours at 4° C. to 10° C., and then centrifuged at 130,000 g to 150,000 g for 60 to 90 minutes to collect the precipitate, which is the royal jelly exosomes.

2. The method according to claim 1, characterized in that The volume ratio of the royal jelly to the PBS buffer in step (1) is 1:20-30.

3. The method according to claim 1, characterized in that: The pH value of step (1) is adjusted to 5.0-6.

5.

4. The method according to claim 1, characterized in that: The pH value of step (1) is adjusted to 5.8-6.

0.

5. The method according to claim 1, characterized in that: The pH value of step (1) is adjusted to 6.

0.

6. The method according to claim 1, characterized in that The low-speed centrifugation parameters in step (2) are: 500-2500 g, 4° C.-10° C., 10-15 minutes.

7. The method according to claim 1, characterized in that The low-speed centrifugation in step (2) is performed three times; the first low-speed centrifugation: 200-500g, 4°C-10°C, centrifugation for 10-15 minutes; the second low-speed centrifugation: 1000-1500g, 4°C-10°C, centrifugation for 10-15 minutes; the third low-speed centrifugation: 2000-3000g, 4°C-10°C, centrifugation for 10-15 minutes.

8. The method according to claim 1, characterized in that: The low-speed centrifugation in step (2) is performed three times; the first low-speed centrifugation: 500g, 4°C, centrifugation for 15 minutes; the second low-speed centrifugation: 1500g, 4°C, centrifugation for 15 minutes; the third low-speed centrifugation: 2500g, 4°C, centrifugation for 15 minutes.

9. The method according to claim 1, characterized in that: The centrifugation parameters of step (3) are: centrifugation at 100,000 g for 1 hour at 4°C to 10°C, and then centrifugation at 135,000 g for 90 minutes.

10. Use of the method according to any one of claims 1 to 9 in the production and processing of royal jelly.

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