Exosome vesicle nano-selenium as well as preparation method and application thereof

Abstract

The invention discloses exosome vesicle nano-selenium as well as a preparation method and application thereof, the exosome vesicle nano-selenium is prepared by adopting a lactic acid bacteria strain YKX which is screened out from a selenium-rich area and has a high nano-selenium conversion rate, and the influence of the exosome vesicle nano-selenium on mouse glycolipid metabolism disorder based on intestinal flora is studied; results show that the nano-selenium slows down weight increase and obesity of mice to a certain extent; the content of total cholesterol, total triglyceride and low-density lipoprotein cholesterol in serum of obese mice is reduced, and the content of high-density lipoprotein cholesterol is obviously increased; secondly, the intervention of the nano-selenium slows down the increase of the abundance of the mouse intestinal tract thick-wall mycophylum induced by high diet to a certain extent at the phylum level, improves the abundance of bacteroides and the overall diversity of the intestinal flora of the mouse, and improves the disorder of the intestinal flora; and finally, the nano-selenium intervenes in a metabolite level of intestinal microorganisms, so that the content of short-chain fatty acid is increased.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of bioengineering and nutrition, in particular to an exosome vesicle nanometer selenium as well as a preparation method and application thereof. BACKGROUND

[0002] Selenium as a trace element has important biological effects and disease prevention effects, but the safety range of traditional selenium supplements is relatively narrow, and it is easy to enter the toxicity range of selenium in application to cause biological toxicity reactions, which greatly limits the applicable field. As a selenium form with the best safety and high bioavailability, nanometer elemental selenium has three synthesis methods of physics, chemistry and biology. Among them, nanometer selenium transformed by microorganisms has special physicochemical properties, strong activity, low toxicity, good absorption, fast conversion, low cost and other characteristics, and has good application prospects. In recent years, exosome vesicles from lactic acid bacteria have been proved to have good intestinal permeability and biocompatibility, and are excellent carriers for drug delivery.

[0003] However, there is almost no relevant product development at present, therefore, the study on the role of nanometer selenium in the biological activity function in vivo will provide a wider idea for discovering new functions, and can provide a new direction for people in selenium supplementation, reasonable diet and treatment of many diseases. SUMMARY

[0004] The present application aims to provide an exosome vesicle nanometer selenium as well as a preparation method and application thereof, so as to solve the problems in the background art.

[0005] In order to solve the above technical problems, the present application provides the following technical solutions:

[0006] An exosome vesicle nanometer selenium is prepared by transforming sodium selenite by Lactiplantibacillus plantarum YKX strain, and the particle size distribution is 800-1200nm, and the surface key stable functional groups of the exosome vesicle nanometer selenium include but are not limited to vesicle active proteins and polysaccharides, and the mass fraction of elemental selenium Se 0 in the exosome vesicle nanometer selenium is not less than 95%.

[0007] A preparation method of an exosome vesicle nanometer selenium comprises the following steps:

[0008] S1, obtaining a lactic acid bacteria strain: obtaining a nanometer selenium high-efficiency transformation strain Lactiplantibacillus plantarum YKX by screening from a natural selenium-rich soil environment;

[0009] S2, activation and culture of lactic acid bacteria: prepare MRS liquid medium according to the MRS medium formula, adjust pH to 6.2-6.4, sterilize at 101 Kpa, 121℃ for 30 min; after sterilization, inoculate Lactiplantibacillus plantarum YKX into the above sterilized MRS liquid medium at an inoculation amount of 5% v / v in a sterile environment, and place it in a 30℃ constant temperature incubator for culture until the OD value is 1.5, and the activated bacteria liquid is obtained;

[0010] S3, selenium-rich fermentation of lactic acid bacteria: reconfigure MRS liquid medium and sterilize, after sterilization, add 3-5 mM sodium selenite to the sterilized MRS liquid medium, inoculate the activated bacteria liquid obtained in S2 at an inoculation amount of 5-10% w / v, and incubate at 30-37℃ until the OD600 value is more than 1.5, and the selenium-rich fermentation liquid containing exosome vesicle nanoselenium is obtained;

[0011] S4, purification of exosome vesicle nanoselenium: exosome vesicle nanoselenium is obtained by sequentially purifying the selenium-rich fermentation liquid in S3 through the following steps:

[0012] Differential centrifugation: centrifuge at 10,000 x g for 30 minutes, then centrifuge at 100,000 x g for 2 hours;

[0013] Sucrose density gradient ultracentrifugation: sucrose mass fraction 20-60%, use SW 32Ti rotor, centrifuge at 100,000 x g for 18 hours;

[0014] ExoQuick-TC precipitation: add ExoQuick-TC reagent at a volume ratio of 1:3-1:5 of the selenium-rich fermentation liquid for precipitation;

[0015] Nanoparticle tracking analysis: confirm the purity of exosome vesicle nanoselenium by nanoparticle tracking analysis, and the particle / protein ratio is more than 3 x 10 10 particles / mg;

[0016] S5, exosome vesicle nanoselenium property analysis: disperse the purified exosome vesicle nanoselenium in deionized water, dilute by 1:10 gradient, ultrasonic treatment at power 300W, frequency 40kHz for 10 min to obtain a uniform dispersion system, and then perform property analysis to confirm that the prepared exosome vesicle nanoselenium meets the preset standard.

[0017] Further, the property analysis in S5 includes but is not limited to dynamic light scattering instrument detection, small-angle X-ray scattering analysis, Fourier transform infrared spectroscopy analysis, X-ray photoelectron spectroscopy analysis, and nanoparticle tracking analysis.

[0018] Use of exosome vesicle nano selenium, which is applied to regulate glycolipid metabolism, and the effective dose of the exosome vesicle nano selenium is 60-400 ug per person per day.

[0019] Compared with the prior art, the beneficial effects achieved by the present application are: the present application uses the lactic acid bacteria strain YKX with high nano selenium conversion rate screened out from a selenium-rich area to prepare nano selenium, and studies the influence of the nano selenium on the glycolipid metabolism disorder of mice based on intestinal flora; the results show that the nano selenium slows down the increase of the body weight and obesity of mice to a certain extent; reduces the content of total cholesterol, total triglyceride and low-density lipoprotein cholesterol in the serum of obese mice, and obviously increases the content of high-density lipoprotein cholesterol; secondly, the intervention of the nano selenium slows down the increase of the abundance of the Firmicutes phylum of the intestinal tract of the mice induced by high diet to a certain extent, increases the abundance of the Bacteroidetes phylum, and improves the overall diversity of the intestinal flora of the mice and the disorder of the intestinal flora; finally, the intervention of the nano selenium improves the content of short-chain fatty acids at the level of the metabolic products of intestinal microorganisms. As a new type of selenium supplement, the nano elemental selenium converted by microorganisms has many advantages, and the continuous expansion of its in-vivo biological activity function will provide a wider idea for discovering new functions, and can provide a new direction for people in the treatment of selenium supplementation, reasonable diet and many diseases. BRIEF DESCRIPTION OF DRAWINGS

[0020] The accompanying drawings are included to provide a further understanding of the present application, and constitute a part of the specification, illustrate the present application together with the embodiments thereof, and explain the present application, but do not constitute a limitation of the present application. In the drawings:

[0021] Figure 1 is a particle size distribution graph of the exosome vesicle nano selenium of the present application;

[0022] Figure 2 is a SAXS scattering spectrum of the exosome vesicle nano selenium of the present application;

[0023] Figure 3 is a stacked FTIR spectrum of the exosome vesicle nano selenium of the present application;

[0024] Figure 4 is a high-resolution XPS Se 3d spectrum of the exosome vesicle nano selenium of the present application;

[0025] Figure 5 is an abundance heat map of the main genera of the intestinal flora of the mice in the nano selenium intervention group of the present application;

[0026] Figure 6 is a graph of the effect of the nano selenium of the present application on the increase of the body weight and obesity of high-sugar high-fat mice;

[0027] Figure 7 is a graph of the influence of nano selenium of the application on food intake of mice induced by high-energy diet;

[0028] Figure 8 is a graph of the change trend of blood glucose value of mice of the application;

[0029] Figure 9 is a graph of the influence of nano selenium of the application on blood lipid indicators of mice induced by high-energy diet;

[0030] Figure 10 is a graph of the difference in OTU distribution between groups of the application;

[0031] Figure 11 is a graph of the Alpha diversity index sparsity curve of the application;

[0032] Figure 12 is a graph of the change in the main door abundance of intestinal flora of mice in different treatment groups of the application;

[0033] Figure 13 is a graph of the influence of nano selenium of the application on the content of short-chain fatty acids of mice induced by high-energy diet; DETAILED DESCRIPTION

[0034] The technical solutions in the embodiments of the application will be described clearly and completely below with reference to the drawings in the embodiments of the application. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the application.

[0035] Please refer to Figures 1-13 , the application provides a technical solution:

[0036] An exosome vesicle nano selenium (EV-Se) is prepared by converting sodium selenite by Lactiplantibacillus plantarum YKX strain, the particle size distribution is 800-1200 nm, the surface key stable functional groups of the exosome vesicle nano selenium include but are not limited to vesicle active protein, polysaccharide, and the mass fraction of elemental selenium Se 0 in it is not less than 95%.

[0037] A preparation method of an exosome vesicle nano selenium, comprising the following steps:

[0038] S1, lactic acid bacteria strain acquisition: a nano selenium high-efficiency conversion strain Lactiplantibacillus plantarum YKX is obtained by screening and separating from a natural selenium-rich soil environment;

[0039] S2, lactic acid bacteria activation and culture: prepare MRS liquid medium according to MRS medium formula, adjust pH to 6.2-6.4, sterilize at 101 Kpa, 121℃ for 30 min for standby; after sterilization, inoculate Lactiplantibacillus plantarum YKX into the above sterilized MRS liquid medium at an inoculation amount of 5% v / v in a sterile environment, and place it in a 30℃ constant temperature incubator for culture until the OD value is 1.5, and the activated bacteria liquid is obtained;

[0040] S3, selenium-rich fermentation of lactic acid bacteria: reconfigure MRS liquid medium and sterilize, after sterilization, add 3-5 mM sodium selenite to the sterilized MRS liquid medium, inoculate the activated bacteria liquid obtained in S2 at an inoculation amount of 5-10% w / v, and incubate at 30-37℃ until the OD600 value is more than 1.5, and the selenium-rich fermentation liquid containing exosome vesicle nanoselenium is obtained;

[0041] S4, exosome vesicle nanoselenium purification: exosome vesicle nanoselenium is obtained by sequentially purifying the selenium-rich fermentation liquid of S3 through the following steps:

[0042] Differential centrifugation: centrifuge at 10,000 x g for 30 minutes, then centrifuge at 100,000 x g for 2 hours;

[0043] Sucrose density gradient ultracentrifugation: sucrose mass fraction 20-60%, use SW 32Ti rotor, centrifuge at 100,000 x g for 18 hours;

[0044] ExoQuick-TC precipitation: add ExoQuick-TC reagent at a volume ratio of 1:3-1:5 of the selenium-rich fermentation liquid for precipitation;

[0045] Nanoparticle tracking analysis: confirm the purity of exosome vesicle nanoselenium by nanoparticle tracking analysis (NTA; Malvern NanoSight NS300), and the particle / protein ratio is more than 3 x 10 10 particles / mg;

[0046] S5, exosome vesicle nanoselenium property analysis: disperse the purified exosome vesicle nanoselenium in deionized water, dilute by 1:10 gradient, ultrasonic treatment for 10 min at power 300 W, frequency 40 kHz to obtain a uniform dispersion system, and then perform property analysis to confirm that the prepared exosome vesicle nanoselenium meets the preset standard;

[0047] Property analysis includes but is not limited to dynamic light scattering instrument detection, small-angle X-ray scattering analysis, Fourier transform infrared spectroscopy analysis, X-ray photoelectron spectroscopy analysis, and nanoparticle tracking analysis.

[0048] As shown in Figure 1 , the dynamic light scattering instrument (DLS, Malvern Zetasizer Nano ZS90) detection showed that the particle size distribution was 800-1200 nm, the dispersion index (PDI) was 0.16±0.03, and the zeta potential was -38.5±2.8 mV (Table 1), proving that it had monodispersity and colloidal stability;

[0049] As shown in Figure 2 , small-angle X-ray scattering (SAXS, Rigaku Nanopix) analysis showed that a characteristic peak (half-peak width ≤0.05 nm -1 ) appeared at scattering vector q≈1.7 nm -1 , corresponding to the interplanar spacing of 0.37 nm, which matched the standard value of the trigonal selenium (JCPDS 73-0465) by >99%. The results confirmed that the nano selenium core was a crystal structure, excluding the existence of amorphous or selenium oxide phase;

[0050] As shown in Figure 3 , Fourier transform infrared spectroscopy (FTIR, Nicolet iS50, ATR mode) characterization showed that there was a characteristic peak of vesicle protein C=O at 1645 cm -1 , and a Se-O-C vibration peak appeared at 1020 cm -1 , indicating that organic selenium coordination mediated surface stability;

[0051] As shown in Figure 4 , X-ray photoelectron spectroscopy (XPS, Thermo Scientific Escalab Xi + ) analysis was performed according to GB / T 19500-2004 standard: the Se 3d3 / 2 binding energy peak was located at 55.2 eV (abundance 97.4%), and the Se 3d5 / 2 was located at 59.1 eV (abundance 2.6%), proving that selenium element was dominated in the form of elemental selenium (Se 0 );

[0052] Nanoparticle tracking analysis (NTA, Malvern NanoSight NS300) combined with BCA protein detection (according to GB / T5009.124-2016) confirmed that the product particle concentration was ≥2.1×10 11 particles / mL, and the particle / protein ratio was >3×10 10 particles / mg (i.e. protein contamination <0.3 μg / mg), meeting the high-purity requirements;

[0053] The amount of sodium selenite, inoculum and culture temperature in S3 were adjusted, and other conditions remained unchanged, as follows:

[0054] Example 1, sodium selenite addition amount 3mM, inoculation amount 5%, fermentation temperature 30℃;

[0055] Example 2, sodium selenite addition amount 4mM, inoculation amount 8%, fermentation temperature 35℃;

[0056] Example 3, sodium selenite addition amount 5mM, inoculation amount 10%, fermentation temperature 37℃;

[0057] After preparation, detection was carried out, and the detection results are shown in Table 1:

[0058]

[0059]

[0060] Table 1. Physicochemical properties of EV-Se under different process conditions

[0061] From the detection results, it can be seen that the exosome vesicle nanoselenium prepared under the process conditions of Example 3 has the best effect, which is the best preparation process condition of exosome vesicle nanoselenium;

[0062] An application of exosome vesicle nanoselenium, the exosome vesicle nanoselenium is applied to regulate glycolipid metabolism, and the effective dose of the exosome vesicle nanoselenium is 60-400ug per person per day (recommended selenium intake of the World Health Organization WHO).

[0063] The influence of exosome vesicle nanoselenium intervention on mouse glycolipid metabolism indicators and intestinal flora abundance was verified by mouse experiments, and the verification method was as follows:

[0064] Animal grouping and treatment: 60 mice with initial body weight of 20±2g were selected, and standard clean grade feed and sterile water were fed for adaptive feeding for 1 week. After the adaptive feeding was completed, the mice were randomly divided into 6 groups, as shown in Table 2, which were blank group, high sugar and high fat group and nanoselenium intervention group, and the nanoselenium intervention group included nanoselenium intervention group 1, nanoselenium intervention group 2, nanoselenium intervention group 3 and nanoselenium intervention group 4.

[0065] Grouping Feeding mode Blank group Feeding normal clean grade feed High sugar and high fat group Feeding high sugar and high fat feed Nano selenium intervention group 1 Feeding high sugar and high fat feed + 2ml 0.8μg / ml EV-Se by gavage Nano selenium intervention group 2 Feeding high sugar and high fat feed + 2ml 1.2μg / ml EV-Se by gavage Nano selenium intervention group 3 Feeding high sugar and high fat feed + 2ml 1.6μg / ml EV-Se by gavage Nano selenium intervention group 4 Feeding high sugar and high fat feed + 2ml 1.2μg / ml EV-Se prepared under the best preparation process condition by gavage

[0066] Note: The mouse gavage dose was calculated from the recommended dose of humans by the body surface area normalization method, and three dose groups of low, medium and high were set. The conversion method was carried out according to FDA “Guidance for Industry: Estimating the Maximum Safe Starting Dose in Adult Healthy Volunteers”.

[0067] Table 2. Mouse experiment grouping and feeding method

[0068]

[0069]

[0070] Table 3. Nutrient composition of high-sugar, high-fat diets

[0071] The control group was fed ordinary clean-grade feed. The high-sugar and high-fat groups and the nano-selenium intervention group were fed high-sugar and high-fat feed for 5 weeks. After the 5th week, as shown in Table 2, the mice in the nano-selenium intervention group were gavaged with nano-selenium every day for nano-selenium intervention.

[0072] After intervention, glucose and lipid metabolism indicators and gut microbiota abundance in mice were measured. The results are as follows: Figure 5 ( Figure 5 The left side of the table shows the main gut microbiota species in mice that caused the changes. The right side shows the abundance fold change axis. (The values ​​are the corresponding abundance fold changes, as shown in Table 4.)

[0073]

[0074]

[0075] Table 4. Effects of EV-Se intervention on glucose and lipid metabolism parameters and gut microbiota abundance in mice.

[0076] The blank group, high sugar and high fat group, and nano-selenium intervention group were selected for further experiments and data were recorded.

[0077] Observe the mice's survival status daily, recording their food and water intake, fur color, mental state, and activity level. Weigh the mice weekly and record their weight. Record the mice's food intake and water consumption every two days, using the formula:

[0078] Energy intake (kcal) = Feed intake (g) × Feed energy content (kcal / g) + Water intake (mL) × Fructose water energy content (kcal / mL)

[0079] Food efficiency ratio (FER) = Weight gain (g) ÷ Energy intake (kcal)

[0080] Calculate the energy intake and food efficiency ratio of mice;

[0081] After recording the mouse weight weekly, the results were as follows: Figure 6As shown, the high-sugar high-fat group mice had obvious obesity and fat accumulation compared with the blank group mice before intragastric administration of nano-selenium at the 5th week, and the fat accumulation degree of the mice was observed between the blank and high-sugar high-fat groups after nano-selenium intervention, indicating that nano-selenium intervention inhibited the fat accumulation of the mice induced by high-energy diet to a certain extent. During the feeding period of the first 5 weeks, the body weight of the high-sugar high-fat group mice showed an obvious upward trend compared with the blank control group. Compared with the high-sugar high-fat group, the nano-selenium intervention slowed down the increase in the body weight of the mice. At the same time, the increase in the body weight of the mice after nano-selenium intervention was significantly lower than that of the high-sugar high-fat group (p<0.05), and the above results indicated that nano-selenium intervention could inhibit the fat accumulation and body weight increase of the mice caused by high-sugar high-fat to a certain extent;

[0082] Because the change in the body weight of the mice was closely related to the food intake, the food intake and water intake of the mice were recorded, and the results are shown in Figure 7 ((a) food intake; (b) water intake; (c) energy intake; (d) food efficiency ratio) As shown, compared with the blank group, the food intake of the high-sugar high-fat group mice was significantly lower than that of the blank group (p<0.05) Figure 7 a), the water intake of each group did not change much Figure 7 b), and the energy intake and food efficiency ratio were significantly higher than those of the control group (p<0.05) Figure 7 c, 7d). After nano-selenium intervention, the food efficiency ratio of the intervention group was significantly lower than that of the high-sugar high-fat group (p<0.05) Figure 7 d), indicating that nano-selenium improved the phenomenon of reduced food intake and increased energy of high-energy diet mice, effectively reducing the food efficiency ratio of the mice;

[0083] Fasting blood glucose determination of mice: once blood glucose determination was performed on each group before nano-selenium intervention as the pre-experiment fasting blood glucose, and blood glucose determination was performed once a week after intervention and the data were recorded; 12 h before determination, the mice were fasted but not watered, alcohol was used for disinfection, then a needle matched with a blood glucose meter was used to prick the tail vein, and the blood glucose test paper was inserted into the blood glucose meter for determination and data recording;

[0084] After the 5th week, the blood glucose of each group was measured when the nano-selenium intervention began, and overall, as shown in Figure 8 compared with the high-sugar high-fat group, the nano-selenium intervention group 4 inhibited the increase in the fasting blood glucose value of the high-diet mice to a certain extent, and the fasting blood glucose value had a downward trend before intragastric administration, indicating that nano-selenium flattened the blood glucose level of the mice induced by high-energy diet to a certain extent;

[0085] Mouse serum sample collection: After 15 weeks, the mice were fasted for 12 hours without water, and the eyeballs were removed to collect blood. The plasma was centrifuged at 5000 r / min at room temperature for 10 min, and the supernatant was collected as mouse serum and stored in a -80°C refrigerator;

[0086] Mouse serum lipid determination: The content of blood lipids in the mouse serum sample obtained by S7 was determined and recorded;

[0087] The total triglyceride, total cholesterol, high and low density lipoprotein cholesterol contents in the serum of each group of mice were detected by kit, and the results were shown in Figure 9 ((a) total triglyceride; (b) total cholesterol; (c) low density lipoprotein cholesterol; (d) high density lipoprotein cholesterol) showed that the total triglyceride, total cholesterol and low density lipoprotein cholesterol contents in the serum of the high sugar and high fat group were significantly increased compared with the blank group (p<0.05), and the high density lipoprotein cholesterol content was significantly reduced (p<0.05). After 3 weeks of intervention of nano selenium, it was found that the total triglyceride content, total cholesterol and low density lipoprotein cholesterol content in the serum of the mice were significantly reduced compared with the high sugar and high fat group (p<0.05), and the high density lipoprotein cholesterol content was significantly increased (p<0.05). The increase of total cholesterol concentration in serum can increase the incidence of cardiovascular and cerebrovascular diseases, and excessive low density lipoprotein cholesterol can cause cholesterol accumulation, and the intervention of nano selenium can reduce these changes and improve the abnormal lipid content in the serum of high dietary mice;

[0088] Small intestine and feces collection: After the mouse serum sample collection was completed, the mouse was dissected, and the mouse feces and small intestine were collected as samples under sterile conditions after dissection. The same group was collected together, labeled with a self-sealing bag, and finally the samples were stored on dry ice and at -20°C;

[0089] Intestinal microbial diversity detection: The microbial richness of the sample was detected by using the macrogene detection method, and the data was recorded after detection;

[0090] Experimental grouping Small intestine Blank group U4 Nano selenium intervention group 4 [S4] High sugar and high fat group <![CDATA[V4]]>

[0091] Table 5 Intestinal microbial diversity detection grouping

[0092] Cluster analysis: After bioinformatics statistical analysis of OTU at 97% similarity level, 1152, 336 and 828 OTUs were obtained in the blank group, the high sugar and high fat group and the nano selenium intervention group 4 respectively. As shown in Figure 10 Compared with the blank group, there were 355 differential OTUs in the nano selenium intervention group 4, and 242 differential OTUs compared with the high sugar and high fat group;

[0093] Alpha diversity analysis: Alpha diversity is often used to measure the richness of species in community ecology, which is a comprehensive index reflecting species richness and evenness. The results are shown in Figure 11 As shown in the figure, the curves of each group tend to be flat, indicating that the amount of sequencing data in the three groups is sufficient to reflect most of the microbial information in the samples, and is suitable for further study;

[0094] Analysis of changes in the composition of intestinal flora at the phylum level: The bacterial abundance of each group of mouse samples at the phylum level is shown in Figure 12 As can be seen from the figure, the intestinal flora of mice at the phylum level is mainly divided into five phyla: Firmicutes, Tenericutes, Proteobacteria, Bacteroidetes, and Actinobacteria. Compared with the U4 group (normal diet group), the abundance of Firmicutes in the V4 group (high-sugar high-fat diet group) increased significantly. Firmicutes has the largest proportion in each group, and a large number of studies have shown that the relative abundance of Bacteroidetes in obese individuals will decrease and the proportion of Firmicutes will increase. As a normal intestinal flora, Firmicutes exists in large quantities, and the increased ability of the body to obtain energy from food can be achieved through the increase of Firmicutes, ultimately leading to excessive accumulation of fat. The intestinal flora diversity of the S4 group that was intragastrically administered with nano selenium increased significantly, and the abundance of Firmicutes decreased significantly, showing a trend of returning to normal levels. This indicates that nano elemental selenium can indirectly regulate fat metabolism in mice to some extent by regulating intestinal flora;

[0095] Intestinal flora metabolite detection: Gas chromatography-mass spectrometry was used to detect the content of short-chain fatty acids acetic acid, propionic acid and butyric acid in mouse feces. After detection, the data was recorded;

[0096] SCFAs are the main bacterial metabolites produced after the fermentation of dietary fiber and resistant starch by specific colon anaerobes, and are closely related to body fat metabolism. They can slow down gastric emptying and increase satiety by regulating intestinal motility, reduce energy intake, and reduce fat accumulation. Their composition mainly includes acetic acid, propionic acid and butyric acid, and propionic acid and acetic acid can promote gluconeogenesis to regulate glucose homeostasis. This design measures the effect of nano selenium on the content of SCFAs in the feces of high-fat diet mice, and the results are shown in Figure 13((a) propionic acid; (b) acetic acid; (c) butyric acid) were significantly decreased (p<0.05) compared with the blank group. Compared with the blank group, the contents of acetic acid, propionic acid and butyric acid in the feces of the nano-selenium intervention group 4 were decreased (P<0.05). Compared with the high-sugar and high-fat group, the contents of acetic acid, propionic acid and butyric acid in the feces of the nano-selenium intervention group 4 were significantly increased (P<0.05). The results showed that the nano-elemental selenium could affect the types and quantities of intestinal flora metabolites in mice. Combined with the above experimental results, it was shown that nano-selenium not only affected the diversity of intestinal microorganisms in mice, but also affected the metabolic products of intestinal microorganisms;

[0097] Statistical processing and result analysis: The obtained experimental data were expressed as Mean ± S, and statistical analysis was performed by SPSS19.0 software. Comparison between groups was performed by one-way ANOVA. P<0.05 was considered to have significant difference, and P<0.01 was considered to have extremely significant difference. Prism 5.0 and Excel software were used for drawing.

[0098] The results showed that nano-selenium slowed down the increase of body weight and obesity in mice to some extent; reduced the contents of total cholesterol, total triglyceride and low-density lipoprotein cholesterol in the serum of obese mice, and obviously increased the content of high-density lipoprotein cholesterol; secondly, the intervention of nano-selenium slowed down the increase of the abundance of Firmicutes in the small intestine of mice induced by high diet to some extent, and increased the abundance of Bacteroidetes and the overall diversity of intestinal flora, and improved the intestinal flora disorder; finally, the intervention of nano-selenium improved the content of short-chain fatty acids at the level of intestinal microbial metabolites.

[0099] Finally, it should be noted that: the above only for the preferred embodiments of the present application, and not for the limitation of the present application, although the present application is described in detail with reference to the foregoing embodiments, for those skilled in the art, it still can be modified to the technical solutions recorded in the foregoing embodiments, or equivalent replacement of some technical features. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A type of exosome vesicle nano-selenium, characterized in that, The exosome vesicle selenium nanoparticles were prepared by converting sodium selenite into Lactiplantibacillus plantarum strain YKX, with a particle size distribution of 800-1200 nm. The key stabilizing functional groups on the surface of the exosome vesicle selenium nanoparticles include, but are not limited to, vesicle active proteins and polysaccharides, and contain elemental selenium (Se). 0 The quality percentage should be no less than 95%.

2. A method for preparing exosome vesicle selenium nanoparticles, characterized in that: Includes the following steps: S1. Acquisition of lactic acid bacteria strains: The highly efficient nano-selenium-transforming strain Lactiplantibacillus plantarum YKX was isolated and screened from natural selenium-rich soil environment; S2. Activation and culture of lactic acid bacteria: Prepare MRS liquid culture medium according to the MRS medium formula, adjust the pH to 6.2-6.4, and autoclave at 101 kPa and 121℃ for 30 min for later use; after sterilization, in an aseptic environment, inoculate Lactiplantibacillusplantarum YKX at a 5% v / v inoculation rate into the above-mentioned sterilized MRS liquid culture medium, and incubate in a 30℃ constant temperature incubator until the OD value reaches 1.5 to obtain the activated bacterial solution; S3. Lactic acid bacteria selenium-enriched fermentation: Prepare new MRS liquid culture medium and sterilize it. After sterilization, add 3-5 mM sodium selenite to the sterilized MRS liquid culture medium, and inoculate the activated bacterial solution obtained from S2 at an inoculation rate of 5-10% w / v. Incubate at 30-37℃ until OD. 600 A value of 1.5 or higher indicates the presence of selenium-enriched fermentation broth containing exosome vesicle nano-selenium. S4. Purification of exosome vesicle nano-selenium: Exosome vesicle nano-selenium was obtained from the selenium-enriched fermentation broth of S3 through the following steps: Differential centrifugation: centrifuge at 10,000×g for 30 minutes, then centrifuge at 100,000×g for 2 hours; Sucrose density gradient ultracentrifugation: sucrose mass fraction 20–60%, centrifuged at 100,000×g for 18 hours using an SW 32Ti rotor; ExoQuick-TC precipitation: Add ExoQuick-TC reagent at a volume ratio of 1:3 to 1:5 of the selenium-enriched fermentation broth to precipitate, thus obtaining exosome vesicle nano-selenium. Nanoparticle tracking analysis: The purity of exosome vesicle selenium nanoparticles was confirmed by nanoparticle tracking analysis, with a particle / protein ratio exceeding 3 × 10⁻⁶. 10 particles / mg; S5. Characteristic analysis of exosomal vesicle selenium nanoparticles: The purified exosomal vesicle selenium nanoparticles were dispersed in deionized water and diluted in a 1:10 gradient. The mixture was then sonicated at 300W and 40kHz for 10 minutes to obtain a uniform dispersion system. Characteristic analysis was then performed to confirm that the prepared exosomal vesicle selenium nanoparticles met the preset standards.

3. The method for preparing exosome vesicle selenium nanoparticles according to claim 2, characterized in that: The characteristic analysis in S5 includes, but is not limited to, dynamic light scattering instrument detection, small-angle X-ray scattering analysis, Fourier transform infrared spectroscopy analysis, X-ray photoelectron spectroscopy analysis, and nanoparticle tracking analysis.

4. An application of exosome vesicle nano-selenium, characterized in that: The exosome vesicle nano-selenium is used to regulate glucose and lipid metabolism disorders, and the effective dose of the exosome vesicle nano-selenium is 60-400ug per person per day.

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