A plum exosome, a preparation method thereof, and an application thereof in preparing a product for relieving constipation

Abstract

This application belongs to the technical field of health foods, and particularly relates to a plum exosome, a preparation method thereof, and an application in the preparation of a product for relieving constipation. The preparation method of a plum exosome provided by this application deeply processes plums, extracts plum exosomes from plums, uses the miRNA nucleic acids contained in the plum exosomes to promote the expression of mucin genes in goblet cells and the proliferation of beneficial bacteria such as Bifidobacterium and Lactobacillus, and uses the antioxidant activity of the plum exosomes to reduce the adverse effects of oxidative stress on intestinal cells and the microbial community, achieving the effect of relieving constipation, thereby solving the technical problem of the lack of deep processing research on plums in the prior art.

Description

Technical Field

[0001] This application belongs to the technical field of health foods, and particularly relates to a plum exosome, a preparation method thereof, and an application in the preparation of a product for relieving constipation and promoting defecation. Background Art

[0002] With the continuous improvement of living standards, people's dietary structure has gradually changed, and the intake of high-calorie, high-fat, and low-fiber foods has been increasing continuously, making problems such as poor defecation and difficulty in controlling weight more and more common. At the same time, consumers' demand for healthy, safe, and effective defecation-promoting and body-shaping products is increasing continuously, prompting them to actively explore safe and effective defecation-promoting and body-shaping ways.

[0003] Dietary fiber in fruits, beans, tubers, and vegetables can increase satiety, help reduce calorie intake, and also promote intestinal peristalsis, help clean the intestines, and is beneficial to weight control. Consuming dietary fiber is an important way to shape the body. Plum is a fruit with a unique flavor, bright color, and rich nutrition, containing organic acids, vitamins, dietary fiber, polyphenols and other nutrients, and has low calories and zero fat. The dietary fiber rich in plums can not only effectively promote intestinal peristalsis, but also increase the frequency of defecation and the softness of feces. It is an edible plant ingredient that can regulate poor defecation, and polyphenols can delay human aging, stimulate heat production, and accelerate energy consumption, thus playing a role in regulating the body's lipid metabolism. Therefore, plums have the application potential for promoting defecation.

[0004] However, the current application of plums is mainly in the forms of direct consumption, squeezing into fruit juice, making into preserved fruits, fermenting into plum fermentation stock solution, etc. There is a lack of in-depth processing research on plums, and it is necessary to further explore the performance of deep-processed plum products. Summary of the Invention

[0005] In view of this, this application provides a plum exosome, a preparation method thereof, and an application in the preparation of a product for relieving constipation and promoting defecation, so as to solve the technical problem of the lack of in-depth processing research on plums in the prior art.

[0006] The first aspect of this application provides a preparation method of a plum exosome. The preparation method includes: extracting the plum juice by ultracentrifugation and density gradient centrifugation in sequence to obtain the plum exosome.

[0007] Preferably, the process of extracting the plum juice by ultracentrifugation and density gradient centrifugation in sequence includes the following steps:

[0008] Step S1: Juicing, standing, and filtering fresh plums in sequence to obtain plum juice;

[0009] Step S2: Ultracentrifuging the plum juice in a way that the centrifugal force increases to obtain a precipitate mixture of plum exosomes;

[0010] Step S3: Add the plum exosome precipitation mixture into a buffer solution for resuspension to obtain a refined plum exosome solution.

[0011] Step S4: Add the refined plum exosome solution to the top of a centrifuge tube pre-filled with a concentration gradient sucrose solution for sucrose density gradient centrifugation to obtain a purified plum exosome solution.

[0012] Step S5: Perform ultra-high-speed centrifugation on the purified plum exosome solution to obtain a plum exosome precipitate.

[0013] Preferably, in step S1, the aperture of the gauze used for filtration is 80 - 120 mesh.

[0014] Preferably, in step S2, the ultra-high-speed centrifugation of the plum juice in a manner of increasing centrifugal force specifically includes the following steps:

[0015] Step S21: Centrifuge the plum juice at 4°C for 5 - 15 minutes at 400 - 600 g to obtain a low-speed centrifugation supernatant.

[0016] Step S22: Centrifuge the low-speed centrifugation supernatant at 4°C for 5 - 15 minutes at 800 - 1200 g to obtain a medium-speed centrifugation supernatant.

[0017] Step S23: Centrifuge the medium-speed centrifugation supernatant at 4°C for 20 - 40 minutes at 2500 - 3500 g to obtain a medium-high-speed centrifugation supernatant.

[0018] Step S24: Centrifuge the medium-high-speed centrifugation supernatant at 4°C for 40 - 80 minutes at 5000 - 15000 g to obtain a high-speed centrifugation supernatant.

[0019] Step S25: Centrifuge the high-speed centrifugation supernatant at 4°C for 80 - 100 minutes at 100000 - 200000 g for ultra-high-speed centrifugation to obtain a plum exosome precipitation mixture.

[0020] Preferably, in step S3, the resuspension process includes: at 4°C, add the exosome precipitate to the PBS buffer solution for resuspension according to a mass ratio of 1:50 - 100 to obtain a refined plum exosome solution.

[0021] Preferably, in step S4, adding the refined plum exosome solution to the top of a centrifuge tube pre-filled with a concentration gradient sucrose solution for sucrose density gradient centrifugation specifically includes the following steps:

[0022] Step S41: Add sucrose solutions with mass concentrations of 60wt%, 45wt%, 30wt%, and 15wt% into centrifuge tubes in a volume ratio of 1:1:1:1 in sequence to obtain centrifuge tubes pre-filled with sucrose solutions of concentration gradient.

[0023] Step S42: Add the exosome extract to the top of the centrifuge tube pre-filled with sucrose solutions of concentration gradient, and centrifuge at 100,000 - 200,000 g for 80 - 100 minutes at 4°C. Take the solution in the 30wt% - 45wt% sucrose solution layer to obtain the purified plum exosome solution.

[0024] Preferably, in step S5, centrifuge the purified plum exosome solution at 100,000 - 200,000 g for 80 - 100 minutes at 4°C to obtain plum exosome precipitate.

[0025] The second aspect of the present application provides a kind of plum exosome, which is prepared by the preparation method described in the first aspect.

[0026] Preferably, the particle size of the plum exosome is 100 - 300 nm.

[0027] Preferably, the particle size of the plum exosome is 150 - 250 nm.

[0028] The third aspect of the present application provides the application of the plum exosome described in the second aspect in the preparation of intestinal laxative products.

[0029] In summary, the present application provides a kind of plum exosome, its preparation method and application in the preparation of intestinal laxative products, which are used to solve the technical problem of the lack of deep processing research on plums in the prior art.

[0030] Compared with the prior art, the plum exosome, its preparation method and application in the preparation of intestinal laxative products provided by the present application at least include the following beneficial effects:

[0031] 1. The preparation method of the plum exosome provided by the present application extracts plum exosomes from plums, realizing the deep processing of plums.

[0032] 2. The application of the plum exosome provided by the present application in the preparation of intestinal laxative products utilizes the excellent properties of the plum exosomes extracted from plums, such as promoting the expression of mucin genes, the proliferation of beneficial bacteria, and antioxidant activity, etc., to achieve the effect of intestinal laxation. Description of the Drawings

[0033] To more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the following will briefly introduce the accompanying drawings required for the description of the specific embodiments or the prior art. Obviously, the accompanying drawings in the following description are some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0034] Figure 1 In the preparation method of a plum exosome provided in Example 1 of the present application, it is a schematic diagram after gradient centrifugation in a sucrose solution with a concentration gradient of 15wt% - 60wt%.

[0035] Figure 2 In the preparation method of a plum exosome provided in Example 1 of the present application, it is a schematic diagram of the particle size distribution of the prepared plum exosome.

[0036] Figure 3 In the preparation method of a plum exosome provided in Example 1 of the present application, it is a transmission electron micrograph of a single prepared plum exosome.

[0037] Figure 4 In the preparation method of a plum exosome provided in Example 1 of the present application, it is a schematic diagram of the potential distribution of the prepared plum exosome.

[0038] Figure 5 In the preparation method of a plum exosome provided in Example 1 of the present application, it is an electrophoresis diagram of the prepared plum exosome protein and plum juice. Figure 5 Among them, M: Maker, protein molecular weight standard, S1: Plum exosome (batch 1), S2: Plum exosome (batch 2), S3: Plum exosome (batch 3), ZZ: Plum juice.

[0039] Figure 6 In the preparation method of a plum exosome provided in Example 1 of the present application, it is a thin layer chromatogram of the lipids of the prepared plum exosome.

[0040] Figure 7 In the preparation method of a plum exosome provided in Example 1 of the present application, it is a diagram of the miRNA content and classification of the prepared plum exosome.

[0041] Figure 8 In the preparation method of a plum exosome provided in Example 1 of the present application, it is a diagram of the antioxidant activity of the prepared plum exosome.

[0042] Figure 9 In Experimental Example 2 of the present application, it is a diagram of the advancement of ink in the small intestine of mice after taking ink and taking or not taking the plum exosome provided in Example 1.

[0043] Figure 10 In Experimental Example 2 of this application, after taking ink and taking or not taking the prune exosomes provided in Example 1, it shows the graph of the propulsion rate of the ink in the small intestine of mice and the time for the mice to defecate the first black stool.

[0044] Figure 11 In Experimental Example 2 of this application, after taking ink and taking or not taking the prune exosomes provided in Example 1, it shows the graph of the number and weight of black stools excreted by mice within 6 hours. Detailed implementation manners

[0045] This application provides a prune exosome, a preparation method thereof, and an application in the preparation of a product for moistening the intestine and relieving constipation, aiming to solve the technical problem of the lack of in-depth processing research on prunes in the prior art.

[0046] Next, the technical solutions of this application will be described clearly and completely with reference to the accompanying drawings. Obviously, the described embodiments are part of the embodiments of this application, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in this application without creative efforts shall fall within the scope of protection of this application.

[0047] In view of the current lack of in-depth processing research on prunes, it is difficult to further explore their application in moistening the intestine and relieving constipation. This application provides a preparation method of prune exosomes, and the preparation method includes: sequentially extracting prune juice by ultracentrifugation and density gradient centrifugation to obtain prune exosomes.

[0048] Studies have shown that exosomes derived from plants such as plums are natural nanoparticles containing proteins, lipids, nucleic acids, and secondary metabolites. They can be taken up by animal cells, including human cells, and exhibit various biotherapeutic and drug delivery functions. At the same time, compared with exosomes derived from cells, plant-derived exosomes show more superior biocompatibility, low immunogenicity, high safety, and green sustainability. They also have functions similar to those of the source plants. Therefore, plum exosomes obtained by deep processing of plums have the potential application of promoting bowel movement. In addition to dietary fiber promoting intestinal peristalsis and helping to clean the intestines, intestinal cells and the microbial community are also closely related to intestinal health. For example, intestinal flora dysregulation caused by reasons such as changes in diet structure can lead to intestinal dysfunction and trigger intestinal diseases such as constipation. The miRNA in the plum exosomes provided in this application can promote the proliferation of beneficial bacteria such as Bifidobacterium and Lactobacillus by regulating signal pathways in the intestine, and can also upregulate the expression of mucin genes in goblet cells, promoting the synthesis and secretion of mucin, thickening the intestinal mucus layer. Moreover, the plum exosomes provided in this application also have antioxidant activity, which can reduce the negative effects of excessive reactive oxygen species and other oxidants produced by the body on intestinal cells and the microbial community. Therefore, the plum exosomes provided in this application play the effect of promoting bowel movement by improving the survival status of intestinal cells and the microbial community, promoting the synthesis and secretion of mucin, and the proliferation of beneficial bacteria, realizing the deep processing research of plums and overcoming the defect of the lack of deep processing research on plums at present.

[0049] Preferably, in the preparation method of the plum exosomes provided in this application, the plum juice is obtained by juicing, standing, and filtering fresh plums in sequence. Standing and filtering can effectively remove the filter residue. Subsequently, the plum juice with the filter residue removed is subjected to ultra-high speed centrifugation in a manner of increasing centrifugal force. Ultra-high speed centrifugation in a manner of increasing centrifugal force can effectively remove unbroken cells, cell debris, cell microparticles, large vesicles, some protein complexes, and large particle impurities in the plum juice, and retain the plum exosome precipitate mixture with a higher density. Then, the refined plum exosome extract after resuspending the plum exosome precipitate mixture is added to the top of a centrifuge tube pre-filled with a sucrose solution with a concentration gradient for sucrose density gradient centrifugation, so that the plum exosomes and other component impurities in the refined exosome extract settle into different sucrose density regions respectively, purifying the refined plum exosome extract to obtain a purified plum exosome solution. Finally, the purified plum exosome solution is subjected to ultra-high speed centrifugation to obtain a high-purity plum exosome precipitate.

[0050] Preferably, in the method for preparing plum exosomes provided by the present application, the aperture of the gauze used for removing filter residue is 80-120 mesh, and the way of increasing centrifugal force for ultra-high speed centrifugation is to carry out centrifugation at 500g / 1000g / 3000g / 10000g / 150000g for 10-90 minutes respectively at 4°C. The buffer used in the resuspension process is PBS buffer, the concentration of the sucrose solution used in the sucrose density gradient centrifugation process is 60wt%, 45wt%, 30wt% and 15wt%, and finally the time for ultra-high speed centrifugation of the purified plum exosome solution is 90 minutes.

[0051] Next, a plum exosome provided by the present application will be specifically described in conjunction with examples and experimental examples.

[0052] Example 1

[0053] The present application provides a method for preparing plum exosomes in Example 1. The preparation method includes the steps of preparing plum juice, ultra-high speed centrifugation, density gradient centrifugation, and ultra-high speed centrifugation.

[0054] Among them, the step of preparing plum juice includes: taking 1500g of fresh plums, removing the cores and juicing them with a juicer for 2 minutes (3 times), standing at 4°C for 15 minutes, and then filtering through an 80-120 mesh gauze to obtain plum juice.

[0055] The step of ultra-high speed centrifugation includes: first centrifuging the plum juice at 500g for 10 minutes at 4°C to obtain a low-speed centrifugation supernatant; then centrifuging the low-speed centrifugation supernatant at 1000g for 10 minutes at 4°C to obtain a medium-speed centrifugation supernatant; subsequently centrifuging the medium-speed centrifugation supernatant at 3000g for 30 minutes at 4°C to obtain a medium-high speed centrifugation supernatant; then centrifuging the medium-high speed centrifugation supernatant at 10000g for 60 minutes at 4°C to obtain a high-speed centrifugation supernatant; next, centrifuging the high-speed centrifugation supernatant at 150000g for 90 minutes at 4°C to obtain a plum exosome precipitate mixture.

[0056] The step of density gradient centrifugation includes: adding PBS buffer at 4°C to the plum exosome precipitate mixture according to a mass ratio of 1:50-100 and mixing well to resuspend, obtaining a refined plum exosome solution. At the same time, prepare sucrose solutions with mass fractions of 60%, 45%, 30%, and 15%, and add them to the bottom of the centrifuge tube in a volume ratio of 1:1:1:1 in sequence; then add the refined plum exosome solution to the top of the solution. The total volume ratio of the concentration gradient sucrose solution in the centrifuge tube to the volume of the added refined plum exosome solution is 4:1. Slowly place it into an ultra-high speed centrifuge and centrifuge at 150000g for 90 minutes at 4°C. Take the solution in the sucrose solution layer with a mass fraction of 30%-45% as the purified plum exosome solution, as Figure 1 shown.

[0057] The steps of ultra-high speed centrifugation include: centrifuging the purified plum exosome solution at 150,000 g for 90 minutes at 4°C to obtain a plum exosome precipitate, adding PBS buffer and mixing well for standby. The dosage ratio of the plum exosome precipitate to the PBS buffer is 40 mg : 1 mL.

[0058] Experimental Example 1

[0059] In this experimental example, the plum exosomes prepared by the method for preparing plum exosomes provided in Example 1 were tested. The tests included using a nanoparticle tracking analyzer and a transmission electron microscope to test the particle size, zeta potential, and vesicle morphology of the plum exosomes, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to test the protein molecular weight distribution of the plum exosomes, using thin layer chromatography (TLC) of lipids to test the lipids of the plum exosomes, analyzing the expression levels and characteristics of miRNAs by small RNA sequencing technology. At the same time, the antioxidant activity was also analyzed by a total antioxidant capacity detection kit (Solarbio, FRAP method) and a free radical scavenging capacity detection kit (Beyotime, DPPH method). The analysis and test results are as Figure 2 - 8 shown.

[0060] From Figure 2 - 8 it can be seen that the particle size range of the plum exosomes extracted by the method for preparing plum exosomes provided in this application is between 100 and 300 nm, the zeta potential is -7.26 mV. At the same time, the SDS-PAGE diagram, the thin layer chromatography diagram of lipids, and the diagram of miRNA types and contents also show that the plum exosomes provided in this application contain rich components such as proteins, lipids, and nucleic acids. Moreover, the plum exosomes provided in this application also have excellent antioxidant activity. Therefore, the plum exosomes provided in this application contain rich proteins, lipids, miRNA nucleic acids, and antioxidant active products. Among them, the miRNAs contained are a type of microRNA, which can regulate the expression of other genes across kingdoms while regulating the physiological functions of plants themselves, and are expected to promote the expression of mucin genes in goblet cells and the proliferation of beneficial bacteria such as Bifidobacterium and Lactobacillus. The antioxidant activity of plum exosomes is expected to improve the adverse effects of oxidative stress caused by excessive production of reactive oxygen species and other oxidants in the body on intestinal cells and the microbial community.

[0061] Experimental Example 2

[0062] In this experimental example, the laxative effect of the plum exosomes prepared by the method for preparing plum exosomes provided in Example 1 was tested.

[0063] The test process of the laxative effect includes:

[0064] Adult male C57 mice weighing 20±2 g were selected. After one week of adaptive feeding, they were randomly divided into three groups: blank control group (CON), model control group (MOD), and plum exosome group (EXO). Each group was given the test sample by oral gavage for 14 days. The blank control group and the model control group were given PBS, and the plum exosome group was given plum exosomes (7.5 mg / kg / d).

[0065] After 14 days, the mice were fasted but allowed to drink water for 16 h. The blank control group was given distilled water by gavage, and the model control group and the plum exosome group were given loperamide (4 mg / kg BW) by gavage. After 0.5 h of gavage, the plum exosome group was given ink containing the corresponding test sample (containing 5% activated carbon powder and 10% gum arabic), and the blank control group and the model control group were given ink by gavage. They were allowed to drink water and eat normally.

[0066] After 25 minutes, half of the mice in each group were immediately sacrificed by cervical dislocation. The abdominal cavity was opened to separate the mesentery, and the intestinal tube from the upper end of the pylorus to the lower end of the ileocecal part was cut and placed on a tray. The small intestine was gently straightened, and the length of the intestinal tube was measured as the "total length of the small intestine", and the length from the pylorus to the ink front was the "advancing length of the ink". The ink propulsion rate of the small intestine was calculated. For the other half of the mice in each group, starting from the administration of the ink, the time of the first black stool excretion, the number and weight of black stools excreted within 6 hours of each animal were recorded to observe its laxative effect.

[0067] The test results of the laxative effect are as Figure 9 - 11 shown. It can be seen from Figure 9 - 11 that compared with the blank control group (CON) and the model control group (MOD), the ink propulsion rate in the small intestine of the mice in the plum exosome group (EXO) was significantly higher. At the same time, the number and weight of black stools excreted within 6 hours of the mice in the plum exosome group (EXO) were also significantly higher than those of the mice in the blank control group (CON) and the model control group (MOD). This shows that the preparation method of the plum exosomes provided in this application, through the deep processing of plums, extracts plum exosomes from plums. The miRNA in the plum exosomes can promote the proliferation of beneficial bacteria such as Bifidobacterium and Lactobacillus by regulating the signal pathways in the intestine, and can also up-regulate the expression of mucin genes in goblet cells, promote the synthesis and secretion of mucin, thicken the intestinal mucus layer. At the same time, the antioxidant activity of the plum exosomes is also utilized to reduce the negative effects of excessive reactive oxygen species and other oxidative substances produced by the body on intestinal cells and the microbial community, so as to avoid the occurrence of oxidative stress phenomena where the body produces excessive reactive oxygen species and other oxidative substances beyond the capacity of its own antioxidant defense system.

[0068] The above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it; although the present application has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. The application of prune exosomes in the preparation of products for promoting bowel movement and relieving constipation, characterized in that, The preparation method of the prune exosomes includes: Step S1: Ultracentrifuge the prune juice in a manner of increasing centrifugal force to obtain a prune exosome precipitate mixture; Step S2: Resuspend the prune exosome precipitate mixture in a buffer solution to obtain a refined prune exosome extract; Step S3: Add the refined prune exosome extract to the top of a centrifuge tube pre-filled with a sucrose solution of a concentration gradient for sucrose density gradient centrifugation to obtain a purified prune exosome solution; Step S4: Ultracentrifuge the purified prune exosome solution to obtain a prune exosome precipitate.

2. The application of the prune exosomes according to claim 1 in the preparation of a product for relieving constipation, characterized in that In step S1, the preparation steps of the prune juice include: juicing, standing, and filtering fresh prunes in sequence to obtain prune juice.

3. The use of the prune exosomes according to claim 2 in the preparation of a product for relieving constipation, characterized in that, In step S1, the aperture of the gauze used for filtration is 80 - 120 mesh.

4. Use of the prune exosomes according to claim 1 in the preparation of a product for moistening the intestine and relieving constipation, characterized in that, In step S1, the ultracentrifugation of the prune juice in a manner of increasing centrifugal force specifically includes the following steps: Step S11: Centrifuge the prune juice at 4°C for 5 - 15 minutes at 400 - 600 g to obtain a supernatant of low-speed centrifugation; Step S12: Centrifuge the supernatant of low-speed centrifugation at 4°C for 5 - 15 minutes at 800 - 1200 g to obtain a supernatant of medium-speed centrifugation; Step S13: Centrifuge the supernatant of medium-speed centrifugation at 4°C for 20 - 40 minutes at 2500 - 3500 g for medium-high-speed centrifugation to obtain a supernatant of medium-high-speed centrifugation; Step S14: Centrifuge the supernatant of medium-high-speed centrifugation at 4°C for 40 - 80 minutes at 5000 - 15000 g for high-speed centrifugation to obtain a supernatant of high-speed centrifugation; Step S15: Centrifuge the supernatant of high-speed centrifugation at 4°C for 80 - 100 minutes at 100000 - 200000 g for ultra-high-speed centrifugation to obtain a prune exosome precipitate mixture.

5. Use of prune exosomes according to claim 1 in the preparation of a product for moistening the intestines and relieving constipation, characterized in that, In step S2, the resuspension process includes: At 4°C, add the prune exosome precipitate mixture to PBS buffer solution for resuspension according to a mass ratio of 1:50 - 100 to obtain a refined prune exosome extract.

6. Use of the prune exosomes according to claim 1 in the preparation of a product for moistening the intestines and relieving constipation, characterized in that, In step S3, adding the refined prune exosome extract to the top of a centrifuge tube pre-filled with a sucrose solution of a concentration gradient for sucrose density gradient centrifugation specifically includes the following steps: Step S31: Add sucrose solutions with mass concentrations of 60 wt%, 45 wt%, 30 wt%, and 15 wt% to the centrifuge tube in a volume ratio of 1:1:1:1 in sequence to obtain a centrifuge tube pre-filled with a sucrose solution of a concentration gradient; Step S32: Add the refined prune exosome extract to the top of the centrifuge tube pre-filled with a sucrose solution of a concentration gradient and centrifuge at 4°C for 80 - 100 minutes at 100000 - 200000 g, and take the solution in the 30 wt% - 45 wt% sucrose solution layer to obtain a purified prune exosome solution.

7. Use of the prune exosomes according to claim 1 in the preparation of a product for relieving constipation, characterized in that, In step S4, centrifuge the purified prune exosome solution at 4°C for 80 - 100 minutes at 100000 - 200000 g to obtain a prune exosome precipitate.

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