Prune exosome, preparation method thereof and application of prune exosome in preparation of bowel relaxing product

Abstract

The invention belongs to the technical field of health-care food, and particularly relates to a prune exosome, a preparation method thereof and application of the prune exosome in preparation of bowel relaxing products. According to the preparation method of the prune exosome, deep processing is carried out on prune, the prune exosome is extracted from the prune, and miRNA nucleic acid contained in the prune exosome is used for promoting expression of mucoprotein genes in goblet cells and proliferation of beneficial bacteria such as bifidobacteria and lactic acid bacteria; the adverse effect of oxidative stress on intestinal cells and microbial communities is reduced by using the antioxidant activity of the prune exosome, and the effect of relaxing bowel is realized, so that the technical problem that deep processing research of prune is lacked in the prior art is solved.

Description

Technical Field

[0001] The present application belongs to the technical field of health food, and in particular relates to a kind of prune exosomes, a preparation method thereof, and an application thereof in the preparation of a laxative product. Background Art

[0002] With the continuous improvement of living standards, people's dietary structure has gradually changed. The intake of high-calorie, high-fat, low-fiber foods has continued to increase, making problems such as poor bowel movements and difficulty in controlling weight more common. At the same time, consumers' demand for healthy, safe and effective bowel movement and slimming products has continued to increase, prompting them to actively explore safe and effective ways to promote bowel movements and slimming.

[0003] Dietary fiber in fruits, beans, potatoes and vegetables can increase satiety, help reduce calorie intake, promote intestinal motility, help clean the intestines, and help control weight. Eating dietary fiber is an important way to lose weight. Prunes are a fruit with unique flavor, bright color and rich nutrition. They are rich in nutrients such as organic acids, vitamins, dietary fiber and polyphenols, and are low in calories and fat. The dietary fiber rich in prunes can not only effectively promote intestinal motility, but also increase the frequency of bowel movements and the softness of stool. It is an edible plant ingredient that can regulate poor bowel movements. Polyphenols can delay human aging, stimulate heat production, accelerate energy consumption, and thus regulate the body's lipid metabolism. Therefore, prunes have the application potential of promoting bowel movements.

[0004] However, the current applications of prunes are mainly direct consumption, squeezing into juice, making into preserved fruits, fermenting into prune fermentation liquid, etc. There is a lack of research on the deep processing of prunes. It is necessary to further explore the performance of deep-processed prune products. Summary of the invention

[0005] In view of this, the present application provides a prune exosome, a preparation method and an application in the preparation of a laxative product, to solve the technical problem of the lack of deep processing research on prunes in the prior art.

[0006] A first aspect of the present application provides a method for preparing prune exosomes, the preparation method comprising: extracting prune juice by ultracentrifugation and density gradient centrifugation in sequence to obtain prune exosomes.

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

[0008] Step S1, squeezing, standing, and filtering fresh prunes in sequence to obtain prune juice;

[0009] Step S2, ultracentrifuging the prune juice in a manner of increasing centrifugal force to obtain a prune exosome precipitation mixture;

[0010] Step S3, adding the prune exosome precipitation mixture into a buffer solution for resuspending to obtain a prune exosome extract;

[0011] Step S4, adding the prune exosome extract to the top of a centrifuge tube pre-filled with a concentration gradient sucrose solution to perform sucrose density gradient centrifugation to obtain a prune exosome purified solution;

[0012] Step S5, subjecting the purified prune exosome liquid to ultrahigh-speed centrifugation to obtain prune exosome precipitate.

[0013] Preferably, in step S1, the pore size of the gauze used for filtering is 80-120 meshes.

[0014] Preferably, in step S2, ultracentrifuging the prune juice in a manner of increasing centrifugal force specifically comprises the following steps:

[0015] Step S21, centrifuging the prune juice at 400-600 g for 5-15 minutes at 4° C. to obtain a low-speed centrifugation supernatant;

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

[0017] Step S23, centrifuging the supernatant of the medium-speed centrifugation at 2500-3500 g for 20-40 minutes at 4° C. to obtain a medium-speed centrifugation supernatant;

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

[0019] Step S25, subjecting the high-speed centrifugation supernatant to ultra-high-speed centrifugation at 100,000-200,000 g for 80-100 minutes at 4° C. to obtain a prune exosome precipitation mixture.

[0020] Preferably, in step S3, the resuspension process comprises: adding the exosome precipitate into PBS buffer at a mass ratio of 1:50-100 at 4°C for resuspension to obtain prune exosome extract.

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

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

[0023] Step S42, adding the exosome extract to the top of a centrifuge tube pre-filled with a concentration gradient sucrose solution, centrifuging at 100,000-200,000 g for 80-100 minutes at 4°C, taking the solution in the 30wt%-45wt% sucrose solution layer to obtain a purified prune exosome solution.

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

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

[0026] Preferably, the particle size of the prune exosomes is 100-300 nm.

[0027] Preferably, the particle size of the prune exosomes is 150-250 nm.

[0028] The third aspect of the present application provides the use of the prune exosomes described in the second aspect in the preparation of a bowel-moisturizing and laxative product.

[0029] In summary, the present application provides a prune exosome, a preparation method and an application in the preparation of a laxative product, which is used to solve the technical problem of the lack of deep processing research on prunes in the prior art.

[0030] Compared with the prior art, the prune exosomes and preparation method thereof provided in the present application and their application in the preparation of a laxative product at least include the following beneficial effects:

[0031] 1. The present application provides a method for preparing prune exosomes, which extracts prune exosomes from prunes and realizes deep processing of prunes.

[0032] 2. The present application provides an application of prune exosomes in the preparation of a laxative product, which utilizes the excellent properties of prune exosomes extracted from prunes, such as promoting the expression of mucin genes and the proliferation of beneficial bacteria, as well as antioxidant activity, to achieve the effect of laxative. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0034] Figure 1 A schematic diagram of a method for preparing prune exosomes provided in Example 1 of the present application, after gradient centrifugation in a sucrose solution with a concentration gradient of 15wt% to 60wt%;

[0035] Figure 2 A schematic diagram of the particle size distribution of prune exosomes prepared in a method for preparing prune exosomes provided in Example 1 of the present application;

[0036] Figure 3 This is a transmission electron micrograph of a single prune exosome prepared in the method for preparing prune exosomes provided in Example 1 of the present application;

[0037] Figure 4 A schematic diagram of the potential distribution of prune exosomes prepared in a method for preparing prune exosomes provided in Example 1 of the present application;

[0038] Figure 5 The electrophoresis diagram of the prepared prune exosome protein and prune juice in the method for preparing prune exosomes provided in Example 1 of the present application; Figure 5 In the figure, M: Maker, protein molecular weight standard, S1: prune exosomes (batch 1), S2: prune exosomes (batch 2), S3: prune exosomes (batch 3), ZZ: prune juice.

[0039] Figure 6 This is a lipid thin-layer chromatogram of prune exosomes prepared in a method for preparing prune exosomes provided in Example 1 of the present application;

[0040] Figure 7 The miRNA content and classification diagram of the prepared prune exosomes in the method for preparing prune exosomes provided in Example 1 of the present application;

[0041] Figure 8 This is a graph showing the antioxidant activity of prune exosomes prepared in a method for preparing prune exosomes provided in Example 1 of the present application;

[0042] Fig. 9 This is a diagram showing the advancement of ink in the small intestine of mice after taking ink and taking or not taking the prune exosomes provided in Example 1 in Experimental Example 2 of the present application;

[0043] Fig.10 This is a graph showing the propulsion rate of ink in the small intestine of mice and the time when the mice excrete the first black stool after taking ink and taking or not taking the prune exosomes provided in Example 1 in Experimental Example 2 of the present application;

[0044] Fig.11 This is a graph showing the number and weight of black stools excreted by mice within 6 hours after taking ink and taking or not taking the prune exosomes provided in Example 1 in Experimental Example 2 of the present application. DETAILED DESCRIPTION

[0045] The present application provides a prune exosome, a preparation method and an application in the preparation of a laxative product, which are used to solve the technical problem of the lack of deep processing research of prunes in the prior art.

[0046] The technical solution of the present application will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present application.

[0047] In view of the current lack of deep processing research on prunes, it is difficult to further explore their application in moistening the intestines and relieving constipation. The present application provides a method for preparing prune exosomes, which comprises: extracting prune juice by ultracentrifugation and density gradient centrifugation in sequence to obtain prune exosomes.

[0048] Studies have shown that exosomes from plants such as prunes are natural nanoparticles containing proteins, lipids, nucleic acids and secondary metabolites, which can be taken up by animal cells including humans, and exhibit a variety of biological treatment and drug delivery functions; at the same time, compared with cell-derived exosomes, plant-derived exosomes show superior biocompatibility, low immunogenicity, high safety and green sustainability, and have similar functions to the source plants. Therefore, the prune exosomes obtained by deep processing of prunes have the application potential of moisturizing the intestines and facilitating bowel movements; in addition to dietary fiber that can promote intestinal peristalsis and help clean the intestines, intestinal cells and microbial communities are also closely related to intestinal health. For example, intestinal flora imbalance caused by changes in dietary structure and other reasons can cause intestinal dysfunction and induce intestinal diseases such as constipation, and the miRNA in the prune exosomes provided in this application It can promote the proliferation of beneficial bacteria such as bifidobacteria and lactic acid bacteria by regulating the signal pathways in the intestine, and can also upregulate the expression of mucin genes in goblet cells, promote the synthesis and secretion of mucin, and thicken the intestinal mucus layer; and the prune exosomes provided in the present application also have antioxidant activity, which can reduce the negative effects of excessive reactive oxygen and other oxidative substances produced by the body on intestinal cells and microbial communities. Therefore, the prune exosomes provided in the present application improve the survival state of intestinal cells and microbial communities, promote the synthesis and secretion of mucin and the proliferation of beneficial bacteria, and play a laxative effect, thereby realizing the deep processing research of prunes and overcoming the current lack of deep processing research on prunes.

[0049] Preferably, in the preparation method of prune exosomes provided in the present application, prune juice is obtained by squeezing fresh prunes, standing and filtering in sequence, and standing and filtering can effectively remove the filter residue; then the prune juice with the filter residue removed is subjected to ultracentrifugation in a manner of increasing centrifugal force, and ultracentrifugation in a manner of increasing centrifugal force can effectively remove unbroken cells, cell fragments, cell particles, large vesicles, some protein complexes and large particle impurities in the prune juice, and retain a prune exosome precipitation mixture with a higher density; then the prune exosome extract obtained by resuspending the prune exosome precipitation mixture is added to the top of a centrifuge tube pre-filled with a concentration gradient sucrose solution for sucrose density gradient centrifugation, so that the prune exosomes and other component impurities in the exosome extract are respectively precipitated into different sucrose density areas, and the prune exosome extract is purified to obtain a prune exosome purified solution; finally, the prune exosome purified solution is subjected to ultrahigh speed centrifugation to obtain a high-purity prune exosome precipitate.

[0050] Preferably, in the preparation method of prune exosomes provided in the present application, the pore size of the gauze used for removing the filter residue is 80-120 mesh, and the ultracentrifugation is performed with increasing centrifugal force at 500g / 1000g / 3000g / 10000g / 150000g for 10-90 minutes 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 ultrahigh-speed centrifugation time of the prune exosome purified liquid is 90 minutes.

[0051] The following is a detailed description of the prune exosomes provided in the present application in combination with embodiments and experimental examples.

[0052] Example 1

[0053] Example 1 of the present application provides a method for preparing prune exosomes, which includes the steps of preparing prune juice, ultracentrifugation, density gradient centrifugation and ultrahigh-speed centrifugation.

[0054] The steps of preparing prune juice include: taking 1500g of fresh prunes, removing the cores and squeezing the prunes with a juicer for 2min (3 times), standing at 4°C for 15min, and filtering through 80-120 mesh gauze to obtain prune juice.

[0055] The ultracentrifugation step includes: firstly subjecting the prune juice to low-speed centrifugation at 500g for 10 minutes at 4°C to obtain a low-speed centrifugation supernatant; then subjecting the low-speed centrifugation supernatant to medium-speed centrifugation at 1000g for 10 minutes at 4°C to obtain a medium-speed centrifugation supernatant; then subjecting the medium-speed centrifugation supernatant to medium-speed centrifugation at 3000g for 30 minutes at 4°C to obtain a medium-speed centrifugation supernatant; then subjecting the medium-speed centrifugation supernatant to high-speed centrifugation at 10000g for 60 minutes at 4°C to obtain a high-speed centrifugation supernatant; and then subjecting the high-speed centrifugation supernatant to ultrahigh-speed centrifugation at 150000g for 90 minutes at 4°C to obtain a prune exosome precipitation mixture.

[0056] The steps of density gradient centrifugation include: adding 4°C PBS buffer to the precipitated mixture of prune exosomes at a mass ratio of 1:50-100, mixing and resuspending to obtain prune exosome extract, and preparing sucrose solutions with mass fractions of 60%, 45%, 30%, and 15%, and adding them to the bottom of the centrifuge tube in sequence at a volume ratio of 1:1:1:1; then adding the prune exosome extract to the top of the solution, and the volume ratio of the total volume of the concentration gradient sucrose solution in the centrifuge tube to the added prune exosome extract is 4:1. Slowly put it into an ultra-high speed centrifuge, centrifuge it at 4°C for 90 minutes at 150000g, and take the solution in the sucrose solution layer with a mass fraction of 30%-45%, which is the purified prune exosome solution, such as Figure 1 shown.

[0057] The ultra-high speed centrifugation step includes: centrifuging the purified prune exosome solution at 150.000g for 90 minutes at 4°C to obtain prune exosome precipitate, adding PBS buffer solution and mixing for later use, and the usage ratio of prune exosome precipitate to PBS buffer solution is 40 mg: 1 mL.

[0058] Experimental Example 1

[0059] In this experimental example, the prune exosomes prepared by the preparation method of prune 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 and potential and vesicle morphology of the prune exosomes, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to test the protein molecular weight distribution of the prune exosomes, and using lipid thin layer chromatography (TLC) to test the lipids of the prune exosomes. The expression level and characteristics of miRNA were analyzed by small RNA sequencing technology. At the same time, the antioxidant activity was analyzed by a total antioxidant capacity detection kit (Solebo, FRAP method) and a free radical scavenging capacity detection kit (Biyuntian, DPPH method). The analysis and test results are as follows: Figure 2-8 shown.

[0060] from Figure 2-8 It can be seen that the particle size range of prune exosomes extracted by the preparation method of prune exosomes provided in the present application is between 100 and 300 nm, and the potential is -7.26 mV. At the same time, the SDS-PAGE image, lipid thin layer chromatography image, and miRNA type and content image also show that the prune exosomes provided in the present application contain rich components such as proteins, lipids, and nucleic acids, and the prune exosomes provided in the present application also have excellent antioxidant activity; therefore, the prune exosomes provided in the present application contain rich proteins, lipids, miRNA nucleic acids and antioxidant activity products, among which the miRNA contained is a type of microribonucleic acid, which can regulate the expression of other genes across boundaries while regulating the physiological functions of the plant itself, and is expected to promote the expression of mucin genes in goblet cells and the proliferation of beneficial bacteria such as bifidobacteria and lactic acid bacteria, and the antioxidant activity of prune exosomes is expected to improve the oxidative stress caused by excessive production of reactive oxygen and other oxidative substances in the body, and the adverse effects on intestinal cells and microbial communities.

[0061] Experimental Example 2

[0062] In this experimental example, the intestinal moisturizing and laxative effect of prune exosomes prepared by the preparation method of prune exosomes provided in Example 1 was tested.

[0063] The testing process of laxative effect includes:

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

[0065] After 14 days, the mice were fasted but not watered for 16 h. The blank control group was gavaged with distilled water, and the model control group and prune exosome groups were gavaged with loperamide (4 mg / kg BW). 0.5 hours after gavage, the prune exosome group was given ink containing the corresponding test samples (containing 5% activated carbon powder and 10% gum arabic), and the blank control group and model control group were gavaged with ink and ate normally.

[0066] After 25 minutes, half of the mice in each group were immediately killed by dislocating the cervical vertebra, the abdominal cavity was opened and the mesentery was separated, the intestinal tube from the upper end of the pylorus to the lower end of the ileocecal region was cut and placed on a tray, the small intestine was gently pulled into a straight line, 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 front of the ink was measured as the "ink advancement length", and the ink small intestine advancement rate was calculated. For the other half of the mice in each group, starting from the injection of ink, the time of the first black stool of each animal, the number and weight of black stools within 6 hours were recorded to observe its defecation-promoting effect.

[0067] The test results of laxative effect are as follows Figure 9-11 As shown, from Figure 9-11 It can be seen that compared with the blank control group (CON) and the model control group (MOD), the propulsion rate of ink in the small intestine of the prune exosome group (EXO) mice was significantly higher, and the number and weight of black stool particles excreted by the prune exosome group (EXO) mice within 6 hours were also significantly higher than those of the blank control group (CON) mice and the model control group (MOD) mice. This shows that the preparation method of prune exosomes provided in the present application, through deep processing of prunes, prune exosomes are extracted from prunes, and the miRNA in prune exosomes can be used to promote the proliferation of beneficial bacteria such as bifidobacteria and lactic acid bacteria by regulating the signal pathways in the intestine, and can also upregulate the expression of mucin genes in goblet cells, promote the synthesis and secretion of mucin, and thicken the intestinal mucus layer. At the same time, the antioxidant activity of prune exosomes is also used to reduce the negative effects of excessive reactive oxygen and other oxidative substances produced by the body on intestinal cells and microbial communities, so as to avoid the occurrence of oxidative stress caused by excessive production of reactive oxygen and other oxidative substances by the body, which exceeds 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 them. Although the present application has been described in detail with reference to the aforementioned embodiments, a person skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some or all of the technical features thereof may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for preparing prune exosomes, characterized in that: The preparation method comprises: Step S1, ultracentrifuging the prune juice in a manner of increasing centrifugal force to obtain a prune exosome precipitation mixture; Step S2, adding the prune exosome precipitation mixture into a buffer solution for resuspending to obtain a prune exosome extract; Step S3, adding the prune exosome extract to the top of a centrifuge tube pre-filled with a concentration gradient sucrose solution to perform sucrose density gradient centrifugation to obtain a prune exosome purified solution; Step S4, subjecting the purified prune exosome liquid to ultrahigh-speed centrifugation to obtain prune exosome precipitate.

2. The method for preparing prune exosomes according to claim 1, characterized in that: In step S1, the preparation step of prune juice comprises: squeezing fresh prunes, standing them, and filtering them in sequence to obtain prune juice.

3. The method for preparing prune exosomes according to claim 2, characterized in that: In step S1, the pore size of the gauze used for filtering is 80-120 meshes.

4. The method for preparing prune exosomes according to claim 1, characterized in that: In step S1, ultracentrifuging the prune juice in a manner of increasing centrifugal force specifically comprises the following steps: Step S11, centrifuging the prune juice at 400-600 g for 5-15 minutes at 4° C. to obtain a low-speed centrifugation supernatant; Step S12, centrifuging the low-speed centrifugation supernatant at 800-1200 g for 5-15 minutes at 4° C. to obtain a medium-speed centrifugation supernatant; Step S13, centrifuging the supernatant of the medium-speed centrifugation at 2500-3500 g for 20-40 minutes at 4° C. to obtain a medium-speed centrifugation supernatant; Step S14, subjecting the supernatant of the medium-high speed centrifugation to high-speed centrifugation at 5000-15000 g for 40-80 minutes at 4° C. to obtain a high-speed centrifugation supernatant; Step S15, subjecting the high-speed centrifugation supernatant to ultra-high-speed centrifugation at 100,000-200,000 g for 80-100 minutes at 4° C. to obtain a prune exosome precipitation mixture.

5. The method for preparing prune exosomes according to claim 1, characterized in that: In step S2, the resuspension process includes: adding the prune exosome precipitation mixture to PBS buffer at a mass ratio of 1:50-100 at 4°C for resuspension to obtain a prune exosome extract.

6. The method for preparing prune exosomes according to claim 1, characterized in that: In step S3, adding the prune exosome extract to the top of a centrifuge tube pre-filled with a concentration gradient sucrose solution for sucrose density gradient centrifugation specifically comprises the following steps: Step S31, adding sucrose solutions with mass concentrations of 60wt%, 45wt%, 30wt%, and 15wt% into a centrifuge tube in a volume ratio of 1:1:1:1 to obtain a centrifuge tube pre-filled with a concentration gradient sucrose solution; Step S32, adding the prune exosome extract to the top of a centrifuge tube pre-filled with a concentration gradient sucrose solution, centrifuging at 100,000-200,000 g for 80-100 minutes at 4°C, taking the solution in the 30wt%-45wt% sucrose solution layer to obtain the prune exosome purified solution.

7. The method for preparing prune exosomes according to claim 1, characterized in that: In step S4, the purified prune exosome solution is centrifuged at 100,000-200,000 g for 80-100 minutes at 4° C. to obtain a prune exosome precipitate.

8. A prune exosome, characterized in that: The invention is prepared by the preparation method according to any one of claims 1 to 7.

9. The prune exosomes according to claim 8, characterized in that: The particle size of the prune exosomes is 100-300 nm.

10. Use of the prune exosomes according to any one of claims 8 to 9 in the preparation of a laxative product.

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